BIOL 2500 - Topic 10 (part 3)

Three main steps of PCR

1.) DNa denaturation (1-2 min)

2.) Primer annealing (1-2min)

3.) Polymerase extension (3-5min)

DNA denaturation step

1.) The helicase activity of the process, where the double helix is unwound by breaking the hydrogen bonds, but it uses heat

2.) The mixture is heated up to 95ºC

Primer annealing step

The mixture temperature is reduced to roughly 45-68ºC, which allows the primers to anneal to the ssDNA template

Temperature of primer annealing step

It depends on the primers, as different primers with different lengths, complexity, and GC content will have different annealing temperatures

Polymerase extension step

The temperature is raised to 72ºC, the optimal temp for the polymerase, allowing it to perform its function of adding the dNTPs to the complementary strand of the template strand

PCR cycles

The three main steps of PCR are repeated over and over again, roughly 25-35 cycles

PCR number of cycles

It depends on the length of the gene of interest, such that short genes require less cycles and long genes require more

Different PCR times and temperatures

Different genes will require different times and temperatures (and number of cycles)

PCR machine

1.) We perform PCR in a thermocycler, aka the thermal cycler

2.) It allows us to program the cycles, times, and temperatures of the PCR conditions needed

How do visualize the fragments of the PCR?

Via gel electrophoresis

Ramp time

The time between the different steps of PCR, where the machine is changing from one temp to another, which is why the reaction is so sensitive to temperature during this time

qPCR

Quantitative PCR, which determines the real-time amount of DNA being produced during each PCR cycle

qPCR assumption

The more gene templates present = the more gene products produced

qPCR use

It is used to compare relative amounts of DNA molecules

Dye used for qPCR

It uses SYBR green instead of ethidium bromide, which is mutagenic and toxic

RT-PCR

Reverse transcription PCR, which is used to detect, amplify, and quantify mRNA, allowing us to understand gene regulation and expression

RT-PCR required step

mRNA is converted into ds cDNA (i.e. the coding part of the sequence), using a reverse transcriptase enzyme

Results of RT-PCRs

1.) The number of cDNA molecules present is proportional to the number of mRNA molecules present

2.) Therefore, we can use this as an indication of expression levels

RT-PCR of upregulated genes

They will have more mRNA (i.e. cDNA), therefore more gene products will be produced from the PCR