A, experimental purposes
1. To master the principle of polymerase chain reaction.
2. Master the basic operation of a pipetting gun and PCR technique.
Second, the experiment principle
The PCR technology, namely the polymerase chain reaction (polymerase chain reaction, PCR) is by the PE Cetus Kary Mullis in 1983 was awarded the Nobel Prize for chemistry (1993). The technique of in vitro after hours response to specific DNA amplification millions of times, this quickly to get a lot of single DNA fragment technology has important significance in the study of molecular biology, greatly promoted the research progress of life science. It is not only the most commonly used DNA analysis technology, and the recombinant DNA and gene expression, structure analysis and feature detection has important application value.
PCR can be considered to be similar to occur in the process of DNA replication in the cell technology, the result is based on the original DNA as a template to create new complementary DNA fragments. Cell DNA replication is a very complicated process. There are many factors involved in replication. PCR was conducted in a test tube of DNA replication, the basic principle and DNA replication in the cell are similar, but the reaction system is relatively simple.
PCR by degeneration, annealing, extension of three basic steps: (1) the denaturation of template DNA: DNA template by heated to 94 ℃ or so after a certain period of time, make double-stranded DNA template or by PCR amplification of double-stranded DNA dissociation, making it the single, so that it combine with primer, preparing for the next round of reaction;
(2) template DNA with primer annealing (compound) : after heating degeneration into a single template DNA, the temperature dropped to about 55 ℃, primers with complementary single-stranded DNA sequence template matching;
(3) the extension of primers: DNA templates, primers combination under the action of Taq polymerase, dNTP as reaction raw material, the target sequence as a template, according to a principle of base pairing and half reserved copy, synthesis of a new and complementary to the template DNA half reserved copy chain.
Recirculation degeneration, annealing, stretches across three process, can get more "half reserved copy chain", and this new chain can be the next cycle of template again. After completing a cycle to 2 ~ 4 minutes, 2 ~ 3 hours to enlarge the purpose gene amplification can be amplified millions of times.