PCR

Question 1

what is the equation for final copy number?

Answer 1

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

Question 2

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

Answer 2

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

Question 3

Tm eq’n?

Answer 3

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

Question 4

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

Answer 4

ya

Question 5

what is the optimal base composition of a primer?

Answer 5

C+G=40-60%

Question 6

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

Answer 6

ya

Question 7

annealing temp 2-5 C below Tm

Answer 7

ya; too low and primers bind non-spec

Question 8

what does annealing time depend on?

Answer 8

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

Question 9

why might high amounts of NTPs be inhibiting?

Answer 9

sequester Mg2+

Question 10

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

Answer 10

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

Question 11

major problems in PCR?

Answer 11

cross-contamination

nons-specific amplification

Question 12

what are some ways to identify PCR amplification products?

Answer 12

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

Question 13

describe TA cloning

Answer 13

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

Question 14

figure out restriction cloning

Answer 14

ya

Question 15

describe allele-specific PCR

Answer 15

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

Question 16

describe long PCR

Answer 16

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

Question 17

describe assembly PCR

Answer 17

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

Question 18

describe gibson assembly

Answer 18

• 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

Question 19

describe RT-PCR

Answer 19

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

Question 20

describe multiplex PCR

Answer 20

figure it out LOL DUNNO–indicates genotype

Question 21

read about emulsion PCR

Answer 21

ya–used to create thousads of rxns in 1 mL

Question 22

normal length of a primer?

Answer 22

17-25 nts

Question 23

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

Answer 23

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

Question 24

taq vs pfu

Answer 24

• 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

Question 25

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

Answer 25

ya