Chapter 7: Techniques of Genetic Analysis Flashcards
In gel electrophoresis, describe how smaller molecules travel on the gel?
faster and appear nearer the bottom of the gel
What is the material analyzed in Southern blots?
DNA
Is electrophoresis required when performing Southern blots?
yes
What is the probe used in Southern blots?
32p-DNA
What is the purpose of using a Southern blot?
method used to detect specific DNA molecules from among a many other DNA molecules.
What is the material analyzed using Northern blots?
RNA
Is electrophoresis used when performing northern blots?
yes
What is the probe used in Northern blots?
32p-DNA
What is the purpose of using Northern blots?
to measure sizes and amounts of specific mRNA molecules to answer questions about gene expression
What is the material analyzed using Western blots?
protein
Is gel electrophoresis used in Western blotting?
yes
What is the probe used for Western blots?
125I- or enzyme-linked antibody
What is the purpose of performing a Western blot?
to measure amount of antigen (proteins) or antibody
What is the material analyzed in a Dot (slot)
RNA, DNA, or proteins
Is electrophoresis required in Dot slots?
no
What is the purpose of using a dot slot?
to detect specific DNA, RNA, protein, or antibody
What are DNA probes?
radioactively labeled single-stranded DNA molecules that are able to specifically hybridize (anneal) to particular denatured DNA sequences
Why is the probe an important part of analyzing any blot?
because the only bands that will appear on the final autoradiogram are those to which the probe has hybridized
What does RFLP stand for?
restriction fragment length polymorphism
What does VNTR stand for?
variable number of tandem repeat sequences
What is a VNTR?
a sequence designates a unit of nucleotides usually between 15 and 60 bp that is repeated in tandem multiple times at a particular location in the DNA
Although the repeated sequence is shared by all individuals the number of repeated units is variable from person to person
What is PCR?
technique in which a selected region of a chromosome can be amplified more than a million-fold within a few hours
What is the major constraint to using PCR?
you must know the nucleotide sequence bordering (flanking) the target region at each of its 3’ ends
What are short tandem repeats? Also how are they useful?
STRs, or microsatelites are repeats of a di to tetranucleotide sequence. They are useful in genetic testing
Describe the steps in PCR?
- Add the sample containing DNA to be amplified
- Add excess amounts of primers complementary to 3’ flanks of the target sequence. This selects the region to be amplified.
- Add a heat-stable DNA polymerase (Taq DNA polymerase) and deoxyribonucleotides (dNTPs) for DNA synthesis.
- Heat the sample to melt the DNA (convert dsDNA to ssDNA)
- Cool the sample to re-anneal the DNA. Because the ratio of primer complementary strands is extremely high, primers bind at the 3p flanking regions.
- Heat the sample to increase the activity of the Taq DNA polymerase. Primer elongation occurs, and new complementary strands are synthesized.
Process repeated for ~20 cycles
What parts of the gene are used in amplification of microsatellite sequences?
VNTRs and STR sequences
How can mutations causing a length difference in PCR be detected?
by gel electrophoresis as a length difference in the PCR product
How mutations causing a sequence difference can be detected in the PCR sequence?
by testing the PCR products with ASO (allele specific oligonucleotide)
What is the difference between dNTPs and ddNTPs?
lacks both the 3’ and 2’ hydroxyl groups in ddNTP
How would you read the sequence of the original strand in DNA sequencing through PCR?
it would be complementary and anti-parallel to the sequence read from the gel
What does ELISA stand for?
enzyme-linked immunosorbent assay
Describe ELISA effectiveness in testing people with HIV?
Elisa has high sensitivity but somewhat lower specificity.
A positive ELISA test is confirmed by what form of genetic testing in regards to HIV? Why?
Western blot for ab that are reactive with specific HIV protein antigens
There are some instances in which ELISA/Western blot is not useful and PCR is the test of choice to detect HIV infection. What are the reasons?
PCR is designed to test for the integrated proviral genome, not for ab to HIV protein ag
Primers that are specific for the HIV provirus are used for the PCR
If the person is infected, the proviral genome will be amplified and detected. If the person is not infected there will be no PCR product detected
2 Important Advantages:
- positive much earlier after infection
- Does not rely on an ab response by the individual
Important situations:
In HIV testing in newborns whose mothers are HIV positive (will always be positive in ELISA/Western blots) and early testing after known exposure to HIV-positive blood (e.g. needlesticks) or other fluid issue
What is an RT-PCR?
detects and can quantify a specific RNA rather than DNA in a sample;
useful in detecting RNA viruses such as HIV and is similar to the Northern blot, determining when a gene is transcribed
What does RT-PCR stand for?
reverse transcriptase PCR
How can RT-PCR specifically aid in measuring the concentration of viral load in AIDs patietns?
- blood sample from HIV individual treated with reverse transcriptase to produce cDNA from any RNA
- The cDNA is subsequently PCR amplified using primers specific for the end sequences of the HIV cDNA
- amplified product is quantitated and, with the use of a standard curve can be related to the original amount of HIV RNA present
