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Course Notes Biology 309

Course Notes Biology 309 Analytical Methods in Molecular Biology Fall 201 6 1

Analytical Methods in Molecular Biology Fall 2016

COURSE INFORMATION

7

LEARNING OBJECTIVES FOR BIOLOGY 309

7

HOW TO DO WELL IN BIOLOGY 309

7

HOW TO USE THESE COURSE NOTES

7

DR. MOFFATTS BEST STUDY STRATEGY FOR BIOL 309:

9

DESIGNING AND EXECUTING EXPERIMENTS IN MOLECULAR BIOLOGY

11

EXPERIMENTAL CONTROLS HELP RESEARCHERS TO DETECT ERRORS AND VALIDATE THEIR

CONCLUSIONS

12

IMAGES REPRODUCED FROM WILEY TEXTBOOKS

14

REFERENCES FOR IMAGES REPRODUCED FROM JOURNAL ARTICLES

16

TUTORIAL #1 OVERVIEW OF COURSE CONTENT AND ESSENTIAL BACKGROUND SKILLS

18

Course overview

18

Review of General Skills

20

MODULE 1 APPROACHES TO STUDYING BIOLOGY AT THE MOLECULAR LEVEL

23

APPLYING GENETIC TOOLS TO THE STUDY OF BIOLOGY

23

Forward versus reverse genetics

26

CRISPR

28

BIOCHEMICAL APPROACHES

29

What is necessary before a scientist can apply a biochemical approach to a problem?

29

Biochemical and genetic approaches are complementary

30

BIOINFORMATICS

30

USING MODEL ORGANISMS TO STUDY MOLECULAR MECHANISMS

34

BUILDING A NETWORK OF INFORMATION HOW ONE ANSWER LEADS TO THE NEXT QUESTION

40

MODULE 2

HOW TO CLONE A GENE: AN OVERVIEW

41

WHAT IS CLONING?

41

The process of cloning

42

Isolation of genomic fragments by library screening

42

Isolation of fragments by Polymerase Chain Reaction (PCR)

44

Conclusion

44

TUTORIAL #2

DESIGNING LIGATION REACTIONS

46

Background information for designing ligation reactions

46

Host restriction-modification

46

Restriction Endonucleases

46

Type I restriction enzymes

47

Type II restriction enzymes

47

Ligation

49

MODULE 3

CLONING: VECTORS & TRANSFORMATION

52

WHAT IS A VECTOR?

53

PROPERTIES OF VECTORS

53

PLASMID VECTORS

54

TRANSFORMATION

57

Measuring competency

59

Recombinant selection/screening

61

VECTORS BASED ON BACTERIOPHAGE LAMBDA COSMIDS & REPLACEMENT VECTORS

62

Packaging of viral DNA

63

Cosmids

64

Replacement vectors

64

Artificial chromosome vectors

65

TUTORIAL #3 – GENERATING TRANSLATIONAL AND TRANSCRIPTIONAL FUSIONS

66

MODULE 4

DNA ISOLATION: PLASMIDS, GENOMIC DNA & CDNA

67

EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS

67

Alkaline denaturation (plasmid purification)

71

Column purification of plasmid DNA

71

DETECTION AND QUANTITATION OF NUCLEIC ACIDS

72

TUTORIAL #4 – DNA QUANTIFICATION AND GEL ELECTROPHORESIS

74

Background information: Gel electrophoresis

74

Analytical versus Preparative electrophoresis

77

MODULE 5

SOURCES OF DNA: GENOMIC AND CDNA LIBRARIES

78

GENOMIC VERSUS CDNA LIBRARIES

78

CREATION OF A GENOMIC LIBRARY

78

Vector choice

79

Fragmentation of genomic DNA

79

Partial or complete genomic digests

80

Construction and evaluation of a genomic library

80

Storage of libraries

81

CDNA LIBRARIES

82

Isolation of total RNA

83

mRNA isolation

83

cDNA synthesis

84

MODULE 6

SOURCES OF DNA: POLYMERASE CHAIN REACTION (PCR)

86

OVERVIEW OF A PCR REACTION

86

Primer design

88

Variations of PCR

91

TUTORIAL #5 –CLONING PCR PRODUCTS

92

Addition of restriction enzyme recognition sites

92

MODULE 7

FINDING THE RIGHT CLONE

93

SCREENING LIBRARIES WITH GENE PROBES: HYBRIDIZATION

93

Hybridization stringency

94

Labelling probes

95

Templates for probe synthesis

96

Incorporation of labeled nucleotides by in vitro DNA synthesis reactions

97

Overview of the key labelling methods

98

SCREENING LIBRARIES WITH ANTIBODIES

101

Preparation of antibodies:

101

CLONING BY FUNCTIONAL COMPLEMENTATION

103

TUTORIAL #6 – RESTRICTION MAPPING

105

 

Double digests

107

SOUTHERN BLOTTING

109

Background information

109

Controls for Southern blot

112

MODULE 8

DNA SEQUENCING – CLONED GENES AND GENOMES

114

Principles of DNA

sequencing

Sequencing

114

Dideoxy (Sanger)

114

Automated sequencing

116

Sequencing larger fragments and genomes: shotgun sequencing and contig assembly

117

Next generation sequencing (NGS)

117

Animation of DNA sequencing

118

MODULE 9

SEQUENCE COMPARISONS

119

Problems encountered in sequencing of whole genomes

120

How do gene-prediction programs “hunt” for genes?

120

TUTORIAL #7 – SEQUENCE ANALYSIS USING BLAST

121

WHAT HAVE WE LEARNED SO FAR?

122

MODULE 10

ANALYSIS OF GENE EXPRESSION – TRANSCRIPTION

123

Describing transcript patterns

123

DETECTION OF TRANSCRIPTS:

124

Northern blotting

125

In situ hybridization

127

RT-PCR

129

QUANTITATIVE RT-PCR (QRT-PCR)

131

TUTORIAL #8 – TARGETED TRANSCRIPT ANALYSES

133

COMPARING TRANSCRIPTOMES

134

EST abundance

134

TRANSCRIPTOME SEQUENCING (REFERRED TO AS DEEP SEQUENCINGOR “RNA-SEQ”)

135

To be covered in class:

136

MICROARRAYS

137

Animation of microarrays

137

Subtractive hybridization

139

TUTORIAL #9 – APPLICATIONS OF TRANSCRIPTIONAL FUSIONS

141

Monitoring transcription initiation

142

Nuclear run-on assays

142

Using transcriptional reporter gene fusions to monitor transcript initiation

143

INVESTIGATING CIS ELEMENTS IN PROMOTERS

145

MODULE 11

ANALYSES OF GENE EXPRESSION – TRANSLATION

149

ANALYSIS OF PROTEIN ABUNDANCE

149

Protein Purification

150

SDS polyacrylamide gel electrophoresis (SDS PAGE)

151

Two-dimensional electrophoresis (2D gels)

153

Western or immunoblotting

156

ANALYSIS OF PROTEIN SUBCELLULAR LOCALIZATION

158

Translational fusions to reporter genes or epitopes

159

Immunolocalization

161

Protein profiling (proteomics)

162

TUTORIAL #10 – IMMUNODETECTION & PROTEOMIC EXPERIMENTS

164

MODULE 12

ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS

165

PROTEIN-PROTEIN INTERACTIONS

165

Pull-down assay using epitope tagged proteins

165

Co-immunoprecipitation

166

Yeast Two-Hybrid Assay

167

TUTORIAL #11 – PROTEIN-PROTEIN INTERACTION ASSAYS

170

MODULE 13

ANALYSIS OF PROTEIN-DNA INTERACTIONS

171

Gel shift (gel retardation assays; electrophoretic mobility shift assay (EMSA)

171

DNase I

footprinting

175

APPENDIX SUMMARY OF KEY PREREQUISITE KNOWLEDGE

177

OVERVIEW OF NUCLEIC ACID STRUCTURE AND GENE EXPRESSION

177

Nucleic acid structure

177

Monitoring DNA denaturation

180

RNA structure

181

Nucleic acid synthesis

182

Coiling and supercoiling of DNA

184

GENE STRUCTURE AND ORGANIZATION

186

Common genetic elements

186

Redefining the term gene…

187

Transcription

188

Translation

188

Gene organization in prokaryote and eukaryote genomes

190

Course Information

Learning objectives for Biology 309

By the end of this course, you should be able to:

1. Explain the use of genetic, biochemical, and bioinformatic approaches to address biological questions at the molecular level.

2. Select appropriate techniques to test experimental hypotheses, as well as analyze and interpret data from these methods.

3. Select positive and negative controls for specific techniques and explain what the controls validate in each case.

4. Describe the procedures of key techniques used to monitor/characterize DNA, RNA, or protein abundance and indicate the benefits and limitations of each technique.

5. Recognize the benefits of working collaboratively to solve biological questions at the molecular level.

How to do well in Biology 309

To be successful on midterms and the final exam you should:

1. Read the textbook before class. There are very few pages to read in each case.

2. Review the learning objectives. They will help you filter the information.

3. Turn off your phone and do not do email or use social media during the class.

4. Attend lectures and take notes by hand rather than photographing powerpoint slides; make an effort to write down what you hear in your own words.

5. Ask questions and participate in class.

6. Use your iClicker – learning is difficult and using iClickers in class can help make the learning process more effective.

7. Regularly make review notes in whatever style works for you.

8. Prepare for the weekly tutorial activities- these are not optional and are worth marks!

9. Self-evaluate your understanding of the course material regularly.

10. Post questions on the message board and answer the queries of your classmates.

11. Come to see me during my office hours if you have further questions.

How to use these course notes

These notes are an outline of the course material for Biology 309. They have been designed to provide you with a useful framework for taking notes in class and a guideline for reading the textbook which is a compilation of the first chapter of “From Genes to

Genomes” 3rd edition by Dale, von Schantz and Plant, and two chapters of “Fundamental Molecular Biology” by Allison. Many of the figures originate from these books and are reprinted here with the permission of the publisher, Wiley. B. Moffatt developed all the other figures in these notes for an online version of a related course, Biol 330, except for those attributed to other sources.

These notes are not a replacement for coming to class, consulting the textbook or making your own notes.

Taking notes is a learned skill and takes practice. It involves more than jotting notes on the side of a powerpoint slide. You will find that you learn better by listening and then recording what you have heard, in your own words. Some students can do this in class whereas others make study notes later on. In either case, try to write your conclusions in your own words. If you find you cannot summarize what you have heard, that means you didn’t fully understand the concept and need more information. There is plenty of research that shows taking hand-written notes improves learning. For example, “A recent study in the journal Psychological Science found university students who were asked to hand-write notes during lectures were better able to answer questions based on the lectures later, compared with those who typed their notes using a laptop. The researchers suggest handwriting required the students to engage more in processing the information, selecting only the most important details, instead of mindlessly transcribing what they heard.” (Wendy Leung, Globe and Mail, August 24, 2014.) I often meet students who haven’t had trouble taking notes until 2 nd or 3 rd year. The Student Success Office has a workshop outlining various options. One popular method is the Cornell System of notetaking.

