T2P2: Polymerase Chain Reaction

What was the problem when searching things up in the Human Genome project? what was the solution?

It is way too big - make so many copies of one sequence that its 99% that one.

Ingredients for the PCR

DNA template, Primers, DNA polymers, dNTPs, Mg2+, and Buffer

Mg2+ in PCR

This is a required cofactor for DNA polymerase activity. It helps coordinate the interaction between the \(3^{\prime }\)-OH of the primer and the incoming dNTP, allowing the polymerase to function correctly and catalyze the synthesis reaction.

PCR steps

1. Denature DNA to break H bond

2. Rapidly cool to anneal primers to complimentary strand and temp depends on number of bonds

3. Extend DNA with DNA polymerase and give it a little bit of extra time jic

taq polymerase origin

thermus aquaticus

RTPCR stands for and goal

reverse transcrition polymerase chain reaction and to measure levels of mRNA

Steps of RT-PCR

1. write mRNA 5’ to 3’

2. use reverse transcriptase with primer to synthesize DNA from RNA with dNTPs

3. PCR it

3 ways to measure RNA

GE; QPCR: SYBR green, Multiplex probe

SYBR Green

increases in fluorescence when it binds to double stranded DNA and the lower the cycle number to increase the fluorescence  means that the concentration was high to begin with

Multiplex Probe

oligonucleotide to bind to its compliment where there is a quencher and a reporter and when it is being replicated the reporter has to move and it glows. It also allows the use of different probes for sim amp of genes using different compliment oligonucleotides.