real time PCR

Question 1

what are the 4 major processes for measurement of HIV RNA levels?

Answer 1

1. specimen preparation to EXTRACT and ISOLATE HIV-1 RNA.
2. Reverse transcription of the target RNA to generate cDNA.
3. simultaneous PCR amplification of target cDNA.
4. detection of cleaved dual-labeled oligonucleotide detection probe specific to the target.

Question 2

what is the purpose of sample preparation?

Answer 2

extract and concentrate the target RNA molecules to make the target accessible for amplification

Question 3

binding buffer that lyse and releases nucleic acids to remove potential inhibitors of amplification from the extract

Answer 3

Chaotropic lysis

Question 4

___ capture technique bound to the surface of the magnetic glass particles

Answer 4

silica-based

Question 5

unbound substances, such as ___, proteins and other cellular impurities, are removed by washing the ____

Answer 5

salts; magnetic glass particles

Question 6

After separating the magnetic glass particles and completing the washing steps, the adsorbed ____ are eluted at ____ temperature with an aqueous solution

Answer 6

nucleic acids; elevated

Question 7

bound nucleic acids are transferred to

Answer 7

96 deep-well plate

Question 8

lyse cells in nuclei lysis buffer and release nucleic acid into the buffer

Answer 8

lysing

Question 9

add magnet beads to the buffer and mix in order to bind the nucleic acid to the coated of beads

Answer 9

binding

Question 10

repeatedly wash the surface of beads to get rid of needless nucleic acid, protein, salts or other impurities

Answer 10

washing

Question 11

move the magnet beads to elution buffer and mix well. the nucleic acid will fall off from the surface of beads and dissolve to the buffer

Answer 11

eluting

Question 12

steps of principle

Answer 12

lysing, binding, washing, eluting

Question 13

target RNA is converted to cDNA by the

Answer 13

reverse transcriptase

Question 14

extended by activity of the thermostable ___ ____

Answer 14

rTth DNA polymerase

Question 15

target sequence region is highly

Answer 15

conserved

Question 16

___ are designed to hybridize to the pol integrase region with the fewest possible mismatches among various target sequence subtypes

Answer 16

primers

Question 17

HIV-1 primers anneal to their respective targets and are extended during a _____

Answer 17

prolonged incubation period

Question 18

what includes in master mix?

Answer 18

deoxyuridine triphosphate (dUTP), AmpErase enzyme

Question 19

dUTP vs dTTP

incorporated into the newly synthesized DNA (amplicon)

Answer 19

dUTP

Question 20

any contaminating amplicons from previous PCR runs are eliminated by the _____

Answer 20

AmpErase enzyme

Question 21

AmpErase enzyme is used during

Answer 21

first thermal cycling step

Question 22

the probes are labeled with target specific _____ reporter dyes hybridization of the probes to the specific _____ DNA template

Answer 22

fluorescent; single-stranded

Question 23

cleavage of the probe by ____ exonuclease activity of the DNA polymerase resulting in _____ of the reporter and quencher dyes and the generation of a fluorescent signal

Answer 23

5’-3’; separation

Question 24

with each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye ______ _____

Answer 24

increase concomitantly

Question 25

each round of thermal cycling-amplification products dissociate to ____ at ____ allowing primer _____ and extension as the temperature is _____

Answer 25

single strand at high temperature; annealing; lowered