This site is 100% ad supported. Please add an exception to adblock for this site.

7. Genetic techniques and molecular medicine


undefined, object
copy deck
What is the function of Type II restriction endonuclease?
Cleaves dsDNA at specific base sequences
What is the function of DNA ligase?
Joins two DNA molecules or fragments
What is the function of DNA pol I?
Fills gaps in duplexes by stepwise addition of NTs to 3' ends.
What is the function of Reverse Transcriptase?
Makes a DNA copy of an RNA molecule (reverse transcptn)
What is the function of a Polynucleotide Kinase?
Adds a phosphate to the 5' OH end of a polynucleotide to label it or permit ligation
What is the function of a Terminal Transferase?
Adds homopolymer tails to the 3'OH end of a linear duplex
What is the function of Exonuclease III?
Removes NT's from the 3' ends of a DNA strand.
What is the function of Bacteriophage lambda exonuclease?
REmoves NT's from the 5' ends of a duplex to expose single stranded 3' ends.
What is the function of Alkaline phosphatase?
Removes terminal phosphates from either the 5' or 3' end
What enzymes are critical tools for molecular analysis of the eukaryotic genome?
Restriction Endonucleases
What kind of sequences do RE's normally cleave?
Palindromic sequences
What is produced as a result of RE cleavage of DNA?
ds-DNA fragments with one of 3 types of ends.
What are the 3 types of ends generated by RE cleavage?
1. Flush "blunt" ends
2. Staggered/sticky 5' ends
3. Staggered/sticky 3' ends
What is the Southern blot used for?
Analysing eukaryotic genomes - Detects DNA
What is a Northern blot for?
What are the 6 steps in Southern blotting?
1. RE cleaves total genome
2. Gel electrophoresis
3. Denature dsDNA w/ Alkali pH
4. Blot on nitrocell. membrane
5. Detect via hybrid probe
6. Wash w/ increasing temp, decreasing salt conc.
What are 3 common types of DNA probes used?
1. Oligonucleotide
2. Genomic DNA probe
3. cDNA probe
When are oligonucleotide probes used?
When only a limited amt of a sequence of the target is known --> i.e. when protein sequence is known.
How could the gene sequence encoding a protein be identified?
-Know the amino acid sequence
-Know the possible codons that encode each amino acid
-Look at regions of minimal degeneracy (only 1/2 codons encoding each amino acid)
What are 2 methods used for generating DNA probes?
1. 5' end labeling with 32P (y - gamma phosphate
2. Backbone 32P (alpha phosphate) Random-primer labeling
What enzyme is used for 5' end labeling?
Polynucleotide kinase
What enzyme is used for generating random-primed lables?
DNA Pol I (Klenow fragment - minus the 5'-3' exonuclease)
...or DNA Pol III would work too
What is RFLP?
Restriction Fragment Length Polymorphism
What does RFLP refer to?
The heterogeneity of the human genome in that it contains both disease-producing and benign mutations.
where do polymorphic genes usually occur in the genome?
In intragenic sequences that don't code for proteins.
How are genomic variations detected?
By examining restriction fragment lengths - RFLPS.
What 2 types of variations result in RFLPs?
1. Single base point mutations in DNA - result in loss/gain of restriction site.
2. Tandem repeats - insertions of variable tandem repeat siequences - unique for individual.
What are 4 uses of RFLP?
1. Detect genetic variations
2. Detect genetic relatedness
3. Link RFLPS w/ gene mutations
4. Direct detection of mutations that cause disease
Are disease-causing mutations commonly causes of RFLPs?
No - it's rare; one example though is Sickle cell disease.
HOW exactly does RFLP detect polymorphism?
Genetic differences cause differences in restriction fragment lengths - this is then detected by southern blotting.
How does southern blotting show the polymorphism?
The position of bands will be different on the blot.
How does size affect a molecule's movement on gel?
Larger fragments move more slowly than small.
What is FISH?
Flourescence in situ hybridization
What is the purpose of FISH?
Visualization of specific DNA sequences in whole chromosomes.
What is an example of FISH?
