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Concept 20.1. DNA Sequencing and DNA cloning are Recombinant DNA molecule
valuable tools for genetic engineering and biological - a molecule containing DNA from two different
inquiry sources, very often different species
C. Using Restriction Enzymes to make a recombinant 2. Annealing – decrease temperature to -68 degrees
DNA Plasmid Celsius
To stick something on something, to put them
Restriction Enzymes together
- were discovered in the late 1960s by biologists You put primer on denatured DNA strand
doing basic research on bacteria
Primer – starts the elongation of
- protect the bacterial cell by cutting up foreign
complementary DNA Strand eventually
DNA from other organisms or phages
replicating DNA, it is a short segment of DNA
- enzymes that cut DNA molecules at a limited
number of specific locations. Primer sticks on very short sequences of bases
Restriction Site (50 – 100 bases), hahanapin ang
- a particular short DNA sequence where complement/counterpart
restriction will start 3. Elongation – increase temperature to 72 degrees
- mostly symmetrical when sequence of Celsius
nucleotides is same on both strands when read New DNA strand elongates
in 5” -3” Complementary strands elongate (started by
Restriction Fragments primer)
- cuts in such a DNA molecule Bases/building blocks will be added to primer
- “All copies of a given DNA molecule always yield Oligonucleotides (DNTPs) – separate
the same set of restriction fragments when
nitrogenous bases
exposed to the same restriction enzyme.”
DNA polymerase gets the nitrogenous bases
Sticky End
and sticks it to the primer, stitches building
- one single-stranded end
blocks (Thermophilus aquaticus -Taq
- These short extensions can form hydrogen-
bonded base pairs with complementary sticky polymerase) – microbe that lives in high
ends on any other DNA molecules cut with the temperature
same enzyme 5’ – 3’ direction of elongation
Gel Electrophoresis Pfu polymerase from Pyrococcus furiosus can
- uses a gel made of a polymer as a molecular also be used but is more expensive than Taq
sieve to separate out a mixture of nucleic acid
fragments by length E. Expressing Cloned Eukaryotic Genes
- used in conjunction with many different
techniques in molecular biology. a. Bacterial Expression Systems
Expression Vector
D. Amplifying DNA: PCR and its use in DNA Cloning - a cloning vector that contains a highly active
bacterial promoter just upstream of a
Polymerase Chain Reaction (PCR)
restriction site where the eukaryotic gene can
- devised in 1985
be inserted in the correct reading frame.
Steps in PCR: Disadvantages:
1. Denaturation – increase temperature to 94 – 96 - Presence of noncoding regions (introns) in
most eukaryotic genes. Introns make gene very
degrees Celsius
long thus, it prevents correct expression by
Breaking bonds
bacterial cells. This can be solved with exons-
Unzipping double stranded DNA
only gene.
Breaks H bond
CRISPR-Cas9
Major Uses of iPs Cells:
a. Act as model cells for studying the disease and C. Pharmaceutical Products
potential treatments - development of useful drugs to treat diseases
- cells from patients suffering from diseases - Pharmaceutical products are synthesized using
have been reprogrammed to become iPS cells methods of either organic chemistry or
b. In the field of regenerative medicine, a patient’s own biotechnology
cells could be reprogrammed into iPS cells and then
used to replace nonfunctional tissues, such as insulin- D. Synthesis of Small Molecules for Use as Drugs
producing cells of the pancreas imatinib (trade name Gleevec)
- small molecule that inhibits one tyrosine kinase.
20.4. Practical Applications of DNA-based technology The overexpression of this kinase, resulting
affect our lives in many ways from a chromosomal translocation, is
instrumental in causing chronic myelogenous
Medical Applications leukemia
- identification of human genes whose mutation plays a
role in genetic diseases E. Protein Production in Cell Cultures
- contributing to our understanding of “nongenetic” - Pharmaceutical products that are proteins are
diseases commonly synthesized on a large scale using
- DNA microarray assays or other techniques to cell cultures
compare gene expression in healthy and diseased - ex. human insulin and human growth
tissues, researchers are finding genes that are turned on hormone
or off in particular diseases. These genes and their - tissue plasminogen activator (TPA). If
products are potential targets for prevention or administered shortly after a heart attack, TPA
therapy. helps dissolve blood clots and reduces the risk
of subsequent heart attacks.
A. Diagnoses and Treatment of Diseases
- use of PCR and labeled nucleic acid probes to F. Protein Production by “Pharm” Animals
track down pathogens Introduce a gene (or other DNA) from an animal
- RT-PCR is often the best way to detect an into the genome of another individual, often of a
otherwise elusive infective agent. different species. This individual is then called a
- genome-wide association studies have transgenic animal.
pinpointed SNPs (single nucleotide Transgene – foreign DNA
polymorphisms) that are linked to disease-
associated alleles G. Forensic Evidence and Genetic Profiles
- prompted improvements in disease - body fluids or small pieces of tissue may be
treatments left at the scene or on the clothes or other
possessions of the victim or assailant
B. Human Gene Therapy and Gene Editing
Gene therapy DNA testing - can identify the guilty individual with a
- the introduction of genes into an afflicted high degree of certainty because the DNA sequence of
individual for therapeutic purposes every person is unique (except for identical twins)
- The aim of this approach is to insert a normal
allele of the defective gene into the somatic Genetic profile (dna fingerprint) - individual’s unique
cells of the tissue affected by the disorder. set of genetic markers
- Bone marrow cells, which include the stem cells
that give rise to all the cells of the blood and Short tandem repeats (STRs) - tandemly repeated units
immune system, are prime candidates. of two- to five-nucleotide sequences in specific regions
H. Environmental Cleanup
- diverse abilities of certain microorganisms to
transform chemicals are being exploited for
environmental cleanup
- Genetically engineered microorganisms may
become important in both mining (especially as
ore reserves are depleted) and cleaning up
highly toxic mining wastes.
- Biotechnologists are also trying to engineer
microorganisms that can degrade chlorinated
hydrocarbons and other harmful compounds
- These microorganisms could be used in
wastewater treatment plants or by
manufacturers before the compounds are ever
released into the environment.
I. Agricultural Applications
- DNA technology enables scientists to produce
transgenic animals, which speeds up the
selective breeding process.
- Agricultural scientists have already endowed a
number of crop plants with genes for desirable
traits
- Crops engineered with a bacterial gene making
the plants resistant to an herbicide can grow
while weeds are destroyed, and genetically
engineered crops that can resist destructive
insects reduce the need for chemical
insecticides.