Methods {polymerase chain reaction}| (PCR) can make many DNA-sequence copies using heat-stable polymerase, 20-base primers complementary to + strand at one sequence end, and 20-base primers complementary to - strand at other end. Synthesized strands are additional templates, so process doubles copies each primer-annealing, strand-elongation, and dissociation cycle.
purpose
PCR can detect defined sequences in DNA samples. PCR can make stutter bands and add bands resulting from extra nucleotide addition by Taq polymerase.
mRNA amplification
DNA has small amounts, but mRNA has much larger amounts. First, reverse transcription converts mRNA to cDNA and then PCR amplifies cDNA (RT-PCR).
DNA amplification
Machines heat DNA double helix to 94 C for several minutes to make single-stranded DNA. Solution contains DNA polymerase from heat-tolerant organisms and the four bases.
When temperature lowers to 30 C to 65 C for 30 seconds, 20-nucleotide primer DNA binds to DNA, outside region to copy. One primer is for 5' strand, and one primer is for 3' strand. Annealing puts complementary 20-base primer at both ends.
Machines raise temperature to 72 C for some minutes, to allow DNA polymerase and bases to extend both primers beyond other primer region. Now both double-helix molecules have primer on one end and extend beyond other primer on other end. Elongating both strands uses heat-stable DNA polymerase, which synthesizes DNA.
Machines heat DNA to 94 C for several minutes again to extend same primers through other primer at strand ends. Now all synthesized-strand lengths are the same, from one primer through other primer. There are now four DNAs.
Cycles make twice as many DNA strands, and process uses new and old strands again, making chain reactions. Repeating process 30 to 60 times makes millions of copies.
primers
Primers can be genome repetitive sequences, such as Alu repeats. Alu repeats are 300 bases, but smaller region varies little in humans. Alu repeats are in both directions.
primers: nested
After one PCR, second primer that binds inside copied sequence {nested primer} can amplify shorter sequences.
primers: concentration
If one primer has high concentration and one has low, system makes mostly single-stranded DNA {asymmetric PCR}, with no chain reaction.
contamination
Contamination with wrong DNA is common. Negative controls make sure correct DNA amplifies.
Biological Sciences>Genetics>Recombinant DNA>Amplification
4-Genetics-Recombinant DNA-Amplification
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Date Modified: 2022.0224