Detection of CML by detecting the philadelphia chromosome.
What causes the philadelphia chromosome?
Reciprocal translocation between bcr-1 on chrom 22 and c-abl proto-oncogene on chrom 9.
How does the Ph' chromsm cause cancer?
The resulting mRNA from the transcribed mutated DNA encodes a hybrid protein lacking normal controls that repress c-abl tyrosine kinase activity.
What is gleevec?
the treatment for CML
How does gleevec work?
INactivates BCR-abl tyrosine kinase activity.
What is Expression profiling?
The use of DNA chips to view the comprehensive set of genes that are expressed during a certain process.
What does expression profiling allow you to see?
A set of genes expressed in one situation, Another set expressed in another situation, and Genes that are common to both. (3 colors result)
What is the process of expression profiling?
1. Generate mRNA via reverse transcription
2. Isolate mRNA from sample
3. Label chip w/ comprehensive array of cDNA oligont's on slide
4. Hybridize mRNA to slide, look at flourescence pattern.
What did Dr. Basile's lab use that was an example of this type of expression profiling?
Microarray - to see the expression pattern of acute renal failure and ischemia.
What are common MARKERS inserted into plasmids, to allow you to tell whether it has been taken up by the intended cells?
Antibiotic resistence genes - their expression allows resistence when placed in a medium containing the drug - ampicillin and tetracycline.
How do you introduce DNA fragments into vectors?
A. Use the SAME endonucleases to make cuts in
1. chromosomal DNA, and
2. The plasmid cloning vector
B. Use ligase to glue the DNA fragment into the vector.
How do you get dna vectors to be expressed in eukaryotic cells? (2 ways)
1. Chemical transfection (to force cellular uptake of vector)
2. Viral uptake - cripple the virus DNA but take advantage of its cell invasion ability.
What are the 3 requirements of a eukaryotic vector EXPRESSION MODULE?
1. Promotor and enhancer elements (usually viral promoters)
2. Termination and Poly A signals (for stability and efficient translation)
3. Splicing signals
What other type of sequence is commonly added to vectors that isn't essential for just expression of the gene?
Selectable markers - i.e. antibiotic resistance genes - to allow for detection of the gene's absence or presence.
What is conditional expression?
Expression of the vector gene under certain controllable conditions.
What are 2 types of conditional expression?
1. Tissue specific
2. Inducible
What causes tissue specific expression?
The insertion of tissue specific promoters - so a protein from that tissue would cause expression of the gene.
What are some common inducible expressors (3)?
1. Metal inducers - for example metallothionein
2. Glucocorticoid responsive - inducd by dexamethasone
3. Tetracycline responsive promoters
In what nature is genetic information introduced into transgenic mice?
Nonhomologously - random; there's no way of knowing where it is going to insert.
What is done to allow for Selecting where in the genome the transgenic vector will insert?
Knockout cell generation
Why is it important to know the exact bp sequence in DNA?
to understand the mechanism of gene regulation and uncover the mechanisms of disease.
What is sanger sequencing?
A technique for determining the DNA sequence.
What is the gist of sanger sequencing?
The use of ddNTP (dideoxy NTPs) to cause the termination of DNA synthesis - they lack the 3' OH and can't form a phosphodiester linkage.
What are the necessary ingredients for sanger sequencing?
-DNA Polymerase
-4 dNTPs
-4 ddNTPs
What happens in sanger sequencing?
-normal dNTPs are incorporated until a ddNTP causes chain termination;
-ddNTPs are labeled w/ dye
-Denaturation produces a bunch of dye-labeled fragments
-Subject fragments to gel electrophoresis, then read.
What is the product of a sanger sequencing?
A computer printout showing the distance each fragment migrated; this is the order of the DNA sequence, because each fragment is specifically labeled for its NT identity.

Deck Info