7. Genetic techniques and molecular medicine
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- 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?
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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?
- RNA
- What are the 6 steps in Southern blotting?
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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?
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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?
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-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?
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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?
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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?
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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?
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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?
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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?
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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)
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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?
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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?
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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)?
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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?
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-DNA Polymerase
-4 dNTPs
-4 ddNTPs - What happens in sanger sequencing?
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-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.