Building on past course content: The prerequisite courses for Biol 309 are Biol 130 and 239. Biol 240 is also highly recommended. You are expected to know the material from the required courses, in particular key concepts relating to mutant isolation, gene expression and DNA replication. There will be a “pre-knowledge” test on the second Monday of term to evaluate how well you understand key concepts. If your grade is not

what you had anticipated or hoped, please come to meet with Dr. Moffatt during office hours to discuss how to get back on track.

Tutorials: We will cover fundamental applications/topics not addressed in class during the tutorials. In this way you will have an opportunity to think more deeply about these methods. You should consider your attendance to be mandatory because you need to hand in your answers to the tutorial questions at the end of each session. Your understanding of these topics will be assessed on the midterms and the final exam too.

The tutorial sessions will be “flipped” classes: you will watch a short video before each tutorial that explains the new material and then work on questions during the tutorial. You must watch the video to be able to do the questions. The teaching assistants will not give any presentations but are instead there to answer questions. Dr. Moffatt will attend the tutorials as well. The questions to be answered will be posted by Tuesday evening each week on LEARN.

Dr. Moffatt’s best study strategy for BIOL 309:

Consider how you take notes, participate in class and study. Each of these has a big impact on your understanding of new information.

My very best learning strategy for this course:

1. Once a week, review the material covered in the past 3 classes, by consulting the

required readings, text on reserve / listening to the summary podcasts. Review the tutorial questions and answers too. Use the learning objectives of each module to focus your efforts.

2. Close all these resources.

3. Pretend you are now the course instructor. What is a straightforward question worth 2 marks that evaluates a key concept in the past week’s course material, including the tutorial. Make your question something that will test fundamental understanding and not memory work. Now think of a longer question, perhaps with

a couple of parts of just generally require more thought or detail (4 marks).

4. you cannot think of any good questions you need to go back and review some more.

If

5. If you have your questions, send them to Dr. Moffatt or better yet, come to office hours. It is really an excellent way to learn to filter the information effectively. This works for lots of courses and helps you have the same perspective on the material as your instructor.

Review the information you learned on these topics in BIOL 130 and 239 as we go. You will then more readily see how you are learning more details rather than make the mistake of thinking you learned the topic already.

Designing and executing experiments in molecular biology

In molecular biology there are some techniques that may be more survey-like (e.g. transcriptome profiling) whereas others test specific hypotheses. Some experiments involve in vitro assays and others involve in vivo measurements. In all cases, it is essential for the researcher to know that their results are valid: that the assay used is providing meaningful results. One way to evaluate whether results are reliable is to replicate the experiment several times. This allows the researcher to estimate the variation in the results and from this be more confident in how to interpret them. Variation in biology experiments arises from technical and biological sources.

To understand the meaning of technical variation, consider weighing a spoonful of sugar 10 times. Even if you try to do this exactly the same way each time, there will still be some small differences in the weights. Now consider doing a biochemical assay that involves multiple components. Each has to be measured carefully as it is added to the reaction vessel. But despite how careful the researcher is, there will be undoubtedly be some variation on each measurement and each contributes to differences in the results of seemingly identical replicates. In addition to this source of variation, there can be differences in the execution of the experiment itself. For example, the quality of reagents; controlling the reaction period and conditions; separating the products of a reaction reproducibly etc. can all contribute to variation between replicate samples. On top of this there are also technical errors due to a lack of knowledge or attention to detail.

Biological differences also contribute to sample variation. Molecular biology analyses often involve assays of subcellular components, such as nucleic acids or proteins. To reduce this variation researchers prepare individual samples from the same tissues of genetically identical organisms of the same age. Both technical and biological variation must be reduced as much as possible for any experiment to give meaningful results.

Aside from reducing unnecessary variation between samples, control samples have to be included along with the test samples, in order to be sure that the experimental protocol is valid. The control samples are what give the researcher confidence that their results mean something. For example, if no difference is observed between two samples, this may be because there really isn’t a difference between them or it may mean there was something

wrong with the assay on that day. Thus, experimental controls are an absolutely essential component of every experiment and must be included every time. If you don’t have enough time or reagent to include the control, you shouldn’t bother with the experiment at all. They are that important.

Experimental controls help researchers to detect errors and validate their conclusions

When carrying out experiments, researchers collect data from the analysis of test samples and then interpret these results. For their conclusions to be meaningful, they have to be sure that test being used is working as expected: that positive and negative test results are a true reflection of what they believe is being measured.

“Controls” are samples that evaluated alongside the test samples and should give predictable results, if the test is working correctly. Positive control: If the assay is working correctly (i.e. measuring what you think it is), these samples will give a positive signal (i.e. you will see a predictable result – e.g. colour change, DNA product etc.). The positive control samples must give the expected signal, if the technique was done correctly.

Negative controls: the negative control samples should not give a signal, if the technique was done correctly.

Ideally one tries to have both types of controls for an experiment but sometimes this is not possible.

Controls may be samples that leave out a key reagent, evaluate a different genotype or tissue or substitute an important component. To design these you first anticipate what your test will show and then try to think of all the ways that a positive or negative result could be misinterpreted. Then you try to design a sample that would test for that error.

The concept of controls is new to many students because it is not something we do in our daily lives often. For example, what do you do when you don’t get an internet signal on your computer? You try to connect to a “reliable site” – this is a positive control – it is a

site that you should be able to connect to if the internet is functioning. As we go along this term, try to think about how each assay or experiment might give invalid results and what samples you should include in your experiment to test for these factors.

Images reproduced from Wiley textbooks

This material is reproduced with the permission of John Wiley & Sons Canada, Ltd:

From “Genes to Genomes” 2 nd edition by Jeremy Dale and Malcolm von Schantz 2007 (ISBN 0-470-01733-3)

Textbook Figure or Table Number

Page number in course notes

3.1

38

3.6

40

4.1

64

From “Genes to Genomes” by Jeremy Dale, Malcolm von Schantz and Nick Plant 2012 (ISBN 978-0-470-68385-9)

Textbook Figure or Table Number

Page number in course notes

2.9

44

2.22

53

2.25

59

2.26

59

2.30

61

2.4

67

2.5

69

4.1

84

4.3

86

3.15

108

6.1

123

13.8

138

1.9

179

1.10

180

1.11

181

From: “Fundamentals of Molecular Biology" by Lisbeth A. Allison 2012 (ISBN 978-1-118-

05981-4)

Textbook Figure or Table Number

Page number in Course notes

Figure 5.20

51

Figure 9.11

24

Figure 8.1

61

Table 8.2

51

Figure 8.9

61

Figure 8.5

97

Figure 8.14

105

Figure 8.7

108

Figure 8.17

114

Figure 16.7

117

Figure 16.8

118

Figure 9.9

129

Figure 9.1

139

Tool Box 9.3 Figure 1c

151

Figure 9.10a

154

Figure 9.14a

163

Figure 9.14c

164

Figure 9.14b

165

Figure 9.13a

169

Figure 9.13c

173

References for images reproduced from journal articles

Cho J, Yu N-Y, Choi J-H, Sim SE, Kang SJJ, Kwak C, Lee S-W, Kim J-I, Choi DII, Kim VN and Kaang B-K. (2015) Multiple repressive mechanisms in the hippocampus during memory formation. Science 350: 82-87.

Foresti O, Rodriguez-Vaello V, Funaya C and Carvalho P (2014) Quality control of inner nuclear membrane proteins by the Asi complex. Science 346: 751-755.

Gamerdinger M, Hanebuth AM, Frickey T and Deuerling E. (2015) The principle of antagonism ensures protein targeting specificity at the endoplasmic reticulum. Science

348:201-207.

Harbauer AB, Opali ń ska M, Gerbeth C, Herman JS, Rao S, Schönfisch B, Guiard B, Schmidt O, Pfanner N and Meisinger C. (2014) Cell cycle–dependent regulation of mitochondrial preprotein translocase. Science 346:1109-1113.

Huarte M. (2016) A lncRNA links genomic variation with celiac disease. Science 352: 43-

45.

Ishimura R, Nagy G, Dotu I, Zhou H, Yang X-L, Schimmel P, Senju S, Nishimura Y, Chuang JH, and Ackerman SL. (2014) Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration. Science 345: 455-459.

Kwon I, Xiang S, Kato M, Wu L, Theodoropoulos P, Wang T, Kim J, Yun J, Xie Y, McKnight SL. (2014) Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science 345: 1139-1145.

Liu X, Feng H, Huang D, Song M, Fan X and Xu G. (2015) Two short sequences in OsNAR2.1 promoter are necessary for fully activating the nitrate induced gene expression in rice roots. Scientific Reports 5: 11950 doi:10.1038/srep11950

Mähönen AP, Bonke M, Kauppinen L, Riikonen M, Benfey PN and Helariutta Y. (2000) A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes & Development 14: 2938-2943.

Marris E. (2015) Geneticists reveal what makes great rice. Nature

doi:10.1038/nature.2015.17918

Matsui K, Umemura Y and Ohme-Takgi M. (2008) AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis. Plant Journal 55: 954–967.

Merchante C, Brumos J, Yun J, Hu Q, Spencer K R, Enriquez P, Binder BM, Heber S, Stepanova AN and Alonso JM. (2015) Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2. Cell 163: 684-692.

Moreno-Risueno MA, Sozzani R, Yard GG, Petricka JJ, Vernoux T, Blilou I, Alonso J, Winter CM, Ohler U, Scheres B and Benfey PN. (2016) Transcriptional control of tissue formation throughout root development. Science 350: 426-430.

O’Malley, RC, Huang S-SC, Song L, Lewsey MG, Bartlett A, Nery JR, Galli M, Gallavotti A and Ecker JR. (2016) Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape. Cell 165: 1280-1292.

Ori A, Toyama BH, Harris MS, Bock T, Iskar M., Bork P, Ingolia NT, Hetzer MW and Beck M. (2015) Integrated Transcriptome and Proteome Analyses Reveal Organ-Specific Proteome Deterioration in Old Rats Cell Systems 1: 224237.

Pennisi E. (2015) Gene turns mosquito into a vampire.

www.sciencemag.org/news/2014/11/gene-turns-mosquito-vampire

Schumacher MA, Tonthat NK, Lee J, Rodriguez-Castañeda FA, Chinnam NG, Kalliomaa- Sanford AK, Ng IW, Barge MT, Shaw PLR and Barillà D. (2015) Structures of archaeal

DNA segregation machinery reveal bacterial and eukaryotic linkages. Science 349:1120-

1124.

