PCR

Question 1

Basic components of PCR

Answer 1

Template DNA
Oligonucleotide primers
Deoxyribonucleotide triphosphates
heat stable dna polymerase
suitable buffer solution

Question 2

alterations to taq

Answer 2

make enzyme more processive(it can then copy longer regions)
more tolerant of sub-optimal conditions(will amplify without needing to determine best conditions)
hot start taq(proper activity at 70˚C)(won’t incorporate nucleotides at 20-40˚C)(enzyme trapped in wax bead or with antitaq antibodies)

Question 3

cycle temps and time

Answer 3

intial denaturation - 95˚C for 1-10 minutes
- additional denaturations - 94-95˚C for 30-120 seconds
annealing - 40 - 65˚C(primer specific) for 30-120 seconds
extention at 68-78˚C(72 is standard) for (product length in kb) seconds
repeat 30-40 cycles, hold at 4˚C

Question 4

Pfu

Answer 4

pyrococcus furiosus(firballs of fury! ༽つ۞﹏۞༼つ ─=≡ΣO)))
more thermostable than taq(18 hour half life at 95˚C)
has 3’ to 5’ exonuclease activity
error rate 1/385,000
no 3’ dA overhangs
amplifies longer products better, more processive and sensitive

Question 5

In selecting a region to be amplified

Answer 5

usually a dna sequence from sequencing you have done

size of amplicon varies from 100-10000 bp

Question 6

in choosing a pair of primers

Answer 6

annealing temps should be ± 2˚
should be 50% G+C
Should have GC clamp
shouldn’t false prime, form hairpins, or primer dimers

Question 7

Tm

Answer 7

temp that a double stranded sequence of the primer would fully melt into single stranded molecules

Question 8

mutagenesis

Answer 8

primer designed with single nucleotide mismatch

- amplified product cloned into vector

Question 9

reverse transcriptase

Answer 9

makes a ssDNA

Question 10

RT-PCR

Answer 10

amplifies an RNA sequence after it is reverse transcribed into DNA(amplifies ssDNA)

Question 11

Quantitative pcr

Answer 11

specialized pcr machines with w/ fluorescent detectors for each sample

Question 12

in quantitative pcr, the product is quantified by

Answer 12

SybrGreen dye: binds dsDNA and fluoresces

primers that fluoresce when bound to template

Question 13

sequence assembly

Answer 13

each piece extends off each other piece, end primers used to amplify only correctly assembled piece