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28 Cards in this Set

  • Front
  • Back
Give the definition for SNPs, STRs, and RFLPs.
Single nucleotide polymorphisms: substitution of a single nucleotide

Short tandem repeats: tandem bi, tri or tetra nucleotide repeats such as (TG)xn (a.k.a. microsatellite markers.

Restriction fragment length polymorphisms: alter fragment lengths by changing a restriction site (palindromic, recognized by restriction enzymes for cutting) or due to internal VNTR (variable number tandem repeats).
What are 3 methods of mutation detection for novel mutations?
Southern blot

Single stranded conformational analysis/Denaturing high performance liquid chromatography

Direct sequencing
What are restriction sites? What is the purpose of restriction enzymes?
Palindromic sequences (ex: AGCT – it’s complementary strand is also AGCT in the 5’→ 3’ direction) recognized by restriction enzymes. A genetic variation at the restriction site can cause gain or loss of the restriction site.

In nature, restriction enzymes are meant to rid the body of foreign DNA. But they’re used in the lab to cut and paste DNA at precise location because each enzyme recognizes a specific sequence.
Describe the method to detect restriction fragment length polymorphisms.
A polymorphism that is located within a restriction site can be detected and genotyped by restricting the DNA. For example: restriction enzyme EcoRI recognizes 5’-GAATTC-3’ wherever it occurs in a double-stranded DNA molecule and cleaves it at that site. There are about 1000 such restriction enzymes known.

If the mutation occurs in the restriction site, the EcoRI will not cut the DNA there any more, so by this you can distinguish alleles.
What is on the filter in a Southern, Northern, and Western blot? Which one is named after an actual person?
Southern – DNA
Northern – RNA
Western – Protein

Southern blot is named after Dr. Ed Southern.
Describe the Southern blotting technique and what it is good for. Is it more or less specific than FISH? How do fragments move on the gel?
Southern blotting is the standard method for analyzing the structure of DNA cleaved by restriction enzymes. It is useful for detecting deletions/insertions/rearrangements on the order of 100-1000 kb. The Southern blot is better than FISH for detecting smaller mutations, but not useful for single deletions.

DNA fragments (all negatively charged) move on the gel based on size. Smaller pieces move faster, charge moves faster (toward positive pole).

Start w/ genomic DNA. → Restrict (cut) the DNA w/ restriction enzymes. → Run it on an agarose gel, end up with a smear of millions of differently-sized fragments. → Denature DNA to make it single stranded → Transfer the smear onto a nylon filter (that’s the “BLOT”) → Probe the filter with the gene of interest. → wash off excess probe. → Result: autoradiograph of your genes of interest. (can be viewed using X-ray or fluorochrome)
What types of deletions would be detected by a Southern blot?
Deletions that affect a restriction site or more commonly involving at least a single exon (several hundred bp or more). It would not be useful for identifying a point mutation (too small) or the deletion of an entire gene (too big, it would just look invisible – there would be no novel “junctional fragment” present on the blot).
_________ is the “molecular Xerox machine” → all you need are ________.
Polymerase Chain Reaction (PCR)
Primers
Describe the PCR process. What is the main advantage of this method?
Start with a “template” (the substrate you use to amplify the DNA) → denature the DNA → put down primers, one going in the forward direction, one going in the other direction on the complimentary strand of DNA → After strands are separated, use DNA polymerase to synthesize a new complimentary strand for the denatured DNA. → After the new complimentary strand is synthesized, denature and place primers again, ad infinitum → Each cycle of PCR doubles the amount of target until all the primers have been utilized.

Main advantage → CREATE A LOT OF MATERIAL FROM A VERY SMALL AMOUNT. Unlike cDNA methods, PCR does not require a lot of DNA material. Can done with minute amounts, as little as the DNA from a single cell. It’s MUCH faster and cheaper, less labor intensive than former methods.
What are some practical applications for PCR?
After amplification, you can use the amplified material for SSCP, oligospecific allele amplification, paternity testing (amplify microsatellites, count number of repeats). ALSO, simple sequence repeats can be used as markers to track a chromosome (see what parent a gene came from).
Describe how PCR can be used in Huntington Disease.
Amplify the CAG repeat sequence. The CAG repeat size can be quantified by sizing the PCR product via electrophoresis.