Service K. (2015) Yeast engineered to brew opiods. Science 350:1458-1463.

Ubeda-Tomás S, Swarup R, Coates J, Swarup K, Laplaze L, Beemster GTS, Hedden P, Bhalerao R and and Bennett MJ. (2008) Root growth in Arabidopsis requires gibberellin/DELLA signalling in the endodermis. Nature Cell Biology 10: 625-628.

Whittington CM, Griffith OW, Qi W, Thompson MB and Wilson AB. (2015) Seahorse brood pouch transcriptome reveals common genes associated with vertebrate pregnancy. Mol Biol Evol 32: 3114-3131.

Zhao D, Ni W, Feng B, Han T, Petrasek MG, and Ma H. (2003) Members of the Arabidopsis-SKP1-like Gene Family Exhibit a Variety of Expression Patterns and May Play Diverse Roles in Arabidopsis. Plant Physiology 133: 203-217.

Tutorial #1 Overview of course content and essential background skills

Course overview

Tutorial #1 Overvi ew of course content and essential background skills Course overview 18
19

Review of General Skills

Along with recalling basic facts about cellular components, it is important to review

general numerical and experimental tools that are essential for molecular research before

starting this course. For this tutorial you should review:

Basic numeracy skills (i.e. arithmetic without a calculator)

Interconverting SI units

Converting moles of DNA to weight

Electrophoresis – watch JoVE video

Centrifugation – watch first 5.4 min of JoVE video

Genome complexity

Experimental controls (pg 9-11 of course notes)

To prepare for the tutorial this week you will review these concepts by doing some

questions after watching videos of the indicated techniques so that you are thoroughly

ready to begin adding more information to your knowledge framework. Take this review

seriously; it is the lack of foundational information that causes most students difficulties in

this course.

To access the JoVE videos you have to either use a campus computer or login in to the library’s proxy server via the “connect from home” link. If you have never done this before, there are instructions on the course LEARN site to take you through the steps.

Connect to JoVE by Browsing the “J” selection in the Research Database:

LEARN site to take you through the steps. Connect to JoVE by Browsing the “J” selection

Select the JoVE link in the list:

Select the JoVE link in the list: Then, select the “Basic Biology” collection 21

Then, select the “Basic Biology” collection

Select the JoVE link in the list: Then, select the “Basic Biology” collection 21

For the tutorial this week you should watch these two videos:

For the tutorial this week you should watch these two videos: Located in: Basic Methods in

Located in: Basic Methods in Molecular and Cellular Biology

Located in: Basic Methods in Molecular and Cellular Biology Located in: General Laboratory Techniques (only need

Located in: General Laboratory Techniques (only need to view first 5.4 min)

Module 1 Approaches to studying biology at the molecular level

Learning Objectives

Explain the applicability of biochemical and genetic approaches to the study of specific biological questions

Describe the types of information gained from using each approach

Identify how the results from using one of these approaches provides tools for the alternate approach

References: class discussion

Two main approaches have been commonly used to address biological questions at the

molecular level:

1. Genetic Approach

2. Biochemical Approach

More recently a third approach is often used too; this involves computer analysis of nucleic

acid or protein sequences accessed from genome-wide experiments. This is called a

bioinformatic approach. We will discuss this more a bit later.

Applying genetic tools to the study of biology

Genetic approaches are used to identify genes important in developmental or physical

aspects of an organism. This is called Forward genetics – find a mutant and then identify

what gene causes the phenotype.

a mutant and then identify what gene causes the phenotype. Two teams of molecular geneticists, working

Two teams of molecular geneticists, working independently, have identified a gene that controls both shape and texture and can be selected for without sacrificing the yield of the crop…The gene can induce

radical changes in shape by promoting longitudinal cell division over transverse cell division. The more copies of a particular version — or allele — of the gene that a variety has, the longer the grain. The gene is dominant, which makes it useful for creating hybrid varieties.

www.nature.com/news/geneticists-reveal-what-makes-

great-rice-1.17918

These are processes that cannot be studied in vitro (which literally means “in glass” to reflect doing experiments in a test tube.)

Once you identify the genes causing these phenotypes, you can be sure they are involved in the process. From there you can isolate the proteins these genes encode and apply biochemical tools to the problem

What is a mutant? (Describe in your own words)

How are mutations induced?

• Exposure to mutagens such as chemicals, radiation (UV, gamma), genetic elements (e.g., transposons, TDNA, CRISPR)

What types of DNA changes might a mutant have?

• Substitution – single nucleotide changes (or single nucleotide polymorphisms SNPs; pronounced ‘snips’)

Small Insertion or Deletion: (called indels) can result in frameshift mutations

Larger Insertions or Deletions or Inversions

1. How might these mutations be used for molecular analyses ?

Research Example using SNPs:

Research Example using SNPs: Gluten triggers inflammation. The action of lnc13 in a normal macrophage or
Research Example using SNPs: Gluten triggers inflammation. The action of lnc13 in a normal macrophage or

Gluten triggers inflammation. The action of lnc13 in a normal macrophage or a macrophage with an active inf ammatoryresponse is shown. A decrease in available lnc13 (due to its increased association with DC2P) is linked to celiac disease. lnc13 function is impaired by the change in RNA sequence caused by a risk-related SNP, rs917997 (red star). Science 352: 43-45.

How are mutants of interest identified?

• Determine what “normal” is for the trait of interest.

• Create a population of mutagenized organisms by exposure of a homozygous wild-type population to a mutagen.

Screen for mutants by looking for those altered in the trait of interest.

• Sometimes can select mutants of interest – if you have conditions where the mutant has a growth advantage.

• Map mutation using molecular markers (Single nucleotide polymorphisms <http://learn.genetics.utah.edu/content/pharma/snips/> or directly sequence the genome.

Consider this situation: You want to identify genes involved in what regulates jumping in frogs. it is impossible to grind up a frog and then purify the proteins that regulate jumping because you can’t assay jumping in an extract. This is when you need to use a genetic approach: you find frogs with altered abilities to jump from within a mutagenized population of frogs and then figure out what genes are mutated.

List 6 types of mutant behaviors that might be useful to identify genes that regulate jumping.

1.

2.

3.

4.

5.

6.

Forward versus reverse genetics

Forward genetics – find a mutant and then identify what gene causes the phenotype.

Reverse genetics – start with a gene of interest; create an organism that expresses that gene abnormally due to the introduction of additional sequences (transgenes) that lead to the degradation of all complementary mRNAs. This technique is called “gene silencing”.

Thus, these transformants can be used to learn the phenotype of an organism with reduced abundance of a specific mRNA (the “target mRNA).

This is exactly what scientists do to silence genes by “reverse genetic” approaches. An additional copy of all or part of the coding region is introduced into the organism so that the antisense strand is transcribed.

The fascinating thing is that plants, fungi and animal cells actually use this type of gene silencing as a method to regulate gene expression!

Learning the mechanisms behind these real-life examples of RNA silencing has allowed scientists to develop novel ways to reduce the expression of target genes of interest. These silencing techniques involve small RNA molecules (siRNAs = short interfering RNAs or miRNAs = microRNAs) which are small RNA molecules complementary to the target mRNA.

are small RNA molecules complementary to the target mRNA . Distinguish, in your own words, the

Distinguish, in your own words, the difference between forward and reverse genetics:

CRISPR

CRISPR CRISPR is a genetic tool developed from an adaptive immune system designed to protect microbes

CRISPR is a genetic tool developed from an adaptive immune system designed to protect microbes from pathogen (e.g. phage) infection. It was discovered in 1993 but its molecular analysis was only first reported in 2005. Since that time, applications of the natural system have led to tools for precise genome editing (mutations), site-specific introduction of heterologous sequences into genomes and RNA editing systems.

The following list of titles is taken from the journal “The Scientist” and lists just a few of the topics addressed using CRISPR that were published in the journal in the previous 2 months!

(Image not available due to copyright restrictions – available for download from LEARN)

< www.the- scientist.com/?articles.list/tagNo/6156/tags/crispr/>

Biochemical approaches

In general terms, biochemistry is the study of proteins and enzyme activities. It includes the study of protein structure, ligands, modifications and subcellular localization. List five types of information that biochemical analysis may reveal about a protein or enzyme under study:

What is necessary before a scientist can apply a biochemical approach to a problem?

Need to prepare an assay mix containing the protein/enzyme of interest in its native, active state. Must consider the appropriate pH buffer and necessary salts, cofactors and protease inhibitors.

Must be able to measure the protein or enzyme activity or developmental trait quantitatively. This is called an assay. The assay is a quantitative measurement. What does this mean? Explain this in your own words.

Amazing assays are developed for specific projects: For example, it is now possible for

researchers to measure the following:

Binding of nucleosomes to nuclear pore proteins,

Epigenomic changes in ancient DNA,

Molecules that preferentially inhibit the splicing of a specific intron in a human cell,

Where ribosomes stall while translating specific mRNAs.

In many of these cases, the assays can be done on the whole genome at one time!

Biochemical and genetic approaches are complementary

Several types of information can be learned from the pure protein that would be helpful

in acquiring the corresponding gene (using genetic approaches or tools). By the time you

are done this course you will be able to list the steps involved in each case (i.e.

biochemical to genetic and vice versa).

Bioinformatics

BEFORE applying either of these approaches scientists now use computer-based or

bioinformatic tools.

Bioinformatics is the combination of biology and computer science that involves the

collection of protein and nucleic acid information into searchable databases that allow

comparative analysis to be simplified.

With the availability of complete genome sequences and nucleic acid and protein

databases it is possible to search for information that may help with future genetic or

biochemical experiments.

Examples:

If a scientist identifies a gene sequence of a mutant these databases might reveal:

Other members of gene family or related genes in same organism

Similar genes in other organisms

mRNA sequences and thus predicted amino acid sequence

Map position

Start site of transcription

Identifies site of mutation relative to wild-type sequence

If a scientist is interested in studying a particular enzyme activity, the complete genome sequence could be scanned for:

A gene encoding this activity based on known sequence of related enzyme from another organism

Presence of cDNA sequences encoding this enzyme based on known complete or partial amino acid sequence

Indicate the presence of other members of enzyme pathway; number of copies of gene encoding this activity

If the information they wanted to know is already determined

There are many databases, but the most comprehensive is Genbank which is maintained by the National Center for Biotechnology Information (NCBI). This database can be accessed at www.ncbi.nih.gov. This is a repository for all publicly available sequences. When a scientist publishes a paper describing a new gene or protein sequence they must make this sequence available to the public by first submitting it to this database.

