PCR principles and practices

Important parameters in the PCR:

1.Template DNA quantity (complexity determines ng) and quality (average length)

While people typically measure DNA quantity in ng, the relevant unit is actually moles, i.e.，how many copies of the sequence that will anneal with your primers are present.Thus, the amount of DNA in ng that you need to add is a function of its complexity. In theory, a single molecule of DNA can be used in PCR but normally people use between 1000 and 100,000 molecules for eukaryotic nuclear DNA.

Example for sorghum (genome size = 760 Mb):

760 Mb = 7.6 x 108 bp x 649 daltons/bp = ~5 x 1011 grams/mole

20 ng = 2 x 10-8 grams/ 5 x 1011 grams/mole = 1 x 10-18 mole

1 x 10-18 mole x 6 x 1023 molecules/mole = 6 x 104 molecules

Example for a 5 kb plasmid clone:

5 kb = 5 x 103 bp x 649 daltons/bp = 3.2 x 106 grams/mole

1 pg = 1 x 10-12 grams/ 3.2 x 106 grams/mole = 3.2 x 10-18 mole

3.2 x 10-18 mole x 6 x 1023 molecules/mole = 2 x 106 molecules

Template quality and PCR product size both affect the amount of DNA you need to add. If your DNA is very high MW, and/or your product length is short (e.g., an SSR)， you can use less DNA because a higher fraction of the molecules will contain the annealing sites for both the forward and reverse primer. If your DNA is degraded and you want to amplify a large product, it may not work, but the same DNA may be fine for amplifying SSRs.

2. Tm of primers

The melting temperature of your primers should be similar and should be as high as possible,within reason, in order to increase specificity. òWithin reasonó means that you don?t want the Tm of the primer to be higher than the reaction temperature of Taq polymerase (72 C).In practice, I usually aim to have my primer Tms around 66 C, and this is usually possible with a primer length of 22 or 23 bases. These primers can frequently be used in 2-step PCR (see below).

To calculate Tm for duplex DNA <50 bp:

Add 2deg. C for each A or T

Add 4deg. C for each G or C

The annealing temperature you use in your PCR should be a function of the Tm of your primers, and it should not be much lower unless you have designed the primer from heterologous sequence, in which case you may want to do a gradient PCR (see below).

3.Mg concentration

Standard Mg++ concentration is 2 mM, but sometimes you may find that the concentration needs to be raised (rarely lowered) to get a PCR to work. Raising Mg lowers specificity, and is roughly comparable to lowering the annealing temperature. It may cause multiple bands to appear (or, occasionally, disappear). In my experience, most troubleshooting is more easily accomplished by playing with other variables (temperature, DNA quality or amount)。

4. Length of expected product

The length of the extension step (72 C) should be a function of the length of the product you are trying to amplify. A general guideline is 1 minute/kb of product length, but in fact this is more than is needed, particularly if you are doing 3-step (i.e., conventional) PCR, as extension will take place during the annealing step and during the ramp time. Taq polymerase is a very fast and very processive enzyme.