Lab 2: the polymerase chain reaction (PCR) Flashcards
What is PCR?
- 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.
Explain the process of PCR. What are the 3 main steps?
(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)
Function of Template in PCR.
- key component
- typically dsRNA, but can also be ssDNA or RNA (converted to DNA using Reverse Transcriptase).
- works on small or complex samples.
Function of oligonucleotide primers in PCR.
- 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.
Function of DNA Polymerase in PCR.
- key component.
- Taq polymerase (remains active at higher temperature).
- often heat stable-enzymes like Pfu polymerase are also used for higher fidelity.
Function of Deoxyribonucleotide Triphosphates (dNTPs) in PCR.
- key component.
- raw materials for building new DNA strands: dATP, dCTP, dGTP, dTTP.
- must be stored on ice to prevent degradation.
Function of Mg2+ in PCR.
- key component.
- essential co-factors for polymerase activity, often added as magnesium chloride or sulfate.
Function of PCR buffer in PCR.
- key component.
- maintains optimal pH, ionic strength, & osmolarity for polymerase enzyme.
What is a master mix? what is the point?
- master mix combines all reagents (except variable components) to reduce error and save time.
What are the 4 main steps in this lab?
(1) Gene fragment amplification.
(2) Genotype determination.
(3) Site-directed mutagenesis.
(4) qPCR (measure primer pair efficiency)
In part 1, there was amplification of gene fragments from Nuclear Pore Protein Nup205. What were the specifics in this section?
- Primers were used
- Nup205 - F1
- Nup205 - F2
- Nup205 - R1
- Nup205 - R2
- Run PCR
- Agarose Gel Electrophoresis - to visualize amplified products and determine fragment sizes.
In part 2, there was sex determination of biological samples using PCR. What were the specifics in this section?
- use PCR to determine the sex of DNA samples by detecting the amelogenin gene.
- primers:
AMEL- F
AMEL - R - run PCR
- Agarose gel electrophoresis
In part 3, site-directed mutagenesis is done. what were the specifics in this section?
- 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).
DpnI
- 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.
In site directed mutagenesis, what do the competent cells look like?
XLI - Blue
What is on the Agar plates?
LB + Amp + X-gal + IPTG
qPCR
- 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
In Site-Directed mutagenesis: what is MR, DC, and TC?
(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.
How was efficiency of mutagenesis calculated?
blue colonies / total colonies
What did the information about the PCR amplified Intron 1 of X-Y homologous amelogenin gene tell us?
- 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)