There are other databases, often specific for one organism or enzyme activity (e.g. Drosophila, mouse, yeast, human genes) that are also useful research tools. These databases often contain datasets of other researchers from large scale microarray experiments, proteomic or subcellular localization studies. The data are made publicly available and anyone can search for information about their “gene of interest” without even starting their experiments.

32
32

Conclusion

By isolating and purifying a protein, a scientist gains information that cannot be acquired

by genetic approaches. This can provide the tools to isolate the corresponding gene and

understanding the genetic signals that control its expression, its gene sequence, number of

introns, map location etc. In fact, almost all projects now involve both genetic and

biochemical approaches. Before either of these are considered, a researcher consults what

information is already known about their system, using literature databases (e.g, PubMed,

Web of Science) and sequence repositories (e.g., NCBI Genbank).

Terminology to master

Genetic approach, biochemical approach, complementary versus complimentary, mutation, mutant, mutagen, screen, selection, genotype, phenotype, dominant, recessive, forward, reverse genetics, siRNA, gene silencing, assay, quantitative, NCBI, Genbank, annotation, accession

Using model organisms to study molecular mechanisms

Learning objectives:

Identify features that distinguish eukaryote genomes.

List the common attributes of model organisms. Explain the importance of each.

List the specific attributes of the key model organisms.

Explain the benefits and drawbacks of using a model organism for molecular genetic studies.

Reference: Focus box 9.1 (pg 74-75 of Required Readings); see also:

S Fields, M Johnston. Wither model organism research. Science (2005) 307-1885-

1886.

This article is available online via UW’s ejournals (instructions for how to connect to an

ejournal are provided on the Biol 309 Learn site). Note: the content of this article will

NOT be tested in the pre-knowledge test but will give you a valuable perspective on the

use of model organisms and be covered on the midterm.

Eukaryote genomes are complex

What does complexity mean with respect to DNA sequences? How is this defined?

Eukaryote genomes vary in their sequence complexity.

Estimating genome information content:

It is helpful as we go along to have a general concept of the coding capacity of various

model organism genomes:

 

Genome

# Protein-

% of

Average size of a gene

Transcripts/cell

size

coding

genome that

(Mbp)

genes

encodes

proteins

 

E. coli

4

4200

90%

1200

bp

 

Yeast

12

6000

70%

2000

bp

 

(only 4%

have introns)

C. elegans

97

19,000

25%

5000

bp

 

(including

introns)

human

3000

~21,000

1-2%

10-20,000*

8-15,000

different transcripts/cell; Each cell may have ~50,000 different polypeptides

It is also helpful to be able to readily interconvert protein size to a corresponding mRNA or gene size:

Example:

“average protein” = 400 aa. Average MW of an amino acid = 100 daltons Average protein MW = 40,000 daltons

Each amino acid requires 3 nt codon Average coding region is 1200nt + several hundred nt for each untranslated region Studies at the molecular level are simplified by using less complex organisms; these are

called model organisms.

It is assumed that the simpler organism has similar features (anatomy, physiology, stress

responses, gene sequences etc.) to those of the more complex organisms so that molecular

information learned from the simpler organism will be applicable to those that are more

complex.

Model organisms have several traits in common. How does each trait aid the study of biology at a molecular level?

Small genome size

Low amount of repetitive DNA

Diploid (not a trait of all model organisms)

Short life cycle

Small, cheap to maintain

Transformable

If an organism isn’t transformable many techniques can’t be used. Keep a list here as we proceed of the methods that require transformation.

Examples of Model Organisms: After reading over the text pages make notes on the key attributes and uses each of these organisms. Also, consider how each is maintained. Which are grown in liquid culture? Which are multicellular organisms?

Escherichia coli

Saccharomyces cerevisiae

Caenorhabditis elegans

Drosophila melanogaster

Danio rerio

Arabidopsis thaliana

Mus musculus

Xenopus laevis

There are numerous benefits to using model organisms. List at least 8.

But there are drawbacks to using them too! List at least 4.

Building a network of information – how one answer leads to the next question

In the case of this course, you will learn the specifics of several new methods. However, when researchers use these methods to address specific questions they first mentally answer the following questions:

1. What do I want to know?

2. What method will provide this information?

3. What do I need to carry out this method? (e.g. biological material, controls, reagents, equipment etc.)

4. What could go wrong in this method and what controls can I use to test for these errors and ensure my results are valid?

5. Assuming that the experiment tells me what I wanted to know, what is the next step?

As you proceed this term, consider the answers to each of these questions for the techniques we cover. This will help you to link the methods and see how they are linked to address research questions involving multiple steps.

Conclusion:

The use of model organisms has led to great technical advancements as well as a wealth

of knowledge that has provided insight to the molecular mechanisms mediating the growth

of more complex organisms including humans. However, model organisms are not ideal for

the study of all problems. For this reason there are many less common model organisms

used to address specific research topics.

Terminology to master:

model organism, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Danio rerio (Zebrafish), Xenopus laevis, Drosophila melanogaster, Mus musculus, annotation, accession, diploid, repetitive DNA, transformable, cis elements, trans factors

Module 2

How to clone a gene: an overview

Learning objectives

Define and describe the terms “a recombinant molecule” and “cloning”.

Explain the steps in generating a recombinant molecule.

Describe how a genomic library is made.

Explain hybridization and how it can be used to screen a genomic library.

Describe the process of a polymerase chain reaction and how it can be used to isolate specific DNA fragments.

Reference: Genes to Genomes 2 nd edition, pg 21-29 (available online)

What is cloning?

Cloning is the asexual propagation of genetically identical organisms; when applied to

molecular biology, the term is used to mean the generation of an organism that contains a

specific piece of DNA. The organism is usually a bacterium, which by replicating generates

many copies of the same DNA fragment.

usually a bacterium, which by replicating generates many copies of the same DNA fragment. Genes to

Genes to Genomes 2nd edition

The process of cloning

The process of cloning involves ligating a DNA fragment “of interest” to a vector DNA molecule, creating recombinant DNA molecules. The ligation products are introduced into the host cell (most often bacteria) in a process called transformation. Once this bacterium replicates for 30 generations (10 h) there will be 10 9 progeny and each will carry a copy of the gene of interest!

There are numerous types of vectors including plasmids and bacteriophages.

There are three steps involved in the process of cloning:

There are three steps involved in the process of cloning: Isolation of genomic fragments by library

Isolation of genomic fragments by library screening

Gene libraries provide a means to isolate specific cloned fragments. A genomic library is created by ligating each of the restriction fragments of a genomic digest into a vector. Each bacterial colony is transformed with a different recombinant plasmid so that ideally every fragment in that genome is cloned.

The transformants are screened with modified fragments specific for a target sequence. These modified fragments

The transformants are screened with modified fragments specific for a target sequence. These modified fragments are called probes.

The probe detects the target sequence in a hybridization reaction.

Question: The probes used for hybridization reactions are generated by in vitro DNA synthesis reactions. In the simplest case these reactions require:

1. DNA template

2. 4 deoxynucleotide triphosphates (dNTPs); one being radioactive.

3. Buffer containing Mg 2+ (to maintain enzyme activity)

4. DNA polymerase

5. DNA primer (to initiate DNA synthesis)

containing Mg 2 + (to maintain enzyme activity) 4. DNA polymerase 5. DNA primer (to initiate
containing Mg 2 + (to maintain enzyme activity) 4. DNA polymerase 5. DNA primer (to initiate

Isolation of fragments by Polymerase Chain Reaction (PCR)

A technique called polymerase chain reaction (PCR) is an alternate way to recover

a specific genomic fragment. PCR is an in vitro DNA synthesis reaction.

fragment. PCR is an in vitro DNA synthesis reaction. Comparison of genomic cloning versus PCR isolation

Comparison of genomic cloning versus PCR isolation of genomic fragments

Genomic cloning

PCR isolation of genomic fragments

Takes about few weeks to do if everything goes well, but can be longer.

Takes 1-2 days to order and receive the primers and a few hours to do the PCR, if genomic DNA is available.

Need a probe

Need sufficient sequence information to design primers; no probe

Usually involves use of radioactivity

No radioactivity needed.

Conclusion

Cloning provides a limitless source of a specific DNA. There are numerous types of cloning vectors and different methods to isolate and detect DNA sequences for cloning. Two commonly used methods are library screening by hybridization, and polymerase chain reactions.

Terminology to master Vector, plasmid, recombinant DNA, cloning versus a clone, ligation, transformation, genomic, library, hybridization, probe, screening, polymerase chain reaction

Tutorial #2

(All tutorials are designed to help with your midterm and exam success. Remember to review these concepts.)

Designing ligation reactions

to review these concepts.) Designing ligation reactions Reference: Required re adings pg 31 - 35 Extra

Reference: Required readings pg 31-35 Extra details: https://www.neb.com/tools-and-resources/usage-guidelines/cloning- guide

Background information for designing ligation reactions

The concept of creating recombinant molecules originated with the discovery of restriction enzymes.

Host restriction-modification

These are naturally occurring enzymes that exist in microbial organisms that function to protect the host cell from foreign DNA. This process is called host restriction-modification. This is a surveillance or protection system that microbial organisms use to protect themselves from invading pathogens. The host genome is “modified” (methylated) at a specific recognition sequence. The host cell also carries an enzyme that can recognize and cut at the same site, when it is not methylated. (See Figure 8.2 in required readings)

Once restriction enzymes were discovered and shown to cut at specific sequences, researchers began to exploit them to map genes and cut DNAs from different organisms. By ligating DNAs from two different sources together they were able to create the first recombinant DNA molecules.

Restriction Endonucleases

Distinguish between the effect of endo versus exonucleases on DNA:

Distinguish between the effect of endo versus exonucleases on DNA: endonuclease treatment exonuclease treatment 46
endonuclease treatment
endonuclease treatment

exonuclease treatment

Type I restriction enzymes Type I enzymes require ATP, S-adenosylmethionine (SAM or AdoMet), and magnesium

Type I restriction enzymes

Type I enzymes require ATP, S-adenosylmethionine (SAM or AdoMet), and magnesium ions as cofactors for activity. The enzyme activities reside in complex of three polypeptides:

one for recognition; one for methylation and one for restriction. Type I enzymes recognize a sequence and cut an undefined (nonspecific) distance from that sequence.

Example: EcoK and EcoB which mediate E. coli host restriction-modification are Type I restriction enzymes. EcoK recognition site is: AACNNNNNNGTGC

Type II restriction enzymes

Type II enzymes are comprised of a two-enzyme complex: the methylase and the endonuclease are in discrete polypeptides that function independently of each other. Type I enzymes recognize a sequence and cut at a specific site relative to that sequence.

Type II restriction enzymes are named based on the microbial host from which they originated. For example:

EcoRI – Escherichia coli RY13 BglII Bacillus globigii

NOTE: The first three letters of a restriction enzyme name are italicized since they are an abbreviation of a Latin name. The rest of the name is not italicized because it designates the different versions of the enzyme that organism has.

