Molecular bio Wk 2

What are the 3 different phases of PCR?

denaturation, annealing, elongation

What is denaturation?

94-98C, causes the double-stranded DNA to denature (separate into 2 single strands), allowing primers to bind to the DNA

What is Annealing?

50-65C, allowing the primers to bind their complementary sequences on the single-stranded DNA templates

What is Elongation?

72C, the enzyme synthesises new DNA strands by adding nucleotides to the 3' end of the primers, extending the strand in the 5' to 3' direction

What is Allele-specific PCR?

-if the allele of interest is present, the primer will bind and allow for amplification -if the mutation or variant is absent, the primer will not bind properly, and amplification will not occur

What is Quantitative Real-Time PCR (qPCR) used for?

to quantify the amount of target DNA in a sample by measuring the amount of fluorescence produced during the amplification process (the more target DNA present, the earlier fluorescence is detected)

What does real-time refer to?

the ability to monitor the amplification as it occurs, cycle by cycle, instead of after the reaction has been completed

What are the mechanisms underlying the two main chemistries used in real-time PCR?

-SYBR green chemistry -TaqMan probe chemistry

What does SYBR Green Chemistry do?

-a dye that binds to double-stranded DNA and fluoresces when excited by light -as the PCR reaction progresses, more double-stranded DNA is formed, leading to an increase in fluorescence -allows monitoring of DNA amplification (drawback is that SYBR Green will bind to any double-stranded DNA)

What is TaqMan Probe Chemistry?

-involves the use of a fluorescently labelled probe that is complementary to the target sequence between the two primers -the probe has a fluorescent reporter dye on one end and a quencher on the other end -during amplification, the polymerase exonuclease activity cleaves the probe, separating the reporter dye and quencher, allowing fluorescence to be detected (MORE SPECIFIC THAN SYBR Green because probe binds only to target sequence)

What is absolute quantitation in real-time PCR?

-involve determining the exact amount of a target RNA/RNA molecule in a sample -a standard curve is generated from known concentrations of a template, and a Ct value from the sample is compared to this curve to determine the absolute amount of target

What is Relative Quantification in real-time PCR?

-measures the relative expression or quantity of a target gene compared to a reference gene -this method does not require a standard curve (results presented as ratios)

What does allelic discrimination PCR do?

used to distinguish between different alleles of a gene, typically in the context of SNPs

What is Fluorescence-based detection (basis of allelic discrimination)?

alleles can be differentiated based on differences in the fluorescence emitted by probes or primers when they bind to the different alleles

What is typical PCR?

-at room temp: DNA double stranded -step 1: 95C (denaturation of DNA) -step 2: ~55C (annealing of primers) -step 3: 72C (extension of primers, synthesis of new DNA)

What is the formula for primer design?

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