PCR

Question 1

What is PCR?

Answer 1

The amplification of DNA based on DNA polymerase being able to synthesize a new strand of DNA complementary to the original template strand

Question 2

What are the 5 reasons that we conduct PCR?

Answer 2

1. Sensitive
2. Specific
3. Cheap
4. Rapid
5. Robust

Question 3

What are the main enzymes needed DNA replication in eukaryotes?

Answer 3

1. Helicase = catalyze the disruption of the hydrogen bonds that hold the two strands of double-stranded DNA together
2. Primase = create RNA primers on the template which bind to DNA
3. Polymerases = catalyze the synthesis of DNA or RNA polymers whose sequence is complementary to the original template
4. Nuclease = catalyze the cleavage of phosphodiester bonds
5. Ligase= joins the Okazaki fragments together
6. ssDNA binding proteins
7. Sliding clamp = holds the polymerase on the ssDNA

Question 4

What are the key components and what are their roles in PCR?

Answer 4

1. DNA Template = single stranded DNA
2. Primers (forward and reverse) = to start synthesis of new DNA strand (small ssDNA molecules -> 18-24 bases)
3. Enzyme = DNA polymerase (copy DNA accurately) + Taq Polymerase (catalyst synthesis of new DNA strand)
4. Magnesium = a co-factor ( needed to enable the activity of the catalysis)
5. Buffer = potassium ions to promote annealing / optimal pH is 8 - 9.5 or Tris-HCl
6. Water = essential for PCR to work

Question 5

What occurs in the first cycle of PCR?

Answer 5

1. DENATURATION = DNA strands are heated up to 90 degrees, bonds are broken and DNA strands are separated
2. ANNEALING = primers are added from 5’ to 3’. Temperature is decreased and bonds form again around 50-60 degrees.
3. ELONGATION = Polymerase runs from 5’-3’ along the DNA forming the double stranded DNA

Question 6

What occurs in the second cycle of PCR?

Answer 6

1. DNA is heated up and denatured again
2. DNA is then cooled down so that the primers can anneal again
3. Short strands begin to appear = stops producing strands which are the right size

Question 7

How can we detect DNA?

Answer 7

Using intercalating agent which binds to the DNA between base pairs = fluoresces under the UV light to show the double stranded DNA

Question 8

How can we detect PCR products?

Answer 8

Run products on agarose gel and use intercalating dye to stain the DNA to determine the size

Question 9

What is the role of reverse transcriptase?

Answer 9

Convert RNA (often mRNA) to cDNA

Question 10

What is the different between end point PCR and real time PCR?

Answer 10

End point PCR:
- cheap
- semi-quantitative
- sequencing, genotyping, cloning
- see result at end

Real time PCR:
- more expensive
- quantity of PCR is proportional to amount of template
- more steps
- measures at exponential phase = more precise

Question 11

What do we need in the qPCR reaction?

Answer 11

1. Good clean material = extraction and purification of DNA
2. Thermos light cycles = detects fluorescence
3. SYBR green or TaqMan = fluorescent
4. Master mix

Question 12

How much cDNA does qPCR produces compared to end point PCR?

Answer 12

Each cycle cDNA amount is double

1 cycle = 2x more DNA
2 cycle = 4x more DNA

Question 13

How does SYBR green fluoresce?

Answer 13

Binds to groove of dsDNA

Question 14

How does TaqMan fluoresce?

Answer 14

Uses probes with fluorescence reporter and quencher

Question 15

What are the steps for PCR-RFLP (restriction fragment polymorphism)?

Answer 15

1. Amplify the substrate = 2 double stranded alleles
2. Take the two alleles and mix with an enzyme -> if Restriction Endonuclease (RE) site in alleles is present = alleles is cut
   RE not present = allele is not cut
3. Put into electrophoresis gel

Question 16

What occurs when RE sites are or aren’t present in electrophoresis gel?

Answer 16

RE site in both = 2 lines (a,b)

RE site in neither = 1 line (a+b)

Heterozygous = 3 lines (a+b, a, b)

Question 17

What are the Pros vs Cons of PCR-RFLP?

Answer 17

Pros:
- cheap
- easy design
- applied to micro ideals and SNPs
- simple resources
- commonly used techniques

Cons:
- only possible if the site has a known RE site
- some REs are expensive
- hands on and time consuming

Question 18

What is the function of ARMS-PCR (amplification refractory mutation system)?

Answer 18

Detects allergic variants using allele-specific primers

Question 19

What are the roles of the allele specific primers (ASP)?

Answer 19

ASP1 binds to allele 1 and leads to amplification
ASP1 binds to allele 2 = no amplification

ASP2 binds to allele 1 = no amplification
ASP2 binds to allele 2 = application

Question 20

What is the mutation that leads to cystic fibrosis?

Answer 20

F508 mutation

Question 21

What is the different between RFLP-PCR and ARMS-PCR?

Answer 21

RFLP:
- uses locus specific primers
- relies on presence or absence of a restriction site to distinguish between variants

ARMS:
- uses allele specific primers
- relies on stringency of the PCR to distinguish between alleles

Question 22

What does quantitave PCR measure?

Answer 22

Measures the abundance of DNA or RNA in a clinical sample

Question 23

What does the Ct value show?

Answer 23

The cycle number at which the fluorescence reaches a threshold value

Question 24

What does a lower Ct value indicate?

Answer 24

Lowe the Ct value = the greater the quantity of DNA/cDNA in the starting template