Type II enzymes recognition sites are palindromic. (See Figure below) Describe what palindromic means in your own words using this example:

Describe what palindromic means in your own words using this example: Figure 2.9 Genes to Genomes

Figure 2.9 Genes to Genomes 3 rd edition

Isoschizomers Isoschizomers are restriction enzymes that recognize the same site. They may cut that site the same way or differently. If they cut the site differently they are called neoschizomers. An example:

differently they are called neoschizomers . An example: Both recognize the same sequence but Sma I

Both recognize the same sequence but SmaI cuts in the middle of the recognition site creating blunt ends whereas XmaI cuts asymmetrically and creates sticky or complementary ends.

Cohesive (or sticky or complementary) ends can have 5' or 3' overhangs:

or complementary) ends can have 5' or 3' overhangs: Some enzymes recognize different sequences but create

Some enzymes recognize different sequences but create the same sticky ends making it easy to ligate them together. When might this be useful?

it easy to ligate them together. When might this be useful? Reprinted from www.neb.com (2016) with

Reprinted from www.neb.com (2016) with permission from New England Biolabs, Inc.

Inactivating restriction endonucleases:

All restriction enzyme require Mg 2+ as a cofactor. They can be inactivated by the addition of EDTA (ethylenediaminetetraceticacid) which chelates (binds) the magnesium. Restriction digests can also be stopped by heat (e.g., 65 o C for 10 min) although some enzymes are less sensitive to this than others. Phenol extraction is also used to inactivate and remove restriction enzymes from a DNA preparation.

Ligation

The joining of two fragments (ligation) is catalyzed by an enzyme called ligase. The most commonly used type of ligase in research is isolated from the bacteriophage T4 and is thus called T4 DNA ligase.

Describe the reaction catalyzed by ligase and cofactors needed.

the reaction catalyzed by ligase and cofactors needed. Note: removal of ATP and Mg 2 +

Note: removal of ATP and Mg 2+ prevents ligation. EDTA chelates Mg so EDTA is often added to stop restriction digests or ligations that require this cofactor.

Companies that sell restriction enzymes have very comprehensive tech support on their web sites as well as web-based tools for analyzing DNAs. New England Biolabs’ web site has a tool called “neb cutter” that is very user friendly. You may want to use this tool for answering tutorial questions. (http://tools.neb.com/NEBcutter2/)

tutorial questions. ( http://tools.neb.com/NEBcutter2/ ) Reprinted from www.neb.com (2016) with permission from New
tutorial questions. ( http://tools.neb.com/NEBcutter2/ ) Reprinted from www.neb.com (2016) with permission from New

Reprinted from www.neb.com (2016) with permission from New England Biolabs, Inc.

“This year biologists in the United States engineered yeast to convert sugar into the makings

“This year biologists in the United States engineered yeast to convert sugar into the makings of opioid painkillers. The feat of biosynthesis, once unique to opium poppies, might lead to better pharmaceuticals—or, more darkly, to home-brewed morphine and heroin. In a bioengineering tour de force, the researchers altered yeast to express an additional 21 genes. Those genes originally came from a diverse set of species, including three types of poppies, a plant called Goldthread, bacteria, and even a rat. The result was an organism that could turn sugar into thebaine, normally derived from poppies—the precursor of synthetic painkillers such as hydrocodone and oxycodone. With two additional genes, the yeast could make hydrocodone as well. The U.S. team and others had previously engineered different yeast strains to carry out either the first or second half of this complex pathway. This year, they took the final steps: performing a single chemical transformation to link the two halves, and putting all the genes together in one strain of yeast. The newly engineered yeasts are far from efficient: Producing one dose of painkiller would probably take thousands of liters of culture. (Reassuringly, the researchers found that a home-brew setup couldn’t make enough opioid to be detectable.) Medicinal chemists are now working to increase the yeasts’ output and trying to tweak their biochemistry to produce safer, more effective drugs.” Science 350, 1458-1463

Module 3

Cloning: Vectors & Transformation

Learning objectives

Explain the basic elements present in plasmid cloning vectors and the role of each in the process of cloning.

Describe the features of vectors that are critical for creating recombinant DNA molecules.

Explain the features of bacteriophage lambda that are exploited in lambda cloning vectors.

Distinguish between translational and transcriptional fusion vectors and what each is used for.

Design experiments using these vectors to address specific research situations.

Explain the features of and uses for BAC and YAC vectors.

Describe the process of transformation by CaCl 2 and electroporation and how transformation efficiency is measured.

Describe how transformants are identified.

References: 195-199; 201-203

There are two JoVE videos on transformation, one is on the heat-shock method and one

is on electroporation. For the first video focus on the segments: “The Heat Shock Reaction”

and “Making Competent Cells”; for the second, watch the segments, “Electroporation,

Basic Principles” and “Electroporating Bacteria”. There is space on pages 52 and 53 of

these notes to add new information from the videos.

What is a vector?

The word vector has several meanings. In molecular biology the term means a nucleic acid molecule used to carry and replicate sequences of interest in a host organism. There are several types of vectors, each of which is used for distinct applications.

Properties of vectors

Features common to all cloning vectors: (rather than just memorizing these, record why each is important.)

Replicate independently

Unique cloning sites

Selectable markers (e.g. kanamycin resistance; ampicillin resistance) – Mode of action information for these markers is available in Table 8.3 – what do you see in common about their mode of action?

Easy to recover from host cell

Plasmid vectors The most commonly used vectors are plasmids. These are circular, supercoiled DNA molecules

Plasmid vectors

The most commonly used vectors are plasmids. These are circular, supercoiled DNA molecules that exist in cells as extra-chromosomal elements (meaning they are not part of the genomic DNA). (Figure 5.20)

elements (meaning they are not part of the genomic DNA). (Figure 5.20) Note the naming convention

Note the naming convention for plasmids:

Features common to many plasmids: Indicate why each trait is important:

Small (often 2-5 kb)

Origin of replication:

Multiple cloning site

kb) • Origin of replication: • Multiple cloning site • Copy number control • Sequence of
kb) • Origin of replication: • Multiple cloning site • Copy number control • Sequence of

Copy number control

Sequence of vector is known

Selectable marker

Other features that may be present: “blue-white screening” Insertional inactivation sites

“blue-white screening” Insertional inactivation sites • lacZ' is a partial b -galactosidase gene from the

lacZ' is a partial b-galactosidase gene

from the lac operon; the other part of the coding region is supplied by the host

genome; this is called a-complementation

lacZ expression is induced by growing the host cells on IPTG (isopropyl-beta-D- thiogalactopyranoside ). In the presence of the substrate Xgal (5-bromo-4-chloro- 3-indolyl-b-D-galactoside) the cells turn blue. Figure 2.22 Genes to Genomes 3 rd edition

What happens if a fragment is cloned into the multiple cloning site within lacZ' ?

Note: Figure 8.6 outlines the steps of using plasmids in a cloning experiment. You should be familiar with all these steps by the time of the midterm, including DNA isolation and evaluation of the transformants.

Transformation

Transformation is the uptake of naked DNA (i.e. not ejected from a viral capsid, for example; free of protein) by a bacterial host. Cells that can take up DNA this way are called “competent”. E. coli is not naturally competent but can be induced to take up DNA by various means.

CaCl 2 treatment followed by heat shock

CaCl2 may affect cell wall; allowing DNA binding to surface; heat shock stimulates uptake

These cells must have a minimum efficiency

of 10 7 transformants per µ g of DNA

added to be effective. Ideally, you want them even higher.

Why is the LB added after the heat shock step?

g of DNA added to be effective. Ideally, you want them even higher. • Why is

57

Electroporation

Wash cells with ice-cold, sterile water to

remove electrolytes

Brief pulse of high voltage causes pores to

open in the envelope allowing the DNA to

enter

These cells take longer to prepare but are

faster to use and have higher

transformation efficiencies (>10 8 per µ g)

transformation efficiencies (> 10 8 per µ g) There are “quick and dirty” methods for transformation

There are “quick and dirty” methods for transformation of E. coli too. The competent cells can be prepared quickly but have low competency. When are these appropriate to use?

Companies are always developing new reagents to allow for faster transformation. (Image not available due to copyright: available for download from LEARN)

58

Measuring competency

Once a set of competent cells is prepared it is necessary to test how effectively they take up DNA. Usually a known amount of an intact supercoiled plasmid is used for this test, and the number of transformants recovered per µ g of DNA is the measure of competency. Question: Only a small amount of supercoiled plasmid DNA is used in this test. Why?

Example calculation:

You add 5 ng of pUC18 to 0.1 ml of CaCl 2 -treated E. coli cells. After the heat-shock you

add 1 ml of LB and grow for 1 h.

selective media and grow overnight (O/N). In the morning you count the number of colonies on each plate.

You plate 50 µ l and 5 µ l of the transformation mix on

When you plated 50 µ l of the transformation mix: 137 colonies When you plated 5 µ l of the transformation mix: 18 colonies

Question: What is the transformation efficiency of this batch of cells? (i.e., how many

transformants did you recover per 1 µ g of added DNA?)

Stepping through this calculation:

(This is the way I solve this calculation but you can use the space above to do it your own way.) (We will use the 50µ l value for our calculation because the number is more reliable)

1. First determine the volume of the transformation mix.

2. Next determine the total number of transformants in the mix. To do this you need to consider how much of the mix was plated in each case. 1.1 ml/ 0.05ml = 22 Therefore, in total the 1.1 ml contains 22 (137) = 3.0 x 10 3 transformants

3.

This is the number of transformants produced when 5 ng of DNA was used in

the reaction. However, transformation efficiencies are expressed as number of

transformants per µ g.

If there are 3.0 x 10 3 transformants when 5 ng of DNA is added, how many colonies would be recovered from 1 µ g of added DNA?

This is 200x more DNA: 200(3.0 x 10 3 ) = 6 x 10 5 colonies would be

recovered. Thus, the cells have a transformation efficiency of 6 x 10 5

colonies/ µ g.

Would you use these cells??

Why does transformation frequency matter?

Recombinant selection/screening

Very few of the competent cells actually take up the recombinant plasmid so it is

necessary to have some kind of selection to detect successful transformants. The selection is

usually based on the expression of a gene on the introduced DNA – this is called the

selectable marker (e.g. ampicillin resistance or kanamycin resistance).

Alternatively, the transgene may expression a gene product that gives the host cell a new

functionality.

In any case, it is essential to select for transformants because the efficiency of recovering

the clone of interest, even when done well, is 1in 100s of thousands if not a million. So you

cannot just test the cells one-by-one.

