01Clean Laboratory Conditions
Since PCR is capable of detecting a single molecule of DNA, it is important to maintain pristine conditions in the laboratory. Always wear fresh gloves; use sterile glassware, tubes, and pipette tips; and clean the work area before beginning work.
Always include control reactions, without template DNA and without enzyme, to ensure the results are truly due to amplification of the right sample.
Most people make sure to have their own set of solutions that are not shared to make troubleshooting easier and designated pipettes are often set aside for PCR only. DNase and RNase-free PCR tubes, aerosol-resistant pipette tips, and working in a fume hood with UV light are also ways to minimize problems.
Also of critical importance is the purity and integrity of template (purified DNA) and primer design. There are several software programs available online for designing primers. The best primers:
- Have 18 to 24 bases
- Have no secondary structure (e.g., hairpin loops)
- Have balanced distribution of G/C and A/T pairs
- Are not complementary to each other at the 3-foot ends
- Have melting temperatures (Tm) about 5-10 degrees Celcius below the annealing temperature, which is usually between 55 and 65 degrees Celcius. The Tm for both primers should also be similar for best results.
03Reaction Mix: Template and Primers
The proportions of the reaction mixture have a huge influence on the quality of PCR results. There is a general formula for concentrations of template, enzyme, primers, and nucleotides to use, but this can be tweaked a little bit.
Optimal primer concentrations are between 0.1 and 0.6 micromoles/L. The amount of template varies depending on the type of DNA (human, bacterial, plasmid).
With very low amounts of template, there are other strategies to improve results, like increased cycle numbers or use of "hot start." Always test new primers with a positive control reaction, to be sure they work under your specific experimental conditions.
04Reaction Mix: Enzyme Activity
The choice of DNA polymerase affects the fidelity of the reaction and quality of the product. The traditional Taq polymerase has been replaced in many labs by higher fidelity enzymes (those that make fewer errors).
The concentration of MgCl2 in the reaction mix can influence the PCR outcome and could play an important role in more fastidious reactions. Magnesium cation forms soluble complexes with dNTPs to produce the actual substrate that the polymerase enzyme recognizes.
Equal amounts of all four dNTPs also help reduce the polymerase error rate. Certain additives such as betaine, BSA, detergents, DMSO, glycerol, and pyrophosphatase can also affect enzyme specificity or reaction efficiency.
05Type of Thermocycler
When shopping for laboratory equipment, bear in mind that some thermocyclers are less precise than others at maintaining exact, desired temperatures.
This is one area where going cheap will not pay off in the long run when you are faced with a finicky reaction or using primers that require a narrow range of annealing temperature.
Thin-walled reaction tubes designed to fit the precise brand of thermocycler you are using also help optimize reaction temperatures.
Reaction cycle lengths, temperatures, and a number of cycles, all have a critical role in determining how well a PCR will work. The initial heating step must be long enough to completely denature the template and cycles must be long enough to prevent melted DNA from reannealing to itself.
Increased yield can be achieved by increasing the extension time about every 20 cycles, to compensate for less enzyme to amplify more template. Usually, less than 40 cycles is enough to amplify less than 10 template molecules to a concentration large enough to view on an ethidium bromide-stained agarose gel.
What's Required for Superior PCR Results
Adjustments to any of the components of a Polymerase Chain Reaction (PCR) reaction can alter the quality of the outcome, either by improving or diminishing the product yield and quality or by improving the reaction specificity and sensitivity. Parameters that influence the end result can be either physical (e.g., temperature, cycle times) or chemical (e.g., template concentration, type of enzyme used). The following are several key factors that influence the success of PCR.