Principles of Biology Unit 2: Cell Division Polymerase Chain Reaction (PCR) Working with DNA -DNA can be used to… -Determine identity (DNA fingerprinting) -Determine relatedness (genetic genealogy) -Create new products (genetic engineering) -Calculate disease risk (genetic testing) -Cure/treat disease (gene therapy) -In order to do these things, researchers need large amounts of specific regions of the DNA that contain the information they need. -A single human cell has over three billion base pairs of DNAs in each strand. Since there are two copies, there are actually six billion base pairs. The average gene that a researcher would need to study is only five thousand base pairs. The five thousand base pairs were extremely difÏcult to locate in the six billion base pairs of human DNA. This was made easier by PCR, or polymerase chain reaction. Polymerase Chain Reaction (PCR) -PCR mimics the process of DNA replication. However, instead of making copies of all the DNA in a cell, it only makes copies of a specific region that has the information the researcher wants to study. The researcher can also use PCR to make as many copied of that one particular piece of information they want (amplification of a particular piece of DNA. -In order for PCR to work, you need a certain number of key components: -A sample of DNA that contains the region of information that you want to study. -Buffers and salts to keep the enzyme in your reaction sustained. -DNA primers that allow you to target the specific region that you want to copy. -A heat-stable DNA polymerase. -A machine to repeatedly raise and lower temperatures. -PCR in Action -Imagine you have a sample of template DNA within which is a piece of information you want to copy. The first step during PCR is you heat a piece of DNA (or your sample) to high temperatures. These high temperatures cause the hydrogen bonds holding the two strands together to break, and the strands to separate. -You will also have DNA primers that will recognize and attach to these template strands at specific locations. These primers are able to recognize specific regions of DNA because they can form base pairs. They can make complimentary hydrogen bonds between the primer and specific regions of DNA in your template strand. These primers will only stick to complimentary regions of DNA. -Now you add DNA polymerase and it can add on to the 3’ end of these primers and use the template strands to replicate a new complementary DNA strand. -Once you’ve done this, you simply raise the temperatures on your samples again. It causes your template strands and the newly produced complimentary DNA strands to separate. -You add more primers and DNA polymerase, and now the new strands you just produced can also serve as templates for replication. -Theoretically, if you repeat this process 32 times, you can produce two billion copies of the specific DNA you want to study, which is plenty for most any study that a researcher would want to conduct. KEY TERMS Amplification: in the context of DNA, a rapid increase in the number of copies of a DNA fragment. Electrophoresis: technique used to separate DNA fragments according to size. Primer: short stretch of nucleotides that is required to initiate replication; in the case of replication, the primer has RNA nucleotides.