CAG 10-26 = normal; CAG 27-35 = at risk for expansion; CAG 35-41 variable penetrance; CAG 42-121 = affected.
What are SSCP gels? What are they good for and how do they work?
Single Stranded Conformational Polymorphism. Used as a supplement to sequencing – a quick and dirty way of discovering NEW DNA POLYMORPHISMS (esp. when you don’t know what you’re looking for → “Mutation Discovery”). Can be used to QUALITATIVELY screen for genetic differences within 100-400 bp PCR fragments. Can detect single nucleotide changes that would be missed as a double strand. It does not indicate the nature of the variant. Sequencing is then required.

DNA is denatured to a SINGLE STRAND. After denaturation, single strands undergo 3-dimensional folding and may assume different conformational states according to DNA sequence. The different shapes travel through gel at different speeds, so the gel separates the different conformations.
DNA sequencing is based on random _____________. It proceeds by making a __________ of DNA off a specified primer and terminating the DNA synthesis when a ______________ is included (these are “sprinkled in” with the mix). While the _______ribonucleoside allows strand extension at the ____ end, _______ribonucleoside prevents it because the _____ group has been removed from the _____ end. Each ________ is indicated by a different ________ and may be read using an _____ instrument; or it may be physically separated in a different lane (back in the olden days) and read from the bottom up. This type of sequencing is known as _________ sequencing.
termination of replication
complementary strand
dideoxy nucleotide
color fluorophore
deoxy
3’
dideoxy
hydroxy (OH)
3’
dideoxynucleotide
ABI
Sanger (also known as “dideoxy sequencing”)
The next generation of sequencing is done by ________. In real time, DNA is synthesized with a different _________ for each nucleotide. Synthesis is captured in real time by ________. An example of this technology is _________. Each run of this type of sequencing generates 2 terrabytes of data (made up of short reads of ____ bp), which must then you must computationally figure out how to overlap/align to generate _______.
Synthesis (REMEMBER: “Sequencing by synthesis” principle)
Fluorephore
Taking pictures of the sequencing
Solexa Sequencing (now known as Illumina)
35
Contigs
If a specific genetic mutation or variation is already known, how can it be detected?
Targeted genotyping of single nucleotide polymorphisms can be done using a variety of different methods.
• Restriction enzyme recognition
• Allele specific PCR
• Allele specific hybridization
• FP TDI (fluorescence polarized di termination reaction)
• Mass spectrometry
• Pyrosequencing
What is allele specific PCR and allele specific hybridization?
Allele specific hybridization: an allele-specific oligonucleotide (ASO) probe will hybridize only to the normal complementary sequence exactly; it will not hybridize to an imperfect complementary sequence. (Alternatively, you could make a mutant ASO to hybridize to a mutant gene sequence.) Mismatches are washed away and you can read out which oligos hybridize to infer the genotype of the patient.

Allele specific PCR is the same idea, but you design a primer in the exact area you’re interested in studying. If there is a mutation in that spot, there is not an exact 3’ sequence match, the DNA polymerase will not function and synthesis will not take place.
How do you make a gene expression profile? What is it and what are they used for? What is the substrate for this type of hybridization?
Affymetrix expression is one famous company that has developed this technique. It is microarray analysis that measures the level of gene expression by hybridizing mRNA (mRNA is the SUBSTRATE, though for stability it is usually converted to DNA via reverse transcription) to oligonucleotides immobilized on an array. By making these profiles, you must measure and compare the activity of TWO DIFFERENT samples of ~30,000 gene arrays at once (for example, low-grade CaP vs. high-grade CaP, good outcome pt vs. bad outcome pt on the SAME ARRAY) to see which genes are more or less activated on which individual. Hot spots (darker color) represent more expression of the gene you’re interrogating on one individual compared to the other. Black spots mean equal expression of the gene between proband and baseline. These profiles may in the future be used to individualize treatment decisions.
How can you use real time PCR to measure gene expression? What else can real time PCR be used for?
rtPCR → reverse transcriptase followed by real time PCR → a.k.a. “quantitative PCR”

This technique measures the amount of PCR product produced in real time, which will increase exponentially with each cell cycle until a plateau is reached (i.e. primer is used up). Based upon how much PCR product is made, one can then calculate back how much of the target was present in the original sample. This can be done in a very targeted way; diagnostic tests and recurrence risk calculations for certain cancers (esp. breast cancer) are now established based on this method.

