Chapter 7

Question 1

What is the general blotting technique?

Answer 1

fragments in material to be analyzed (DNA, RNA, protein) are separated by gel electrophoresis

–small molecules travel faster and are at bottom of gel

–bands are blotted to surface of a membrane

–membrane is incubatedwith (radioactive) labeled probe

–visualize labeled probe will reveal which bands interacted with prboe

Question 2

What is the difference between DNA, RNA and protein blotting?

Answer 2

DNA is cleaved by restriction endonucleases and restriction fragments analyzed

RNA and protein: not cut up; analyzed directly

Question 3

What is the difference between Southern, Northern, Western and dot (slot) blots?

Answer 3

Question 4

What are probes?

Answer 4

radioactively labeled single stranded DNA molecues that hybridize (anneal) to particular denatured DNA sequence

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

Question 5

What is a southern blot?

Answer 5

determine which restriction fragment length polymorphisms are associated with genes

–variable number tandem repeats (repetitive sequences contribute to RFLPs in centromeric and telomeric regions)

RFLPs can be used in genetic testing to infer presence of disease causing allel in family with genetic disease

Question 6

What do northern blots analyze?

Answer 6

RNA extracted from tissue

determine which genes are being expressed

variaility in lengths of mRNA transcribed from single gene may be hte result of alternative splicing

Question 7

What is Fragile X syndrome?

Answer 7

inherited mental retardation

SYMPTOMS: large ears, elongated face, hyperombile joints, macroochidism in postpubertal males

FMR1 on long arm of X chromsome

Question 8

What is gene expression profiling (microarrays)?

Answer 8

embed probes for many different mRNA in multi-well gel or on chip to simultaneously determine whether hundreds of genes are expressed in a particular tissue

Question 9

What are western blots (immunoblots)?

Answer 9

separted proteins by gel electrophoresis and use 125I labeled probe antibodies to detect proteins (antigens)

–presence of antibodies to HIV virus in HIV testing

–identify whether protein is in cell

–test gene expression at level of translation

Question 10

What is polymerase chain reaction (PCR)?

Answer 10

selected region of chromosome amplified; small samples of DNA to be used for further testing

–must know nucleotide seuqnce bordering (flanking) target region at each of it 3 prime ends

Question 11

What can PCR be used for?

Answer 11

–comparing DNA samples in forensic cases paternity testing

–direct mutation testing

–diagnosing bacterial and viral infection

–HIV testing when Ab tests are uninformative (infants whose mothers are HIV positive)

Question 12

What aer short tandem repeats (STR, microsatellites)?

Answer 12

repeats of a di- to tetranucleotide sequence in spacer region and gene regions

–used in genetic testings

Question 13

How do you do paternity testing?

Answer 13

PCR amplification of microsatellite sequences

–primers overlap 3’ flanking regions adjacent to repeat analyzed

–amplify single locus sequences that are highly polymorphic within population

–9-10 different polymorphism to indicate a match

Question 14

How do you use PCR in direct mutation testing?

Answer 14

mutations causing a length difference can be detected by gel electrophoresis as lenght difference in PCR product

–mutations causing a sequence difference can be detected by testing the PCR products with ASOs

Question 15

How do you sequence DNA for direct mutation testings?

Answer 15

1. use PCR to amplify region and sequence one of the two strands to determine whether it has mtuation
2. many copies of primer that bind only to one strand
3. sample of DNA to be sequence is put in each of four reaction mixes containing DNA polymerase and all necessary dNTPs required to make new DNA
4. each test tube has different dideoxynucleide triphosphate that lack 3’ and 2’ hydroxyl groups

–ddNTP inserted into growing chain of DNA but then anyh subsequence elongation is stopped

5. separated by gel electrophoresis
6. sequence of new strand can be read from smallest to largest fragments on the gel (from bottom to top)
   - original strand (template in sequencing procedure) is complemetnary and anti-parallel to sequence read from gel

Question 16

How do you ELISA for HIV testing?

Answer 16

test for antibodies to HIV virus

high sensitivity, low specificity

postive result confirmed with western blot for Ab that are reactive to HIV protein antigens

Question 17

How do you use PCR for HIV testing?

Answer 17

test for integrated proviral genome

primers are specific for HIV provirus

if person is infected, proiral genome amplified and detected

–ositive early after infection and doesn’t rely on antibody response by individual

USED WHEN: newborn whose mom is HIV positive; early testing after known exposure to HIV positive blood or fluids/tissues

Question 18

How do you measure viral load in HIV patients?

Answer 18

RT-PCR detects and quantifies RNA

–measure concentration of active circulating virus in blood of AIDS patients

*status of infection and infection’s response to antiviral drugs