MAKING RECOMBINANT DNA MOLECULES：使重组DNA分子.pptVIP

Polymerase Chain Reactions (PCR):“Molecular Photocopying” A fast, inexpensive, and simple way to amplify (make lots of copies of) a small sample of DNA- invented in 1985 Inventor (Kary Mullis) was awarded Nobel Prize in Chemistry in 1993 Used for Human Genome Project, DNA fingerprinting, diagnosis of genetic disorders, etc. 1.Add Target DNA to a test tube which has: DNA polymerase (TAQ*) Short oligonucleotide** primer Copies of the four nucleotides *TAQ- Thermus aquaticus , heat stable DNA polymerase (from hot springs, like Yellowstone) **A molecule usually composed of 25 or fewer nucleotides; used as a DNA synthesis primer Heat to 95oC to denature DNA to single strands Cool to between 50o and 65 oC to allow primers (synthetic sequences of DNA) to join to target site. To “anneal” is to form hydrogen bonds between primer and DNA Warm to 72oC. Taq DNA Polymerase binds to the primers and selectively copies the target region with complimentary nucleotides. Warm to 95oC again to separate newly formed and original DNA strands. Then, REPEAT! After 30 cycles (a few hours) one billion copies are attained! Some Animations PCR: /webcontent/anisamples/molecularbiology/pcr.html /content/labs/pcr/ PCR Dash Timekeeper starts clock (3 minutes) 95°C player cuts apart DNA template TC gives template pieces to 55°C player 55°C player matches complimentary primer to each strand and attaches them with tape The TC takes the primed strands to the 72°C player 72°C player matches the complimentary polymerase product to each DNA strand and tapes it to the primer The TC then takes the finished double stranded products back to the 95°C player who denatures (cuts) them The cycle is then repeated as many times as possible before time runs out!