PCR optimization. Primers – design must be good but influenced by template sequence Quality of template DNA/impurities Components of PCR may need to be.

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Presentation transcript:

PCR optimization

Primers – design must be good but influenced by template sequence Quality of template DNA/impurities Components of PCR may need to be optimized –MgCl 2 concentration affects DNA polymerase activity Settings on the PCR machine PCR does not always work the first time. Change one variable at a time to get PCR to work

DNA template Need good quality DNA Old DNA may be degraded –May have DNA from a crime scene stored for many years –May be looking at ancient DNA

Primers Flank the region of interest There are two primers – usually called the forward and reverse primer One is complementary to sequence in the beginning; one is complementary to sequence at end on other strand They go in opposite directions but both are 5’ to 3’

Melting temperature Denaturation and annealing depends on GC content More GC, the higher the melting temperature –How many hydrogen bonds between G and C? –How many hydrogen bonds between A and T? The longer the DNA, the higher the melting temperature Tm is dependent on the length and base pair composition of the primers

Primer design The melting temperatures (Tms) = temperature at which half of the primer has annealed to the template (guide for determining annealing temp. on the PCR machine) Annealing temp is usually 3-5 degrees lower than the Tm –The Tm for the two primers should be similar primers should be bases in length

Primer design Optimal annealing temperature needs to be tested –If it is too low, primers may bind non specifically –If it is too high, primers may not bind or may not bind well the GC content should be 40-60%; purine: pyrimidine content should be about 1:1 primers should end (3') in a G or C, or CG or GC

Things to avoid Complementarity between the 5’ and 3’ ends of the primers You do not want hairpin structures to form

Things to avoid Primer-dimer Do not want primers to anneal to each other

Cycling parameters Temperature of the extension step determined by the type of DNA polymerase used Time at each step influenced by the length of template –Short sequences = shorter times The number of cycle is dependent on the quality and amount of starting template

Many computer programs exist to design primers Stay tuned for biocomputing These programs will take the above factors into consideration. First, you will need the sequence of the gene Go to www. pubmed.org (ncbi site) Then, you will go to this site:

First, go into ncbi to find the sequence of the gene that you are interested in. Go to Go under nucleotides Enter gene name Half way down = protein sequence Find DNA sequence at bottom of page Copy and paste into primer program

Primer design program Copy your sequence Can change a few parameters depending on application Product length = %GC = min. 50, max = 60 Pick about 200 primers so that you cover a large part of the gene (number to return) Then pick a few pairs – can you find them on your sequence.