Vectors based on bacteriophage lambda – cosmids & replacement vectors

When the lambda genome is packaged into the capsid is a linear molecular of about 50,000 bp that has 12 bp cohesive ends.

molecular of about 50,000 bp that has 12 bp cohesive ends. Figure 2.25 Genes to Genomes
molecular of about 50,000 bp that has 12 bp cohesive ends. Figure 2.25 Genes to Genomes

Figure 2.25 Genes to Genomes 3 rd edition

Bacteriophage lambda is a temperate virus. This means that there are two possible outcomes of a lambda infection; the phage may become lysogenic (dormant in the host genome; genetically silent) or it may produce a lytic infection (infects, multiplies and then kills the host cell by lysis). (Note: there is a nice animation of lambda life cycles on LEARN –optional learning tasks) Lytic cycle:

of lambda life cycles on LEARN –optional learning tasks) Lytic cycle: Figure 2.26 Genes to Genomes

Figure 2.26 Genes to Genomes 3 rd edition

of lambda life cycles on LEARN –optional learning tasks) Lytic cycle: Figure 2.26 Genes to Genomes

62

To detect lambda infection, the phage and host cells are mixed together and incubated briefly
To detect lambda infection, the phage and host cells are mixed together and incubated briefly

To detect lambda infection, the phage and host cells are mixed together and incubated briefly to give the phage a chance to attach to its receptor and inject its DNA. These infected cells are mixed with more host cells and plated on solid LB agar plate. The initial infected cell liberates viral particles, which then infect and lyse the surrounding cells. This creates zones of clearing called plaques. If this plate is left long enough or if many infected cells are initially plated then all the bacteria will be lysed and the plate will be totally clear.

Packaging of viral DNA

The viral packaging machinery recognizes the cos sites on concatamers and makes a cut in the DNA, creating a cohesive end and then stuffs a “headful” of DNA into the pre-formed capsid. Normally the head is full at the same time the next cos site on the concatamer is reached.

Deletion mutants of lambda are used as cloning vectors; these vectors were created by identifying lambda genes that are not essential for the lytic cycle. Genomes from 75- 105% of the wild type size can be successfully packaged; smaller sizes produce unstable capsids and larger sizes don’t fit.

In vitro packaging It is possible to package the recombinant phage molecules using extracts of lambda- infected cells that contain heads and other extracts that just make tails. By mixing the

ligated DNA with these extracts, the cos sites in the phage are recognized by packaging enzymes and the recombinant molecule is packaged into the capsids and then the tail is added.

Cosmids

A plasmid with a packaging site (cos site = cohesive ends site)

This site is recognized by the lambda packaging machinery

Inserts must be 32-45 kb to be packaged

Ligation is adjusted to generate multimers (mimics normal lambda packaging) but only

generate multimers (mimics normal lambda packaging) but only • monomers get edition packaged Replacement vectors •

monomers get

edition

packaged

Replacement vectors

Minimum size of lambda that can be packaged is 37 kb

Genes that are not essential for lytic growth are removed but are replaced by the “stuffer” so that modified phage can be packaged.

Allows larger inserts (22 kb)

Arms alone can ligate but not package

Figure 2.30 Genes to Genomes 3 rd

• Allows larger inserts (22 kb) • Arms alone can ligate but not package Figure 2.30

64

Artificial chromosome vectors

Given the large size of eukaryote genomes, such as the human genome and the relatively small insert size limits of cosmids and plasmid vectors, it became necessary to develop a new suite of vectors that would accept larger inserts. There are several types of these vectors. These are called BACs or YACs depending upon whether they replicate in bacterial or yeast hosts, respectively. (See table on page 51).

Terminology to master:

Native protein, recombinant protein, endogenous gene, expression vector, fusion protein, expression vector, cosmid, bacteriophage lambda, replacement vector, cos site, packaging extract, lytic, lysogenic, YAC, BAC

Tutorial #3 – Generating translational and transcriptional fusions

(All tutorials are designed to help with your midterm and exam success. Remember to review these concepts.)

Learning Objectives

Remember to review these concepts.) Learning Objectives 1. Distinguish between translational and transcriptional

1. Distinguish between translational and transcriptional fusion vectors and what each

is used for.

2. Design an appropriate strategy to address a specific question using gene fusions.

3. Recognize the key criteria in designing these constructs.

4. Explain the function of reporter genes in these vectors; describe at least one example of a reporter gene and how it is detected.

Draw a schematic diagram of a transcriptional fusion vector with an insert:

Draw a schematic diagram of a translational fusion vector with an insert:

Module 4

DNA isolation: plasmids, genomic DNA & cDNA

Learning objectives

Explain the steps in the isolation and purification of nucleic acids.

Describe the role of key reagents in these protocols.

Compare different methods for quantifying nucleic acids including genomic DNA, RNA and plasmid DNAs

Explain how nucleic acids are separated, detected and sized by agarose and acrylamide gel electrophoresis

References: 198; lecture resources; consult material on reserve for more details

Extraction and purification of nucleic acids

Many techniques start with the isolation DNA or RNA. The protocol used depends on the

how pure the nucleic acid needs to be for the specific technique and the starting material.

Generally the fastest, cheapest method is chosen.

The basic steps involved are:

1. Collect the tissue/cells containing the nucleic acid of interest.

2. Homogenize tissue and lyse cells.

The following reagents are often present in extraction buffers. Why? EDTA (ethylenediaminetetraacetic acid)

SDS (sodium dodecyl sulphate or sodium dodecyl sulfate)

What specialized treatments are used to lyse the following cell types? Bacterial cells:

Animal or plant tissue:

Yeast:

Nucleic acids are transferred using micropipettors.

Yeast: Nucleic acids are transferred using micropipettors. 3. Isolate the desired nucleic acid away from other

3. Isolate the desired nucleic acid away from other cellular components.

There are many constituents in the lysate (proteins, genomic DNA, membranes, unbroken cells, large protein complexes (e.g., ribosomes), –the protocol varies

depending on whether you want to isolate plasmid DNA, genomic DNA or mRNA. There are different methods depending on the purity needed for the subsequent steps.

Describe the following purification steps:

RNA removal

DNA removal

Protein removal

purification steps: RNA removal DNA removal Protein removal Figure 4.1 Genes to Genomes 2nd edition Figure

Figure 4.1 Genes to Genomes 2nd edition

removal DNA removal Protein removal Figure 4.1 Genes to Genomes 2nd edition Figure 2.4 Genes to

Figure 2.4 Genes to Genomes 3 rd edition

This is a widely used method for concentrating DNA or changing the solution it is dissolved in. There are also commercial kits that can be used for this. In those cases, the DNA is poured over a matrix and then eluted with a small amount of water.

over a matrix and then eluted with a small amount of water. Example of a microfuge

Example of a microfuge and microfuge tubes. See JoVE video on centrifugation for details.

Plasmid purification

Cesium chloride gradients: high salt solution creates a density gradient when centrifuged at high speed (eg >100,000xg). DNAs form bands consistent with their density.

a density gradient when centrifuged at high speed (eg >100,000xg). DNAs form bands consistent with their

Ethidium bromide is added to visualize the DNA and aids separation of genomic versus plasmid DNAs. Plasmid DNAs are more dense and thus separate from the genomic DNA.

Alkaline denaturation (plasmid purification)

genomic DNA. Alkaline denaturation (plasmid purification) Figure 2.5 Genes to Genomes 3 rd edition Column

Figure 2.5 Genes to Genomes 3 rd edition

Column purification of plasmid DNA

The cells containing the plasmid are often broken by alkaline lysis and the plasmid DNA is recovered from the supernatant by column chromatography instead of alcohol precipitation.

Detection and Quantitation of nucleic acids

The purine and pyrimidine bases absorb light maximally at 260 nm. Thus the amount of nucleic acid contained in a solution can be quantified by measuring the amount of 260 nm light the solution absorbs (i.e. following Beer’s law, nucleic acid concentration is proportional to absorbance.) This is called a spectrophotometric quantification of nucleic acid. The presence of contaminating protein or small molecules makes these measurements less accurate.

or small molecules makes these measurements less accurate. The amount of nucleic acid is quantified using
or small molecules makes these measurements less accurate. The amount of nucleic acid is quantified using

The amount of nucleic acid is quantified using the ratios:

1 0D 260 = 50 µ g ml -1 (DNA)

1 0D 260 = 40 µ g ml -1 (RNA)

Question: How might the absorbance reading give you a false measurement? (Hint:

consider what would happen if the plasmid DNA was contaminated with other cellular constituents.) How can error be avoided?

Limitations of this method:

1.

2.

3.

4.

Ethidium bromide staining is also commonly used to monitor the quality of nucleic acids and monitor their concentration. This is most often done to nucleic acids that have been first separated by electrophoresis. This is an intercalating dye:

separated by electrophoresis. This is an intercalating dye: When exposed to invisible UV light the dye

When exposed to invisible UV light the dye absorbs energy and emits this (fluoresces) in the form of visible red-orange light. This dye will also bind to the user’s DNA so it is important to wear gloves when handling ethidium bromide and dispose of the stained gels safely. Other, safer dyes have been developed recently (e.g. GelRed).

Animations on aspects of cloning http://www.dnai.org/b/index.html

Tutorial # 4 – DNA quantification and gel electrophoresis (All tutorials are designed to help

Tutorial #4 – DNA quantification and gel electrophoresis

(All tutorials are designed to help with your midterm and exam success. Remember to review these concepts.)

Background information: Gel electrophoresis

Reference: (Tool box 8.6) and JoVE video: Electrophoresis Electrophoresis is the movement of a charged particle in response to an electrical field. DNA is negatively charged (an anion). Thus it migrates towards the positive electrode or anode (“runs to the red”; “anions migrate towards the anode”).

to the red”; “anions migrate towards the anode”). Used with permission of the author, Richard Bowen,

Used with permission of the author, Richard Bowen, Colorado State University.

Nucleic acids can be separated electrophoretically by migrating through different types of gels. Small molecules migrate faster than larger molecules.

The two most common gel types are:

Agarose

types of gels. Small molecules migrate faster than larger molecules. • The two most common gel

74

Used with permission of the author,Richard Bowen, Colorado State University.

Polyacrylamide

Polyacrylamide gels are used for methods that require high resolution (distinguishing fragments that differ by only a single nucleotide) for example, DNA sequencing.

These gels are also used for monitoring protein-DNA interactions (gel shift assay) and protein electrophoresis.

Rate of migration of a molecule in an electric field depends on Strength of the field

Viscosity of the gel (drag on the molecule)

Presence of bound molecules

electric field depends on Strength of the field Viscosity of the gel (drag on the molecule)

Conformation

Conformation Used with permission of the author, Richard Bowen, Colorado State University. Special cases: RNA gels
Used with permission of the author, Richard Bowen, Colorado State University.
Used with permission of the author, Richard
Bowen, Colorado State University.

