PCR

Question 1

What is PCR?

Answer 1

DNA amplification detected by gel electrophoresis or probes

Question 2

What we need

Answer 2

Template DNA, primers, free dNTPs, Buffer, Polymerase enzyme + MgCl2

Question 3

3 cycle steps

Answer 3

Denaturation= 95 °C
Annealing = 60°C
EIongation = 72°C

Question 4

Analysis after PCR

Answer 4

Buffer covers gel, current applied, DNA migrate to +ve electrode

Question 5

ARMS PCR requires

Answer 5

precise base matching at 3’ end

Question 6

PCR for SNP

Answer 6

Selective amplification : Primer matches or mis-matches at 31and

Question 7

ARMS PCR Principle

Answer 7

2 reactions with 3 primers :
1 primer complementary in Both reactions= shows Reaction is working (+ ve control)
others differ at 3’ end for normal or mutant ( 1 in each tube)
match= extension

Question 8

ARMS Result

Answer 8

Homozygous= Amplification in only 1 tube
Herero= Both tubes
control = always +ve
Blank = always -ve

Question 9

+ve control requirements

Answer 9

Diff size to products so it is visible on gel

Question 10

Taq DNA Pol

Answer 10

Lacks 3’ to 5’ exonuclease proofreading activity = doesn’t correct mis-matched primer

Question 11

why use blank?

Answer 11

shows no contamination

Question 12

PCR for CF

Answer 12

ARMS PCR (2 arms=2 tubes)

Question 13

Designing primer

Answer 13

Always 5’ to 3’ direction

Question 14

calc Size of product

Answer 14

No. Of bases from From start of forward primer to start of reverse Primer

Question 15

Prevent carryover x4

Answer 15

filter tips+ designated Pipettes, uv irradiation, uracil N glycosylase, Aliquot reagents

Question 16

Real time PCR Prevent carryover

Answer 16

Sealed reaction+ no post-PCR manipulation

Question 17

Uni - directional workflow

Answer 17

from clean (pres to dirty (postPCR) = Prevents contamination

Question 18

UraciI N -glycosylase principle

Answer 18

dUTP used instead of dTTP, enzyme removes uracil = Not used as template

Question 19

Reverse Transcriptase PCR

Answer 19

measurers MRNA expression :
1) Reverse transcription= RT enzyme produces cDNA,
2) PCR Primers bind target = elongation, gel electrophoresis

Question 20

oligo dT primers

Answer 20

Specificity to MRNA, allow many targets on CDNA

Question 21

Random primers

Answer 21

Synthesise large pools of CDNA by annealing throughout, Commonly qPCR

Question 22

3 PCR graph phases

Answer 22

Exponential : 100%. efficiency (doubling)
Linear: components being used up/ degrading
Plateau : End Point =stopped

Question 23

q (Real time) PCR Principle

Answer 23

Detection during exponential phase by Reporter fluorescence

Question 24

signal

Answer 24

Directly proportional to no. of amplicons

Question 25

qPCR method

Answer 25

same as PCR but with Probes uses DNA or CDNA

Question 26

SYBR Green detection

Answer 26

Binds any dSDNA, Product formed= increased fluorescence

Question 27

real Time disadvantage

Answer 27

No gel run= assume correct DNA

Question 28

Melt curve analysis

Answer 28

Product slowly heated + sequence determines temp = determines correct DNA

Question 29

Melt curve results

Answer 29

1 peak= 1 product

Question 30

incorrect products examples

Answer 30

Non specific products= diff target Primer dimers (short) identify by running an gel

Question 31

Probe (Taqman)

Answer 31

3' quencher 5' reporter Reporter cleaved Relies on NO exonuIease activity

Question 32

Real Time probes bind when

Answer 32

During extension

Question 33

Threshold Cycle ( CT)

Answer 33

Cycle where amount of reporter fluorescence > threshold lower CT = Higher starting no. of copies

Question 34

Absolute Quantitation uses

Answer 34

standard curve

Question 35

Relative Quantitation ( Delta CT)

Answer 35

Differences in expression level of gene between diff samples uses control (housekeeping) gene =fold change

Question 36

Delta CT

Answer 36

CT (target)- CT ( Ref)

Question 37

D D CT

Answer 37

D CT (treated sample) - D CT (untreated)

Question 38

Fold change calc

Answer 38

2 ^ -D D CT

Question 39

uses of q PCR example

Answer 39

HIV monitoring