This technique can also be used to measure viral load (esp. HIV and hep C viral load).
What is RNA In Situ Hybridization? What does it allow you to do?
This is a type of hybridization that uses a DNA or (more commonly) RNA probe to localize a specific sequence to cells in a portion or section of tissue.

It allows you interrogate where within a heterogeneous tissue the gene of interest is expressed.
What is a Western blot? What is an example of its use?
Western blot allows for the detection of specific PROTEINS in a cell. A protein mixture is electrophoresed through a gel and transferred to a nylon membrane then hybridized with a primary antibody that specifically recognizes the protein to be analyzed. A second antibody that is tagged with a traceable substance detects the interaction between the primary antibody and its antigen. The second antibody amplifies and visualizes the signal.

A key example of its use is to detect the presence or absence and, if present, the size of the muscle protein DYSTROPHIN in patients with X-linked muscular dystrophy.
What is the term for using antibodies on a slide of tissue to determine where within the tissue the protein is located.
Immunocytochemistry
What is CGH and what types of mutations can it be used to assess?
Comparative genomic hybridization.

A molecular-cytogenetic method for measuring differences in copy number or DOSAGE of a particular chromosomal segment between TWO different DNA samples. One sample is labeled red, the other is labeled green. The ratio of red-to-green fluorescence in the FISH signal should be 1:1. When it’s off that ratio, you’ve got deletions or insertions (represented by an off-center peak in the graph).

This technique can be used to measure LARGE deletions or insertions of 3 Mb or more. Note: CANNOT detect BALANCED translocations.

This is often done in germline tissue. Also useful in TUMOR TISSUE to detect low levels of mosaicism.
Recombination during meiosis is a function of _________. In general there is a greater chance for recombination if the loci are _______.
The distance between two loci
Far apart (b/c more real estate there to recombine between the two of them).
What is linkage analysis?
Linkage analysis is a way of measuring the distance between genes. It is based on measuring the FREQUENCY with which two genes remain together (“linked”) through meiosis as they are passed from one generation to the next. Genes that are closer together are more likely to stay linked. Genes that are farther apart are more likely to recombine. FREQUENCY OF RECOMBINATION IS A PROXY FOR DISTANCE. LOW FREQUENCY = CLOSE TOGETHER.
How is linkage analysis done?
When you are trying to find a gene responsible for an inherited disease, you will look at genetic markers in relation to affected and non-affected individuals OF THE SAME FAMILY to try to identify possible candidate genes. Markers close to the gene of interest are “linked” and will not segregate independently during meiosis. Markers far away from the gene have a greater chance of recombination and independent segregation (they will distribute more randomly). Among the children, you would look for markers from the affected parent that show consistently on affected individuals AND are consistently absent on non-affected individuals (i.e. “tight coupling” → the markers segregate WITH the disease). Once you identify such markers, you have an idea of where the pathogenic genes are located.
What are genetic markers?
Linkage analysis relies on Genetic Markers, which are any characteristic (often SNPs) located at the same place on a pair of homologues chromosomes that allows you to distinguish one homolog from the other. **Note: The markers themselves are NOT PATHOLOGICAL. Markers that segregate with the phenotype are likely to be located in close proximity to the pathogenic gene.
Linkage can be used to identify a DNA segment in which there are no _________. All the genes within that _________ segment can then be identified and screened for _______.
Recombinations
Non-recombinant
Mutations
Explain Linkage Disequilibrium
Based upon genetic evolution, certain genetic variations may be physically close to each other and rarely recombine and are said to be in linkage disequilibrium. For this reason, genetic variations said to be “associated” with disease may not themselves be the pathogenic variants but may simply be in linkage disequilibrium with the disease causing variant.