Lab 2: the polymerase chain reaction (PCR)

Question 1

What is PCR?

Answer 1

• it is a technique for amplifying specific DNA fragments, making them detectable without the need for purification or cloning.
• uses primers (short DNA sequences) that flank the DNA to be amplified, and DNA polymerases (vital enzyme that synthesizes new DNA strands) and co-factors to create copies of the target DNA.

Question 2

Explain the process of PCR. What are the 3 main steps?

Answer 2

(1) Denaturation:
- at 95C
- DNA strands separate into single strands (sec-2 mins)

(2) Annealing:
- (45C - 70C)
- DNA primers binds to complementary sequences on the template DNA (1-2 mins)

(3) Extension:
- at 72 C
- DNA polymerase extends the primers, creating new DNA strands.
- exponential amplification occurs as the process repeats, doubling the DNA amount with each cycle.
- PCR can be automated and completed in a few hours.
- depends on fragment lengths (reaction can be repeated 10-40X)

Question 3

Function of Template in PCR.

Answer 3

• key component
• typically dsRNA, but can also be ssDNA or RNA (converted to DNA using Reverse Transcriptase).
• works on small or complex samples.

Question 4

Function of oligonucleotide primers in PCR.

Answer 4

• key component
• short DNA sequences (20-25 nucleotides) that bind to target DNA.
• designed to be specific to target sequence to ensure accuracy.
• G + C content around 50%.
• Melting temperature (Tm) is between 45-70C.

Question 5

Function of DNA Polymerase in PCR.

Answer 5

• key component.
• Taq polymerase (remains active at higher temperature).
• often heat stable-enzymes like Pfu polymerase are also used for higher fidelity.

Question 6

Function of Deoxyribonucleotide Triphosphates (dNTPs) in PCR.

Answer 6

• key component.
• raw materials for building new DNA strands: dATP, dCTP, dGTP, dTTP.
• must be stored on ice to prevent degradation.

Question 7

Function of Mg2+ in PCR.

Answer 7

• key component.
• essential co-factors for polymerase activity, often added as magnesium chloride or sulfate.

Question 8

Function of PCR buffer in PCR.

Answer 8

• key component.
• maintains optimal pH, ionic strength, & osmolarity for polymerase enzyme.

Question 9

What is a master mix? what is the point?

Answer 9

• master mix combines all reagents (except variable components) to reduce error and save time.

Question 10

What are the 4 main steps in this lab?

Answer 10

(1) Gene fragment amplification.
(2) Genotype determination.
(3) Site-directed mutagenesis.
(4) qPCR (measure primer pair efficiency)

Question 11

In part 1, there was amplification of gene fragments from Nuclear Pore Protein Nup205. What were the specifics in this section?

Answer 11

• Primers were used
• Nup205 - F1
• Nup205 - F2
• Nup205 - R1
• Nup205 - R2
• Run PCR
• Agarose Gel Electrophoresis - to visualize amplified products and determine fragment sizes.

Question 12

In part 2, there was sex determination of biological samples using PCR. What were the specifics in this section?

Answer 12

• use PCR to determine the sex of DNA samples by detecting the amelogenin gene.
• primers:
  AMEL- F
  AMEL - R
• run PCR
• Agarose gel electrophoresis

Question 13

In part 3, site-directed mutagenesis is done. what were the specifics in this section?

Answer 13

• generate mutant DNA molecules with specific changes to study their function.
• primers - contain desired mutation at centre - flanking mutation site.
• use linear amplification (no experimental growth of DNA).

Question 14

DpnI

Answer 14

• restriction enzyme
• used to digest parental (methylated) DNA, leaving behind daughter (un-methylated) DNA intact for transformation.
• after digestion with dpnl, transform mutagenized DNA into competent bacterial cells to select for mutated clones.

Question 15

In site directed mutagenesis, what do the competent cells look like?

Answer 15

XLI - Blue

Question 16

What is on the Agar plates?

Answer 16

LB + Amp + X-gal + IPTG

Question 17

qPCR

Answer 17

• allows real time quantification of DNA during amplification by measuring fluorescence generated by SYBR Green I dye, which binds to dsDNA.
• Fluorescence increases during amplification and is monitored after each PCR cycle.
• qPCR mix (has DNA polymerase & dNTPs)
  -> primers
  —> Fwd and Rev for GAPDH
  —> Few and Rev for p22

Question 18

In Site-Directed mutagenesis: what is MR, DC, and TC?

Answer 18

(1) MR (mutagenesis reaction)
- primers & DpnI - 103 colonies
- successful B-gal gene repair (mutants)
- 97% efficacy

(2) DC (digestion control)
- no dpnI
- no colonies
- complete digestion or input plasmids

(3) TC (transformation control)
- no dpnl
- 1038 while colonies
- non-mutant plasmids transformed successfully
- around 206x more colonies than MR plate, showing lower amplification efficiency in MR sample.

Question 19

How was efficiency of mutagenesis calculated?

Answer 19

blue colonies / total colonies

Question 20

What did the information about the PCR amplified Intron 1 of X-Y homologous amelogenin gene tell us?

Answer 20

• Band 1 (496 bp): in all samples (X-amelogenin gene)
• Band 2 (347 bp): ony in samples B & C (Y-amelogenin gene)
• size difference (149 bp) aligns with 177 deletion in Y-amelogenin intron.
• A & D - female (only X - amelogenin - one band)
• C & B - male (X & Y - amelogenin - 2 bands)