Chapter 2: Degenerate PCR

What did we do after extracting gDNA?

We took it and used it for the first round of PCR so that we can amplify GAPC gene, but we use degenerate PCR.

What is degenerate PCR?

It is a type of PCR that uses degenerate primers. The primers gave N bases, meaning they can be any of the four bases. It gets used when exact DNA sequence is not known. It helps narrow down the genes that work as DNA template for nested PCR.

What are degenerate primers used for?

They are used when you don’t know the exact DNA sequence, but you know some parts of it because of there species. These primers have N bases so that it can match with all possible versions of the genes. Even if the DNA is not a perfect match, the primer can still bind and copy, helping amplify possible GAPC genes (GAPC, GAPC2, GAPCP1, GAPCP2, non-specific products).

What products can you get from degenerate PCR?

GAPC, GAPC2, GAPCP1, GAPCP2, non-specific products.

What could the PCR bands look like for degenerate?

There could be multiple bands since the degenerate primer might amplify non-specific.

What are the steps of PCR?

Denaturation, Annealing, Extension.

What are PCR products?

They are always double stranded DNA because both strands get copied.

What does the denaturation cycle do and what is the temp. for it?

The temp. should be 95 C. In this step, the double stranded DNA unwinds and becomes single stranded.

What if the temp. is low for denaturation step?

There will be no separation with the double stranded DNA.

What if the temp. is high for denaturation step?

The DNA could degrade.

What is the annealing step and the temp?

The annealing temp for degenerate PCR is 52 C and it is what lets primers bind to the DNA sequence that is complementary.

If the annealing temperature is too low, what happens?

The temp. being low will cause primers to bind randomly.

If the temp. is too high, what happens for annealing?

If it is too high the primers might not bind so there would not be amplification.

What is the extension step and the temp for it?

The temp for the extension step is 72 and it is when the taq polymerase adds dNTPs to build the new strand.

What if the extension temp is too low?

Then the taq polymerase would not work as fast as we would want it to cause less production of DNA.

What if the extension temp is too high?

The taq polymerase would lose its function.

What are the reagents in PCR?

double stranded DNA (gDNA) because it has the gene that we want to amplify. if missing there would not be a gene to amplify. primers which are the single stranded sequences that have to be complimentary to a part of target DNA these also have to be forward and reverse primers. if there were no primers there would not be any amplification. dNTPs are building blocks they are the nucleotides A, T, G, C and if there is not anything new strands can’t get made. There is also buffer which maintains pH level which is important for enzyme activity. Taq DNA pol because that is the enzyme that builds new strand, if there isn’t no DNA would be made.

What are the controls of PCR?

It is negative and positive control. The negative control is water to test for contamination; no DNA. Then Arab gDNA is a positive control and is our known for comparison for the Jacaranda (0.5-2.5). The pGAP is to make sure the reagents and thermocyclers are working(1).

If no bands for samples show does it mean nothing was amplified?

No, more PCR rounds should happen.