PCR

Question 1

What is PCR

Answer 1

Amplification of a specific segment of DNA achieved by the use of specific primers designed to target the sequence of the action of DNA polymerase

Each PCR has three stages

1. Denaturation 94C results in separation of the double stranded DNA
2. Annealing 55-65 target specific primers bind to the sequence. Two primers are required which flank the region being amplified
3. Extension 72C new strands of DNA are made via DNA polymerase Taq polymerase using the original strands as templates

In a typical PCR there will be 25-30 cycles, this results in exponential amplification of the target sequence

Question 2

qPCR (quantitative)

Answer 2

Quantitative PCR allows for the quantification of nucleic acid in the biological sample

The technology allows quantification of PCR products in real-time due to incorporation of fluorescent molecules in the reaction

The reduced times facilitated by simultaneous amplification and visualisation of DNA amplicons means that qPCR can provide fast, high throughput detection as well as quantification of target sequences

Cross contamination of the sample is avoided as further manipulations are required after the amplification stage

Question 3

RT-qPCR

Answer 3

Reverse transcription of qPCR implies that the starting sample was RNA which in turn requires reverse transcription to cDNA prior to performing qPCR

Question 4

Fluorophores

Answer 4

Fluorophores absorb light at a particular wavelength then re emit at a different wave length

Question 5

SYBR Green

Answer 5

High binding affinity for double stranded DNA

It can bind inside the minor groove of DNAs double helix

Upon binding the SYBR Green molecules fluoresce much more strongly due to the increased stability when constrained inside the DNA

Non specific

Cheaper

Question 6

Taqman DNA probe

Answer 6

Quencher and Reporter

These probes consist of a short stretch of single stranded DNA which two additional molecules bound to each end, one is a fluorophore known as a reporter which will fluoresce when exposed to specific light wavelengths

The other molecule is a quencher which absorbs most of the emitted fluorescence before it can be detected. Therefore when the two molecules exist next to each other overall fluorescence is lowered

Taq polymerase degrades and releases reporter and quencher free in solution

Seperated from the quencher molecule, the reporter molecule fluorescence is no longer dampened. the observed fluorescence greatly increases. In each round, Taq polymerase will degrade more probes. Therefore fluorescence will increase proportionally

Specific and anneals to DNA

Expensive