PCR基础知识[PCR basics]

The PCR steps are all carried out, one after the other, in bouts of cycling. Cycle 1 is as follows:

• During denaturation (about 1 min at 95°C), the DNA strands separate to form single strands.

• During annealing (about 1 min at temperatures ranging between 45°C and 60°C), one primer binds to one DNA strand and another binds to the complementary strand. The annealing sites of the primers are chosen so that they will prime DNA synthesis in the region of interest during extension.

• During extension (about 1 min at 72°C), the DNA synthesis proceeds through the target region and for variable distances into the flanking region[Flanking regions: The DNA sequences extending on either side of a specific gene or locus.], giving rise to 'long fragments' of variable lengths


When the second cycle starts, there are effectively two types of template: (1) the original DNA strands; and (2) the newly synthesised DNA strands, consisting of the target region and variable lengths of the flanking region at the 3' end. When the latter template is used in this cycle, only the target region is replicated.

In the third cycle, the newly synthesised target region DNA (i.e. without flanking regions) acts as template. The original DNA molecule is still present, and will be until the end of the reaction. However, after a few cycles, the newly synthesised DNA fragment quickly establishes itself as the predominant template. Cycles are typically repeated 25 to 45 times. Standardisation of the thermocycler's running conditions is essential for the reproducibility of results.

In the initial denaturation step, complete denaturation of the DNA template at the start of the PCR reaction is essential. Incomplete denaturation of DNA will result in the inefficient use of the template in the first amplification cycle and, consequently, poor yield of PCR product.
The annealing temperature may be estimated as 5°C lower than the melting temperature of the primer-template DNA duplex. If non-specific PCR products are obtained in addition to the expected product, the annealing temperature can be optimised by increasing it stepwise by 1-2°C.
Usually, the extension step is performed at 72°C and a 1-min extension is sufficient to synthesise PCR fragments as long as 2 kb (kb = kilobase = 1000 bp). When larger DNA fragments are amplified, time is usually extended by 1 min per 1000 bp.The number of PCR cycles will basically depend on the expected yield of the PCR product.
After the last cycle, samples are usually incubated at 72°C for 5 min to fill in the protruding ends of newly synthesised PCR products.

PCR procedures: conditions for the
reaction mixture
Contamination of the DNA must be prevented by:
! Separating the areas for DNA extraction andPCR
! Using sole-purpose laboratory equipment
! Autoclaving and aliquoting
! Adding a control reaction

Some useful tips:
• DNA extraction and PCR reaction mixing and processing should be performed in separate areas.
• Use of sole-purpose vessels and positive displacement pipettes or tips for DNA sample and reaction mixture preparation is strongly recommended.
• All solutions, except dNTPs, primers and Taq DNA polymerase, should be autoclaved. Where possible, solutions should be aliquoted in small quantities and stored in designated PCR areas.
• A good practice, to confirm absence of contamination, is to add a control reaction without template DNA.

PCR procedures: components

Many PCR machines are now available in 48-, 96- or 384-well formats. This, combined with the use of multichannel pipettors, can greatly increase the number of reactions that can be done simultaneously. If several reactions need to be simultaneously prepared, a master mix should be used as follows: water, buffer, dNTPs, primers, MgCl2 and Taq
DNA polymerase in a single tube. This will then be aliquoted into individual tubes.
Considerations:
Template DNA. Nearly any standard method is suitable for template DNA purification. An adequate amount of template DNA is between 0.1 and 1 μg for genomic DNA for a total reaction mixture of 100 μl. Larger template DNA amounts usually increase the yield of non-specific PCR products.