Special cases:

RNA gels RNA solutions are heated and fast cooled before being loaded onto agarose gels containing formaldehyde. Why are these modifications needed?

Separating chromosomes or very large DNAs:

agarose gels containing formaldehyde. Why are these modifications needed? Separating chromosomes or very large DNAs: 76

76

Analytical versus Preparative electrophoresis

By electrophoresing nucleic acids on gels the integrity (intactness) can be assessed as well as the presence of contaminating genomic DNA or tRNA in plasmid preps.

integrity (intactness) can be assessed as well as the presence of contaminating genomic DNA or tRNA

Module 5

Sources of DNA: Genomic and cDNA libraries

Learning objectives

Compare and contrast the information content of genomic libraries and cDNA libraries and how each library is created.

Explain the factors involved in choosing a vector for genomic and cDNA libraries.

Outline the steps involved in creating genomic and cDNA libraries, evaluating their quality and storing them.

Outline how cDNA is synthesized and the steps that cause artefacts.

Reference: pg 206

Genomic versus cDNA libraries

Use genomic libraries to:

- Isolate specific pieces of genomic DNA (e.g. a gene of interest)

- Isolate fragments adjacent to a specific fragment (e.g. isolate a promoter for a cDNA that you have)

- Gene mapping & genome sequencing

Use cDNA libraries to isolate:

- DNA copies of mRNAs (open reading frame of a gene) which can be used for overexpression, reporter fusions etc.

Creation of a genomic library

A genomic library is a collection of clones that in total contain all the fragments of a

particular genome. In reality this is rarely the case since telomeres, centromeres and other

regions containing highly repetitive DNA are difficult to clone. But a good library will

cover the vast majority of a genome.

There are several steps involved in the process of creating a genomic library.

1. Choose the vector.

2. Fragment the genome into pieces small enough to fit into the vector.

3. Ligate the vector and genomic DNAs; transform into host cells.

4. Evaluate quality of the library.

5. Store library.

Vector choice

Question: What factors must be considered in choosing a vector?

The library is a collection of random fragments and the more members of the library we examine the greater the chance that we find similar or identical members!

Lambda vectors are often used in place of bacterial vectors for the construction of genomic libraries because the phage are easy to screen at high density (bacterial colonies will grow together); also phage are easier than bacteria to transfer to the membrane for hybridization. Also there are many viral particles (each with an insert) in each plaque.

Fragmentation of genomic DNA

with an insert) in each plaque. Fragmentation of genomic DNA Genomic digests create many fragments that

Genomic digests create many fragments that range in size. When these digests are electrophoresed, they create a ladder of fragments that looks like a smear of DNA.

Partial or complete genomic digests

Construction and evaluation of a genomic library

The steps involved in constructing a genomic library are basically the same as those covered previously for ligating one fragment of interest into a vector. Sometimes one can buy a precut vector to be more confident of its quality. It is also possible to buy pre-cut lambda arms (free of the stuffer fragment) for convenience.

Usually the vector will be treated with phosphatase. Why?

If one is using a bacterial vector then the ligation products are introduced into the host cells by chemical transformation or electroporation. These cells are then plated on selective media.

If one is using a lambda vector it is necessary to incubate the ligation mixture with a commercial lambda packaging extract which provides the enzymes to generate cos sticky ends, the proteins for assembly of the viral capsids and tail as well as package the vector DNA.

If using a yeast vector, the ligation mixture is introduced into yeast cells by electroporation.

The first step is to determine how good your library is. Why?? To evaluate the library you count the number of colonies on the original transformation plates. This tells you the complexity of your library (the number of independent clones).

Next you need to know how many of these clones contain an insert and the average size of the inserts. This involves isolating DNA from a representative number of clones and determining the size of the insert in each clone.

Storage of libraries

Bacteria and phage stocks are stored long term at -80 o C. The ice crystals that form cause protein denaturation and damage membranes. Bacteria can be protected by these effects by the addition of glycerol; phage are protected by the addition of dimethylsulfoxide (DMSO). In either case, the stock should not be frozen and thawed because this leads to cell death/phage inactivation. For this reason to sample the culture one takes a small frozen piece from the stock. To further avoid freeze/thaw damage, the original stock can be aliquoted (divided into fractions of equal size) before freezing.

cDNA libraries

Very little (about 1.5%) of the human genome, encodes protein coding sequences (similar to other eukaryotes).

Protein coding regions in genomic libraries contain introns, which interrupt the ORF.

Genomes also contain pseudogenes, which are not expressed; these can confuse molecular studies.

To avoid these complications, it is possible to generate libraries based on mRNAs.

No introns, only expressed sequences (no pseudogenes); no junk, repetitive DNA to be screened.

But there are limitations to their use…

Ref: Tool Box 8.2

Question: What are the limitations of using cDNA libraries? What sequences cannot be

recovered from them? (See learning task on this topic for a review)

1

2

3

4

5

Unspliced transcripts not commonly recovered.

6

Non-polyadenylated mRNAs are not usually recovered.

Isolation of total RNA

The RNA-containing extract is produced by homogenizing the tissue, organ or organism of interest in conditions that inactivate RNases. Often the tissue is flash frozen in liquid nitrogen (temperature of -176 o C) to reduce RNase activity and then homogenized in buffer that denatured protein (e.g. phenol-containing buffer). The cellular debris is removed by centrifugation, leaving a solution that contains cellular DNA and RNAs.

mRNA isolation

To make a cDNA library (DNA copies of mRNAs) you need a method that purifies or captures only the mRNAs from the cell extract (i.e., removing DNA, tRNA and rRNA. This is done by exploiting the presence of polyA tail on mRNA.

Column purification:

mRNA can be isolated by preparing a column of the oligo-dT or (dU) beads and pouring an extract containing the RNAs over the column. The bound mRNAs are eluted by lowering the salt and raising the temperature. Why?

the RNAs over the column. The bound mRNAs are eluted by lowering the salt and raising

Points to keep in mind:

The tissue must be homogenized very rapidly to limit RNase activity.

Often the tissue is frozen in liquid nitrogen and ground in a solution containing phenol and salts that cause protein denaturation.

The cellular debris is removed by centrifugation and then the extract is ready for column purification of the RNA.

cDNA synthesis

cDNA often made directly using the cell extract (i.e., without purification of the mRNA). The polyA tail is again the basis for selective copying of only mRNA. An oligonucleotide of poly(dT) complementary to this tail is used as a primer for the enzyme reverse transcriptase (RT), which synthesizes a strand of DNA complementary to the mRNA to which the primer is bound. Thus RT is classified as a RNA-dependent (or directed) DNA polymerase. RT catalyzes the polymerization of deoxyribonucleotides in the 5to 3direction. There can be technical problems associated with the RT reaction; these can limit the quality of the recovery of some sequences from the library.

The product of the reaction is an RNA-DNA complex. There are several different techniques for replacing the RNA strand with DNA. One involves degrading the RNA and then relying on the inherent complementarity of the DNA strand to form a hairpin, which can act like a primer for a partial complementary strand. Another involves making nicks in the RNA strand with RNase H and then using one of those RNAs as a primer for the synthesis of DNA.

Terminology to master genomic vs. cDNA libraries, reverse transcriptase, partial digest, oligo-dT, cDNA

Module 6

Sources of DNA: Polymerase Chain Reaction (PCR)

Learning objectives:

Illustrate how PCR leads to the amplification of specific sequences.

Explain the parameters that can be modified in the reaction and the basics rules for primer design.

Outline how PCR products can be verified.

Describe how PCR products can be cloned.

Outline different applications of PCR.

Reference: pg 205-206 (Tool Box 8.3)

This technique has revolutionized molecular genetics by providing an approach for the

isolation of nucleic acid sequences and their analysis. PCR makes is possible to study rare

genes, amplify small amounts of DNA and generate specific fragments of DNA easily. It is

also very useful for cloning because any restriction site can be added to the ends a

sequence, via the primers.

Overview of a PCR reaction

PCR is an in vitro DNA synthesis reaction: A thermostable DNA polymerase catalyzes

multiple rounds of DNA synthesis, starting from two primers that flank a region of interest

within a target DNA template.

flank a region of interest within a target DNA template. Video: learn.genetics.utah.edu/content/labs/pcr/ Figure 4.1

Video: learn.genetics.utah.edu/content/labs/pcr/

a target DNA template. Video: learn.genetics.utah.edu/content/labs/pcr/ Figure 4.1 Genes to Genomes 3 rd edition 86

Figure 4.1 Genes to Genomes 3 rd edition

This is an in vitro DNA synthesis reaction and thus requires:

In general the cycles are designed as follows:

25-40 cycles

2 min 95-98 o C (initial denaturation)

1 min 95-98 o C (denaturation)

0.5-1 min 50-60 o C (annealing temp; a few degrees below Tm of primers)

1

min 72 o C (extension, 1 min/kb of product but this depends on the polymerase; many are faster)

5

min 72 o C

Leave at 4 o C

Annealing conditions

Optimized for maximum primer binding to template

Primer design

Indicate the sequence of the forward and reverse primers:

Indicate the sequence of the forward and reverse primers: Figure 4.3 Genes to Genomes 3 rd

Figure 4.3 Genes to Genomes 3 rd edition

Thermostable polymerase

This allows the reaction to cycle many times through 94 o C and still keep polymerase active

Different companies make their own versions isolated from different heat-stable organisms or mutated to give added specificity

Features of specialized thermostable polymerases (eg Pfu, Pwo):

long-range (are more processive and will amplify 10-30 kb);

higher proof-reading;

blunt ends: some thermostable polymerases add an extra A at the end of the product in template-independent manner

dNTPs

Buffer

The buffer is simple: Tris-HCl pH 8.4-8.8 & KCl

MgCl 2 concentration can affect the specificity of the primer binding

Controls

Tris-HCl pH 8.4-8.8 & KCl • MgCl 2 concentration can affect the specificity of the primer
Tris-HCl pH 8.4-8.8 & KCl • MgCl 2 concentration can affect the specificity of the primer

Benefits

Limitations

Explain how each of the following can be used to verify the identity of a PCR product. Gel electrophoresis

Restriction digestion

Digest the PCR product with restriction enzymes known to cut the expected product (i.e. test for presence predicted restriction fragments in product)

Sequencing

The gold standard – sequence the PCR product to verify it is correct. This is essential when using the fragment for subsequent experiments since random mutations can eliminate expression of the gene product or change critical cis elements

Variations of PCR

Reverse-transcription PCR (to be discussed in detail in module 10)

cDNA is used as a template for PCR

The cDNA is diluted so the amount of PCR product is dependent on the amount of the starting cDNA template; therefore the intensity of the PCR product reflects the abundance of the corresponding mRNA

This is a very sensitive method that is particularly useful for discriminating between similar transcripts (e.g. different members of a gene family).

