Chapter 7: Techniques of Genetic Analysis

Question 1

In gel electrophoresis, describe how smaller molecules travel on the gel?

Answer 1

faster and appear nearer the bottom of the gel

Question 2

What is the material analyzed in Southern blots?

Answer 2

DNA

Question 3

Is electrophoresis required when performing Southern blots?

Answer 3

yes

Question 4

What is the probe used in Southern blots?

Answer 4

32p-DNA

Question 5

What is the purpose of using a Southern blot?

Answer 5

method used to detect specific DNA molecules from among a many other DNA molecules.

Question 6

What is the material analyzed using Northern blots?

Answer 6

RNA

Question 7

Is electrophoresis used when performing northern blots?

Answer 7

yes

Question 8

What is the probe used in Northern blots?

Answer 8

32p-DNA

Question 9

What is the purpose of using Northern blots?

Answer 9

to measure sizes and amounts of specific mRNA molecules to answer questions about gene expression

Question 10

What is the material analyzed using Western blots?

Answer 10

protein

Question 11

Is gel electrophoresis used in Western blotting?

Answer 11

yes

Question 12

What is the probe used for Western blots?

Answer 12

125I- or enzyme-linked antibody

Question 13

What is the purpose of performing a Western blot?

Answer 13

to measure amount of antigen (proteins) or antibody

Question 14

What is the material analyzed in a Dot (slot)

Answer 14

RNA, DNA, or proteins

Question 15

Is electrophoresis required in Dot slots?

Answer 15

no

Question 16

What is the purpose of using a dot slot?

Answer 16

to detect specific DNA, RNA, protein, or antibody

Question 17

What are DNA probes?

Answer 17

radioactively labeled single-stranded DNA molecules that are able to specifically hybridize (anneal) to particular denatured DNA sequences

Question 18

Why is the probe an important part of analyzing any blot?

Answer 18

because the only bands that will appear on the final autoradiogram are those to which the probe has hybridized

Question 19

What does RFLP stand for?

Answer 19

restriction fragment length polymorphism

Question 20

What does VNTR stand for?

Answer 20

variable number of tandem repeat sequences

Question 21

What is a VNTR?

Answer 21

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

Question 22

What is PCR?

Answer 22

technique in which a selected region of a chromosome can be amplified more than a million-fold within a few hours

Question 23

What is the major constraint to using PCR?

Answer 23

you must know the nucleotide sequence bordering (flanking) the target region at each of its 3’ ends

Question 24

What are short tandem repeats? Also how are they useful?

Answer 24

STRs, or microsatelites are repeats of a di to tetranucleotide sequence. They are useful in genetic testing

Question 25

Describe the steps in PCR?

Answer 25

1. Add the sample containing DNA to be amplified
2. Add excess amounts of primers complementary to 3’ flanks of the target sequence. This selects the region to be amplified.
3. Add a heat-stable DNA polymerase (Taq DNA polymerase) and deoxyribonucleotides (dNTPs) for DNA synthesis.
4. Heat the sample to melt the DNA (convert dsDNA to ssDNA)
5. 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.
6. 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

Question 26

What parts of the gene are used in amplification of microsatellite sequences?

Answer 26

VNTRs and STR sequences

Question 27

How can mutations causing a length difference in PCR be detected?

Answer 27

by gel electrophoresis as a length difference in the PCR product

Question 28

How mutations causing a sequence difference can be detected in the PCR sequence?

Answer 28

by testing the PCR products with ASO (allele specific oligonucleotide)

Question 29

What is the difference between dNTPs and ddNTPs?

Answer 29

lacks both the 3’ and 2’ hydroxyl groups in ddNTP

Question 30

How would you read the sequence of the original strand in DNA sequencing through PCR?

Answer 30

it would be complementary and anti-parallel to the sequence read from the gel

Question 31

What does ELISA stand for?

Answer 31

enzyme-linked immunosorbent assay

Question 32

Describe ELISA effectiveness in testing people with HIV?

Answer 32

Elisa has high sensitivity but somewhat lower specificity.

Question 33

A positive ELISA test is confirmed by what form of genetic testing in regards to HIV? Why?

Answer 33

Western blot for ab that are reactive with specific HIV protein antigens

Question 34

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?

Answer 34

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:

1. positive much earlier after infection
2. 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

Question 35

What is an RT-PCR?

Answer 35

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

Question 36

What does RT-PCR stand for?

Answer 36

reverse transcriptase PCR

Question 37

How can RT-PCR specifically aid in measuring the concentration of viral load in AIDs patietns?

Answer 37

1. blood sample from HIV individual treated with reverse transcriptase to produce cDNA from any RNA
2. The cDNA is subsequently PCR amplified using primers specific for the end sequences of the HIV cDNA
3. amplified product is quantitated and, with the use of a standard curve can be related to the original amount of HIV RNA present