Lectures 24 and 25: Molecular Biology Methods I and II (Genetic and Genomic)

1. What is a Restriction Endonuclease? What is its function, and what does it specifically
recognize before doing that function?

Restriction Endonuclease’s are enzymes that can cut DNA molecules into fragments. Its
function is to cut the DNA into fragments. It specifically recognizes a DNA sequence, cuts both
strands, and leaves the ends with an either blunt end or overhanging end.

2. What two types of enzymes are used in recombinant DNA technology to “cut and paste” two
DNA segments together?

Restriction enzymes are used to cut the DNA segments and DNA ligases are used to past
them together.

3. What is a plasmid? What is it used for? What sequences are necessary in a plasmid to put the
plasmid in bacteria and have the bacteria overexpress a protein?

A plasmid is a circular DNA. It is used as a cloning vector meaning a DNA fragment that needs
to be cloned is put into it. The Plasmid is cut by Restriction Nuclease and the new DNA
fragment is added by DNA ligase. The new Plasmid is introduced to the bacteria cell and
grown in the presence of an antibiotic to force retention of the plasmid.

4. What are libraries? What are the two most commonly used libraries? How are they made?

Libraries are a collection of all nucleic acid sequences in a genome. The two most commonly
used libraries are Genomic DNA (gDNA) and Complementary DNA (cDNA). gDNA includes all
DNA in the genome (Regulatory regions, introns etc.) and cDNA includes expressed sequence
in the genome. DNA -> Restriction Nuclease -> DNA fragments -> DNA cloning -> gDNA. DNA
-> Transcription -> RNA -> RNA splicing -> mRNA -> Reverse Transcript -> cDNA fragments -
> DNA cloning -> cDNA

5. What does a Northern and Western blot have in common? What’s different about how the
specific macromolecules are detected in these procedures?

Both use gel electrophoresis for detection, Northern detects RNA sequences while Western
detects proteins. Western uses antibodies to tag a protein and a Northern blot uses
hybridization.

6. What is the major difference between Northern and Southern blotting? What information does
it provide?

Northern detects RNA and is geared towards gene expression using formaldehyde to denature
RNA and Southern detects DNA.

7. How is hybridization used in Northern and Southern blots?

Hybridization Is used by binding two strands, one with a chemical dye with sequence
homology. It allows to visualize the location and amount of DNA/RNA on a blot or in a cell and
detects highly specific nucleic acids in a complex mixture.
8. What is a “probe”? How is it made?

A probe is a single stranded DNA fragment with a chemical marker used to identify certain
nucleic acids in solutions. It is made when pure DNA restriction fragment is denatured and
annealed with hexanucleotides, markers for the probe. DNAp and labeled nucleotides are
added and incorporated, leaving DNA with labeled examples of all sequences in both strands.

9. Why is it necessary to use a DNA polymerase that is resistant to heat inactivation in order to
do a Polymerase Chain Reaction (PCR)?

It is necessary to use a DNAp that is resistant to heat inactivation in order to do a PCR

because the strands that are heated to separate are then cooled so primers can bind to the
strands.

10. What are the three “steps (cycles)” used in PCR? What happens at each step?

1. Heat to separate strands – strands are separated

2. Cool to anneal primers – Primers are added onto the DNA
3. DNA synthesis – DNAp + nucleotides are added to create two double stranded DNA
molecules

11. What are expression vectors? What are they used for?

Expression vectors are plasmids that have been designed to produce a large amount of stable
mRNA that can be efficiently translated into protein when the plasmids are introduced into
bacterial, yeast, insect, or mammalian cells. They are used for introducing a specific gene into
a target cell and using that cells protein synthesis to overproduce the protein. Proteins that do
not fold properly are expressed in bacteria usually.

12. How does enzymatic DNA sequencing work? What special reagent is used to make it work?

Enzymatic DNA sequencing uses DNAp along with chain-terminating nucleotides

(deoxyribonucleoside triphosphate) to make partial copies of DNA fragments to be sequenced.
When incorporated into a growing DNA stand they block further elongation of the strand.

13. What is Shotgun Sequencing? How are the four bases “read” in Illumina Sequencing?

Shotgun sequencing is when the DNA is fragmented into small pieces and a genomic library is
constructed, typically using plasmids and bacteria. The nucleotide sequence of tens of
thousands of individual clones is determined and the full genome sequence is reconstructed
using overlaps between the clones. Works well for small genomes (viruses and bacteria) that
lack repetitive DNA. In illumina sequencing the four bases are read by having different colored
fluorophores on them which are picked up when pictures are taken of the DNA.

14. What is RNA-seq used for? Why is it considered more comprehensive than a Northern blot?

RNA sequencing is used for isolating mRNA, converting it to cDNA by RT enzyme for strand
specific sequencing. It is considered more comprehensive than a Northern blot because it
helps to study the complete set of RNA transcripts that are produced by the genome using
micro arrays. Micro arrays can analyze more genes in a quicker amount of time than N
blotting.
15. What is difference between Forward Genetic Screens and Reverse Genetics Screens?

Forward screen is when you broadly change the genes and select the phenotype you want to
study; Reverse genes is when you select the gene you want to modify and observe the
phenotypes that may result.

16. Describe the process of how you can change an amino acid found in a protein.

EG: a gene can be fused to the gene for a fluorescent protein. When this altered gene is
introduced into the genome, the protein can be tracked in the living organism by monitoring its
fluorescence. A genome can be altered so that the expression of any gene can be switched on
or off by the experimenter.
Reverse Genetics: Change the sequence of DNA in the test tube and insert the altered DNA
back into the animal, directly changing the gene you want to in a controlled way.

17. How can recombinant DNA technology introduce DNA into cells and change the sequence of a
chromosome?

Recombinant DNA technology can be used to delete, alter, or introduce DNA into the cells and
change the sequence of a chromosome by replacing the gene with a mutant gene, having the
gene knock out the mutant gene, and by having both the mutant and normal genes.

18. How are transgenic mice made?

Transgenic mice are made by adding an altered version of the target gene into E5 cells in a
culture. The colony with one copy of the target gene replaced by the mutant gene is injected
into an early embryo. Then the offspring are tested for presence of altered gene and bred until
a transgenic mouse is created.

19. What guides the Cas9 protein (used in the CRISPR approach) to its targeted double-stranded
DNA? Why is this an improvement over previously used proteins that can create double
stranded breaks in DNA?

A sgRNA (single guide RNA) composed of a scaffold and spacer guides the Cas9 protein to
the targeted DNA. The sgRNA’s can be customized by researches to target genes-of-interest.

20. What style of genetic screen (forward, reverse) could CRISPR approaches be more easily be
used with? Why?

Reverse screen would be used for CRISPR because a new mutated gene could be introduced
to replace the old one.