How does PCR work?
PCR is an in vitro adaptation of DNA replication and thus uses many of the same components and conditions as in vivo DNA replication. Basically, instead of duplicating an entire chromosome, we target a small region of the chromosome and replicate repeatedly. This results in a doubling of the target DNA each time the cycle is repeated.
The three basic steps of a PCR reaction are
- denature the DNA template (PCR uses heat instead of helicase)
- anneal primers (designed to anneal to a specific sequence and synthesized in vitro)
- synthesize a copy of the DNA starting from the 3' end of the primers (i.e. extend the primer using a heat stable DNA polymerase)
This process is repeated 30 - 40 times and, theoretically, at the end of n cycles, there would be 2n copies of the target DNA for every starting chromosome target.
A key step in PCR is primer design. A PCR reaction requires a pair of primers, one primer annealing to each DNA strand. The primers are designed so that the product produced will have a finite size. Since DNA synthesis is directional (5' to 3'), the 3' ends of the primer pair must 'face' each other when they are annealed to opposite strands.
There are several programs that are helpful for designing primers; however, it is crucial that you know what to look for when choosing primers. A good set of primers is one that allows you to specifically amplify the DNA target you are after, without amplifying unwanted sequences and without producing excessive primer dimers.
You can specifically amplify a single target sequence in the human genome (~3.1 billion base pairs) with primers as little as 20 bases in length. However, not all 20 base sequences would amplify specific sequences. For example, a primer of 20 Ts would give not target a single site. So what do you look for in a good primer?
There are several ‘rules’ that are helpful in choosing good primer. Keep in mind that you cannot always follow all the rules closely because of limitations dictated by your target sequence.
- Primers should be 22-26 bases in length (with 24 being a good target size).
- The melting temperature (Tm)Tm The temperature at which half the DNA duplex is single stranded. should be 58 – 66 °C (60 – 64 °C is ideal). Primer finding programs will calculate the Tm using a complicated algorithm, but for a quick and dirty estimate Tm= 2*(A+T) + 4* (G+C).
- Both primers of a set should have a similar Tm (+/- 2 °C).
- Keep the GC content between 40 and 60% (keep at 50% if possible).
- Avoid repetitive sequences (e.g. AAAA, or ATATAT) as they can cause miss-priming.
- GC content in the 5 most 3’ bases should be 25 - 50% to prevent miss-priming. Priming is most critical at the 3’ end. In fact, if the primer does not H bond at the 5’ end, you can still successfully get product as long as the 3’ end anneals well . Thus a primer that has high GC content at its 3' end is more likely to cause miss-priming// (we actually exploit this in some protocols to find closely related sequences)//.
- Avoid primers that could easily have intra or inter base pairings of more than 3 consecutive bases. Primers that can extensively self anneal (form hairpins) and primers that can extensively anneal with their partner (form primer dimer) may do so instead of annealing to the desired target sequence, thus producing little or no product. A little intra or inter base pairing will not usually mater because the Tm of short matches is considerably lower than the lowest temperature you will use for the PCR reaction.
- Finally, check your primers against a database to make sure they are only targeting the sequence you are interested in (depending on the source of your target DNA you can limit the check to a single species). NCBI's Primer Blast is an excellent tool for this job.