PCR is the breaking apart of DNA double helixes (denaturation), the combining of primers to specific parts of a DNA strand (annealing), and the adding of nucleotides to the primer by polymerase (extension), allowing you to double the DNA at a specific location (in our case, a specific gene) in one thermal cycle.

1: Denaturation is the breaking of the hydrogen bonds that hold DNA strands together, forming the double helix. Hydrogen bonds are very strong, an electronegative element covalently bonded to a hydrogen will 'hog' the electrons, creating a dipole. The exposed proton (hydrogen) has a strong attractive force to electronegative elements. This can be broken with heat. We use 98ºC as it's enough heat to break the bonds, but low enough temperature that the enzyme (polymerase) won't denature.

2: Annealing is the act of joining a primer to the DNA strand. A primer is a synthetically-derived string of nucleotides (A, G, T, C bases) can be used as a template. At an appropriate temperature, that template will only fit onto the DNA strand at the specific sequence of nucleotides. We can bond, or anneal, the primer to a sequence of nucleotides just before a gene, allowing us to amplify (copy) that gene. Annealing is an art, not a science, as different temperatures will permit the annealing to occur at innappropriate sites. Annealing typically occurs between 45º and 60ºC. If the temperature is too cold, the primer can stick anywhere. If the temperature is too hot, it won't stick at all. We can predict appropriate temperatures based on the sequence, as A-T bonds contain two hydrogen bonds, and G-C contain three hydrogen bonds. This gives us information about how much energy is required to make the primer stick to the original DNA strand.

3a: Extension (short) is the lengthening of our primer. After we make the primer stick, we can use an enyzme that will walk along the bonded DNA and primer, adding appropriate nucleotides to the primer. DNA has direction, much as humans understand left and right. DNA is extended (nucleotides are added) in the 5' to 3' direction (five prime to three prime). Enzymes require very specific temperatures to function, and our DNA polymerase works at a predictable speed at 72ºC.

3b: Extension (long) is when we let our polymerase add enzymes for longer than 30-60 seconds. We can end up with copies of DNA 10,000 base pairs long or longer.

By using specific sequences in the DNA, we can anneal our primers to target points, allowing us to amplify a specific gene.

PCR generally goes for 25-35 cycles. You can figure out how many copies you have made using 2ⁿ where n is the number of cycles. For this class we used 35 cycles, yielding a calculation of 2³⁵ (34 billion) copies of each original strand of DNA (at the site of the gene selected by the primer). The cycles are continuous, in the above graph we have isolated a single thermal cycle.

Extension can last for variable time. The polymerase enzyme we used adds about 1kp (1000 base pairs, ie. the A, G, T and C nucleotides) per 30 seconds. Thus by letting the cycle run for 5 minutes, we are creating 10kb fragments of DNA. Manipulating the time for extension allows us to customize our gene fragment lengths.

In addition to adding the DNA to the PCR plate well, we have to make a small broth of chemicals (PCR mix) to make our PCR function.

Buffer: provides a suitable environment (pH, etc.) for PCR.

MgCl₂: polymerase requires specific orientation of the nucleotides during extension, the Mg²⁺ ion helps

dNTP: the A, G, T and C nucleotides which polymerase can grab to extend the primer

Primer 1: A sequence (in this case, 20bp long) that attaches just before a gene

Primer 2: A sequence (in this case, 20bp long) that attaches right after a gene.

Polymerase: The enzyme which attaches dNPTs to the primer

DNA template: The strand of denatured DNA we extracted from an organism, which the primer attaches to, allowing us to extend in the 5' to 3' direction creating a complementary DNA strand