PCR

Question 1

What’s PCR & E.g

Answer 1

a method used to amplify DNA from small amount to large

e.g. ID genetic mutations, forensic testing

Question 2

What must you do /not do when doing PCR?q

Answer 2

• Wear PPE
• Clean tubes & pipette tips
• Don’t talk over tubes
• Minimise touching things w/ gloved hands
• Separate areas for different stages of PCR procedure

Question 3

PCR theory (in vitro)

Answer 3

1. DNA denature @ 95ºC = disrupt H bonds => dsDNA -> ssDNA
2. Annealing @ ~55ºC = primers bind to ssDNA
3. Extension @ 72ºC, DNA polymerase extends complementary strand - add dNTP @ 3’ OH of primer
4. steps repeated in many cycles

Question 4

PCR components?

Answer 4

1. reaction buffer
2. (MgCl2) > require Mg 2+ as a cofactor for Taq polymerase
3. hpH2O
4. All dNTPS
5. a pair of primers
6. DNA to be amplified
7. Taq (DNA polymerase) [most expensive]

Question 5

What types of controls you may need to set up for a PCR?

Answer 5

1. Pos. control: sample contains same region of interest to be amplified = should be 1 band > checks for validity
2. *Neg. : contains all components except DNA > ensures reagents are not contaminated OR check DNA is not amplified non-specifically
3. Amplification: uses a 2nd set of primers - not specific to region of interest but are specific for a region in sample (for reference) > ensures you have the sample in the rxn & rxn works (important for neg. controls)

Question 6

Typical reation volume for PCR is

Answer 6

20-50uL

Question 7

What do you do if your positive control is not working or has multiple bands?

Answer 7

• Invalid
• Perform PCR again
• Check expiry dates

Question 8

What do you do if your neg control is not working or has multiple bands?

Answer 8

Everything you prepped is invalid = throw everything away & start again

Question 9

After PCR what is usually done? & why

Answer 9

Run on gel to check:
• if you have PCR products
• expected PCR product => then can be used for sequencing, cloning, restriction digest

Question 10

typical PCR program

Answer 10

1. initial denaturation
2. denaturation
3. Annealing
4. Extension
5. Reapeat steps 2-4 in cycles
6. Final extension > ensure all PCR product are same length

Question 11

Factors affecting PCR

Answer 11

• polymerase used: w/ or w/out (incorporate wrong dNTP) proofreading activity
• Primer design
• Annealing temperature

Question 12

How might [Mg2+] affect PCR?

Answer 12

• if Mg2+ too much = more non-specific products (stringency dec)
• from primers binding non-specifically
• must be Hi enough that’s >[dNTP]

Question 13

How might the polymerase used affect PCR?

Answer 13

• Taq not have proofreading ability => can insert incorrect dNTP = can cause mutation
• terminal transferase > make sticky overhangs
• pfu & Vent polymerase - polymerase w/ proofreading ability & make blunt ends > for determine SEQUENCE of DNA

Question 14

Difference b/w FWD primer & RVS primer

Answer 14

• FWD primer: uses RVS template strand => forward strand

- RVS primer: uses FWD template strand => RVS strand

Question 15

How might the primer design affect PCR?

Answer 15

• primer length = more dNTP = more H+ bonds = bind to self
• nucleic acid composition: G-C have 3x Hbond Hi annealing temp than A-T have 2x Hbond
• Sequence of homology: mismatch of bases = less H bond

Question 16

what factors must be considered when deigning a Primer

Answer 16

• ~3-5ºC below melting temp of primer of lowest Tm of primer pair

Question 17

HOw can you determine the primer Tm’s (what equation)

Answer 17

= 4(G+C) + 2(A+T)

*add up bases in sequ.

Question 18

How can you determine optimal annealing temp.

Answer 18

use thermal cycler w/ gradient PCR: test which temp. allows optimal annealing

Question 19

Relationship b/w temp & stringency (specific binding)

Answer 19

• low temp = more likely to bind = but binds less specific= lo stringency
• Hi temp = less likely to bind but binds more specific = Hi stringency

Question 20

What makes an ideal primer?

Answer 20

• 18-25bp long
• has a G-C of ~50% (30-70%) > want try h ave equal C-G & A-T = keep Tm close
• Tm’s ~55-60ºC
• primer’s shouldn’t be complimentary to each other = primer dimers

Question 21

When setting up a hot start PCR why do we set up on ice?

Answer 21

reduce activity of Taq = mispriming

Question 22

How is allel-specific PCR used?

Answer 22

• detect SNP mutations

- mismatch @ 3’ end dNTP = won’t amplify

Question 23

Describe mutagenic PCR or the FDB assay

Answer 23

• have Primers not complementary to target
  = intoduce mutations in PCR products
  = introduce R. sites
  = digested = mutant

Question 24

Describe isothermal PCR

Answer 24

• use DNA polymerase that can denature DNA while amplifying it (e.g. Bst polymerase)
• PCR rxn occur @ single temp
  = lower cost & equipment

Question 25

describe Loop-mediated isothermal amplification (LAMP)

Answer 25

- DNA initially denatured & chilled - Bst polymerase added & incubated @ single temp *uses @ least 4 primers in single rx = form dumbell structure = exponential amplification of DNA

Question 26

What is the approximate Tm of the following primer? 5'-AGCTTACGGATCCAATGCAA-3' *

Answer 26

~58ºC

Question 27

You perform a PCR for which you are expecting a single band. Instead, you get multiple bands. Which of the following would potentially solve this issue?

Answer 27

restart w/ new primer (bc maybe there is multiple primers?)

Question 28

What is the extension rate of a standard Taq polymerase?

Answer 28

~1kb /min

Question 29

You perform a PCR for which you are expecting a single band. Instead, you get no bands. Which of the following would potentially solve this issue?

Answer 29

restart w/ new reagents