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1、polymerase chain reaction (PCR),Definition of PCRThe basic principle of PCRPCR Frequently Asked QuestionsApplication of PCR,Definition of PCR,PCR(polymerase chain reaction),is under the catalytic of DNA polymerase,to mother chain DNA as a template, with specific primers for the the starting point of
2、 extension, through the degeneration, annealing, extending steps, with the parent chain template DNA replicate the complementary DNA child chain in vitro .It is one of the amplification technique of DNA synthesis in vitro, can quickly specific amplificate any purpose DNA in vitro.,The basic principl
3、e of PCR,1.PCR reaction components (1) template DNA (2) Primer (3) four kinds of deoxyribonucleotides (4) DNA polymerase (5) Reaction Buffer, Mg2+,2. PCR reactions basic steps(1) Denaturation , The hydrogen bonding between the template double chain DNA ruptured by heating, and double chain apart int
4、o a single chain.(2) Annealling , As the temperature falls, the primer and complementary area of template DNA combined into hybrid molecules.,(3) Extension, In the presence of DNA polymerase, dNTPs,Mg2+, DNA polymerase catalytic primers outspread according to the 5 to 3 direction, synthesis DNA chai
5、n that complementary with template DNA chain. The above three steps for a cycle, the product of each loop can be used as a template for the next cycle .After n cycles, the purpose of DNA plus in the form of 2n.,GGATCTAGCGTATGCTTGAAA,CCTAGATCGCATACGAACT T T,GAACTTT,GGATCTA,5,5,3,5,3,3,5,3,primer1,pri
6、mer2,Template DNA,Picture1, PCR primers combined with template,PCR Frequently Asked Questions,A. No amplification products1. The cycle temperature: denaturation temperature and annealing temperature2. The primer design3. The activity of DNA polymerase4. The inhibitory ingredients (protease and nucle
7、ase)5, DNA samples,B. The specific product and electrophoresis coating shaped1. The concentration of Mg 2+2. Adjust the dosage of the primer, templates, and polymerase3. Decrease the cycle number4. Increasing the annealing temperature, annealing or extension of time,Application of PCR,1. The gene diagnosis of genetic disease2. The diagnosis of infectious diseases (hepatitis virus)3. The cancer gene detection4. The application of forensic science (paternity test)5. DNA sequencing6. Gene cloning7. Introduction of gene mutation point, gene fusion, etc,
