BIOS 390 Week 1 LAB 1

BIOS 390 Week 1 LAB 1 LAB 1: Polymerase chain reaction Polymerase chain reaction (PCR) is a laboratory technique used to increase, or make many copies of, a specific target region of DNA in vitro. The goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For example, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments. PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus). T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active around 70°C (a temperature at which a human or E. coli DNA polymerase would be nonfunctional). This heat-stability makes Taq polymerase ideal for PCR because high temperatures are used repeatedly in PCR to denature the template DNA, or separate its strands. For tag polymerase to make DNA it must first be given a primer, a short sequence of nucleotides that provide a starting point for DNA synthesis. The primer is a short piece of single-stranded DNA determined by the scientist from a certain region of DNA. Two primers are needed in each PCR reaction and they are given sequences that will make them bind to opposite strands of the DNA template by complementary base pairing. Once the primers are fixed to the template, they can be extended by the polymerase, and the region that lies between them will get copied. The main ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized. The basic steps are: 1. Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step. 2. Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA. 3. Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA. This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours, depending on the length of the DNA region being copied. When successful, the target region can go from just one or two copies to billions of copies of DNA. There are many copies of the