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Flashcards in PCR Deck (25)
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1
Q

what is the equation for final copy number?

A

Nf=N0(1+Y)^n
where N0=initial copy number
n= number of cycles
Y=efficiency of amplification per cycle
–>no bigger than 1, or 100% efficient
–>decreases as time goes on, because there is a lower ratio of primers to templates, enzymes to templates, etc

2
Q

what is one of the most improtant factors for successful PCR?

A

primer design –> length, nt sequence, Tm, etc

3
Q

Tm eq’n?

A

Tm=(4x(G+C)]+(2x(A+C)]

4
Q

important to remember than when using two primers (heteroprimers) it is important that Tm and annealing temp are similar

A

ya

5
Q

what is the optimal base composition of a primer?

A

C+G=40-60%

6
Q

antisense primer binds to the coding sequence shown while sense binds to the complementary sequence of the one shown

A

ya

7
Q

annealing temp 2-5 C below Tm

A

ya; too low and primers bind non-spec

8
Q

what does annealing time depend on?

A

the length and complexity of primers–>usually fixed though (60 seconds)

9
Q

why might high amounts of NTPs be inhibiting?

A

sequester Mg2+

10
Q

what are some additives to PCR rxn mixture and what is their purpose?–additives help to increase rxn speed or specificity

A
  • dimethyl sulfoxide–facilitates extension through template secondary structures–>disrupts H-bonds–>easier denaturing
  • glycerol–ezyme stabilizer
  • specificity enhancers
11
Q

major problems in PCR?

A

cross-contamination

nons-specific amplification

12
Q

what are some ways to identify PCR amplification products?

A

sequencing
size–expected size? has insertions?
restriction analysis–mutations resulting in restriction sites

13
Q

describe TA cloning

A

Taq tends to add overhanging A onto end of PCR product–clone into vector with overhanging T to basepair–>creates sticky ends

14
Q

figure out restriction cloning

A

ya

15
Q

describe allele-specific PCR

A

two alleles have at least one nt difference (at end of primer)–>design a primer to adhere perfectly to one allele but not the other–>the one that matches perfectly is amplified while the other is not; do not use an pol with exonuc activity, will correct the mismatch

16
Q

describe long PCR

A

use 2 thermostable pols–>one that is error prone to carry out most of the synth (Taq) and one with exonuc activity to correct the mismatches (Pfu)–>allows for synth of longer regions–>can synth up to 20-30kb

17
Q

describe assembly PCR

A

start with a mutated forward and reverse primer, synthesize; overlap mutated regions of the new templates, synthesize; then use non-mutated F and R primers to carry out amplification

18
Q

describe gibson assembly

A
  • for synth and cloning without restriction sites–assemble many fragments together
  • -overlap two ds seqs you want to anneal; chew back with 5’ 3’ exonuc; anneal (at 50)repair with Phusion pol and Taq ligase
19
Q

describe RT-PCR

A

mRNA with poly-A tail; use polyT oligo primer–>use RT enzyme

20
Q

describe multiplex PCR

A

figure it out LOL DUNNO–indicates genotype

21
Q

read about emulsion PCR

A

ya–used to create thousads of rxns in 1 mL

22
Q

normal length of a primer?

A

17-25 nts

23
Q

Equation for Tm (primers 14-70 nt, [M+] ≤ 0.5M)

A

Tm = 81.5 ̊C + 16.6(log10[M+]) + 0.41(%[G+C]) - 0.61(% formamide) - (675/n)

24
Q

taq vs pfu

A

• Taq

– Faster extension i.e. synthesis
– More tolerant of inhibitory substances
– Tends to add an overhanging adenosine (can be used in TA cloning)
• Pfu
– Lower error rate due to 3’-5’ exonuclease
activity
– Can amplify longer fragments
– Stronger thermal stability
25
Q

• Phusion
– Modified Pfu-like enzyme (adding a double- stranded DNA binding domain)
– Much enhanced extension speed – Can amplify longer DNA fragments

A

ya