Lecture 4 Slides 8, 9, 16+

Question 1

amplicon

Answer 1

ssDNA that is amplified via DNA Polymerase

Question 2

Exonuclease

Answer 2

Enzyme that clips off any unpaired bases “proof reading”

Question 3

Primer

Answer 3

short, single strand of RNA that locates the start and end of a replication region for PCR

Question 4

Probe

Answer 4

piece of RNA or DNA labeled with Reporter that locates a specific region of DNA by hybridization

Question 5

Reporter

Answer 5

a signal, e.g. Radioactive or Fluorescent signal that is detectable

Question 6

Exogenous DNA

Answer 6

DNA outside a cell or an organism

Question 7

Cycle

Answer 7

one cycle of PCR replication

Question 8

PCR

Answer 8

Polymerase Chain Reaction process of replicating DNA

1. 95 C melting
2. 50 C annealing
3. 70 C Extension

Question 9

QPCR

Answer 9

quantitative PCR – monitored signal during reaction (once per cycle)
kinetic information
leverages real time PCR results

Question 10

RT-PCR

Answer 10

Reverse Transcriptase PCR, starting material RNA

Question 11

ICC-PCR

Answer 11

integrated cell culture PCR

- usually applied to detecting viral species in large samples

Question 12

TaqMan Probes

Answer 12

FRET pair, disassemble after extension, not reusable

Question 13

Molecular Beacons

Answer 13

FRET pair, stay intact after extension, re-usable

Question 14

Scorpions

Answer 14

FRET pair, complementary sequence that binds the amplicon and fluoresces

Question 15

SYBR Green

Answer 15

inexpensive, easy to use and sensitive, binds only double stranded DNA (desired and undesired products), an “intercalating dye” (not a FRET pair)

Question 16

Real Time PCR feature

Answer 16

• permits detection during PCR process
• reveals kinetic information on the reaction
• does not require post handling of the sample (ie transfer to an electrophoresis apparatus)
• can detect more than one target in a sample
• works with any PCR reagents