Applications of PCR There are new uses of PCR being developed all the time. Several key uses that you should be aware of are:

Probe synthesis

Cloning strategies

Analysis of ligation products and transformants

Detection of rare genetic events

Detection of splicing variants Example Date

• Detection of rare genetic events • Detection of splicing variants Example Date Science 345: 1139-

Science 345: 1139-1145

Tutorial #5 –Cloning PCR products

Tutorial # 5 –Cloning PCR products Addition of restriction enzyme recognition sites • Sequences of restriction

Addition of restriction enzyme recognition sites

Sequences of restriction enzyme recognition sites are added to the ends of primers, along with a few extra nucleotides because restriction enzymes don’t like to cut right at the ends of DNA fragments.

Example:

A schematic diagram of a cDNA is shown below. The ORF is the solid line. If you wanted to amplify the ORF so that the product was flanked by EcoRI sites, you would add the sequence for the EcoRI recognition site to the ends of each primer.

for the EcoRI recognition site to the ends of each primer. Terminology to Master Polymerase Chain

Terminology to Master Polymerase Chain Reaction (PCR), Thermus aquaticus (Taq), RT-PCR, primer, cycle, nested PCR, thermostable

Module 7

Finding the right clone

Learning objectives

Differentiate between the various methods to identify recombinant clones and the experimental effort involved in each case.

Explain how to synthesize radioactively labeled or chemically labeled probes.

Explain how a degenerate oligonucleotide probe is designed.

Predict the steps involved in verifying a clone of interest by subcloning and restriction mapping, antibody detection and functional complementation.

Reference: pg 207-218

Screening libraries with gene probes: hybridization

Genomic and cDNA libraries are large collections (often tens of millions) of clones; these

must be screened to find the clones that carry the fragment of interest. This is most

commonly done using a modified DNA or RNA fragment (called a probe); this fragment is

capable of hybridizing to the target sequence and once bound, easily detected. The

design of probes and hybridization conditions are thus critical to successful library

screening.

are thus critical to successful library screening. The strategy you use to identify the clone of

The strategy you use to identify the clone of interest depends on what you know about the

target sequence and what tools you have available (e.g. antibodies, nucleic acid probes).

The approach also depends on the expertise of the research team. Ultimately additional

screening may be necessary to identify a clone that has a specific insert in a particular

orientation in the vector.

Hybridization stringency

The process of hybridization was described in Module 2. In the case of screening a library this involves:

transferring thousands of colonies or phage of the library to a membrane;

denaturing the DNA;

fixing the denatured DNAs to a membrane so they are capable of binding to a complementary fragment;

the probe is prepared and denatured;

the membranes are put in a seal-a-meal bag or a roller bottle, along with a labeled probe in a salt solution that favours reannealing;

the mixture is incubated overnight at a temperature suitable for hybridization of the probe to the target sequence within the library, and

unbound probes or those bound only non-specifically are washed off and the bound probe is detected.

The ultimate goal is to bind the probe to perfectly matched target sequence or perhaps to sequences of lesser identity. Stringency is a measure of how restrictive the hybridization conditions are. The degree to which your probe sequence matches the target sequence in the library determines how stringent you make the hybridization conditions. Modifying the salt concentration and the temperature controls stringency. Formamide can also be added to lower the temperatures needed for high stringency.

Question: Explain the basis of these effects:

94
94

Labelling probes

A critical feature of a probe is that its presence be easily detectable. Since the target sequence in a library is a very small proportion of the mixture, there will be relatively little probe bound. So the detection method must be very sensitive.

The classical way to generate probe is to label it with a radioactive isotope, most commonly 32 P. The membrane with the associated bound probe is placed on a piece of X- ray film; the radioactive particles from the probe will activate the silver grains of the film just as light does, creating a black spot or band. More recently sophisticated machines called Phosphor Imagers have been developed which use a phosphor-screen that contains molecules that fluoresce when activated by the radioactivity. This is detected by placing the screen in a phosphoimaging machine that creates a digital image of the screen, thereby locating the radioactivity on the membrane.

There are several non-radioactive labeling methods which are more safe to handle, can be quickly detected and are more stable so the probe can be stored for long periods of time. Modified nucleotides containing specific epitopes (eg. biotin or digoxigenin (DIG)) can be purchased along with antibodies that detected the epitope. This antibody itself carries an enzyme such as alkaline phosphatase or horseradish peroxidase. By adding the substrate of the enzyme, a localized colorimetric or fluorescent reaction in the region of the bound probe occurs.

the substrate of the enzyme, a localized colorimetric or fluorescent reaction in the region of the
the substrate of the enzyme, a localized colorimetric or fluorescent reaction in the region of the

Templates for probe synthesis

There are two types of probes:

Homologous

Heterologous

Another consideration for probe design is whether you want it to bind to a specific gene or to all members of a gene family.

What is a gene family?

What parts of gene are distinct (i.e. would differ between genes that code for very similar proteins or RNAs?)

There is an optional learning task for this topic on LEARN. Be sure to try it out.

Incorporation of labeled nucleotides by in vitro DNA synthesis reactions

The modified nucleotides are incorporated via in vitro DNA synthesis reactions. Recall that DNA polymerases need a primer, a template, four types of deoxyribonucleotides (dNTP) and the appropriate buffer to synthesize DNA. In these labeling reactions, one of the 4 dNTPs is radiolabeled or chemically labeled.

The radioactive phosphate must be in the alpha position of the nucleotide. Why?

must be in the alpha position of the nucleotide. Why? There are numerous methods for incorporating

There are numerous methods for incorporating these modified nucleotides, whether radioactive or chemically modified, into the probe sequence. It is useful to know the basics of each method although the first three listed below are less commonly used now.

Nick translation

Random priming

Filling in ends of restriction sites

Riboprobes (uses α-labelled ribose triphosphates; in vitro RNA synthesis)

Labelling with T4 polynucleotide kinase (requires γ-labelled dNTP donor)

Labelling during DNA synthesis by PCR

Overview of the key labelling methods Random priming (commonly used to label purified fragments which are about 1 kb)

used to label purified fragments which are about 1 kb) Filling ends of restriction enzyme sites
used to label purified fragments which are about 1 kb) Filling ends of restriction enzyme sites

Filling ends of restriction enzyme sites

(may be used to label short purified fragments)

ends of restriction enzyme sites (may be used to label short purified fragments) Labeling during DNA
ends of restriction enzyme sites (may be used to label short purified fragments) Labeling during DNA

Labeling during DNA synthesis by PCR

ends of restriction enzyme sites (may be used to label short purified fragments) Labeling during DNA
ends of restriction enzyme sites (may be used to label short purified fragments) Labeling during DNA

98

Labelling with T4 polynucleotide kinase (commonly used to label short fragments or oligonucledies)

polynucleotide kinase (commonly used to label short fragments or oligonucledies) In vitro transcription – RNA probes

In vitro transcription – RNA probes

polynucleotide kinase (commonly used to label short fragments or oligonucledies) In vitro transcription – RNA probes
polynucleotide kinase (commonly used to label short fragments or oligonucledies) In vitro transcription – RNA probes

Complete the following table:

 

RNA probes

Random

Fill-in

T4 kinase

PCR

priming

ends

labeling

Methods that can be done using genomic DNA as the template

No

       

Methods requiring a purified fragment as the template

No, need a fragment cloned downstream of a promoter

       

The best method to create a sensitive single- stranded probe

Yes

       

Method to label oligonucleotides

No

       

Method to label RNA

Yes

       

Screening libraries with antibodies

If a cDNA library is created in an expression vector so that the cDNA is inserted under the control of a promoter that will be active in the host cell, then each transformant will express a recombinant protein corresponding to the cDNA it contains.

The proteins are transferred to the membrane but are not denatured. The membrane is incubated with an antibody specific for the protein of interest. The bound antibody is detected by:

the protein of interest. The bound antibody is detected by: Preparation of antibodies: Reference: Tool Box

Preparation of antibodies:

Reference: Tool Box 9.4 Antibodies may be generated by injecting a purified protein into a bunny or chicken and isolating the serum from the bunny’s blood or from the egg white of the chicken’s eggs.

Antibodies can also be generated based on the amino acid sequence of a gene of interest. The polypeptide (12-20 aa) is synthesized commercially, conjugated to a larger protein to make it more antigenic and injected into either rabbits or chicken.

Secondary antibodies Binding of a primary antibody is not visible by eye. Instead, a secondary (2 o ) antibody specific for the constant region of the primary antibody and conjugated to either an enzyme or a fluorescent or radioactive tag is used. The 2 o antibody binds to the primary and this binding is detected by the signal produced by the tag. Multiple 2 o antibodies can bind to the primary, providing an increase in sensitivity.

However, it is essential to know the source of the primary antibody (i.e., raised in rabbit, chicken, donkey etc.) before being able to use it in an immunoblotting experiment.

Question: Screening with antibodies is great when it works, but there are several reasons why it is a challenging technique. Describe 4 of these.

1.

2.

3.

4.

Cloning by functional complementation

Sometimes it is possible to clone by complementation of a mutant phenotype in vivo e.g. complementation of a Drosophila mutant with a cDNA expression library. This is often done to clone receptors or transporters.

This is also used to verify that a clone identified by an alternate method actually encodes the proposed activity in vivo.

Basically you transform the mutant with either a cDNA library cloned so the inserts will be expressed in the host or you transform with just one specific cDNA. Then look for transformants that have the WT phenotype.

What do you see as limitations of this method?

Example data To be covered in class:

Purpose of experiment:

Mutant transformed:

cDNA library used:

Phenotype tested:

Proc Natl Acad Sci (USA) 990:10484

Proc Natl Acad Sci (USA) 990:10484

So you have identified a putative positive clone, what’s next?

The first step is often evaluating which colonies have inserts and the size of those inserts.

which colonies have inserts and the size of those inserts. Digest plasmid DNA isolated from individual

Digest plasmid DNA isolated from individual transformants to release the insert

inserts and the size of those inserts. Digest plasmid DNA isolated from individual transformants to release

104

Tutorial #6 – Restriction mapping This is used to identify clones for which a restriction

Tutorial #6 – Restriction mapping

This is used to identify clones for which a restriction map is known; this is more often done when cloning fragments into a vector and you care about the orientation of the insert in the vector. Describe two examples of this.

Examples of restriction mapping:

two examples of this . Examples of restriction mapping: Assume you have cloned the 1 kb

Assume you have cloned the 1 kb PstI fragment shown at the left into the 3 kb vector. The only additonal information you have is the location of the EcoRV sites in the vector and the insert. Draw the two possible restriction maps for transformants that contain only one insert. Assume that the EcoRV site in the plasmid is adjacent to the PstI sit