Molecular Genetics

• The standard polymerase chain reaction (PCR) amplifies a specific DNA segment many fold.
• It uses two oligonucleotide primers complementary to opposite strands of a duplex DNA. The primer 3' ends when annealed to the target DNA are closer to one another than the 5' ends are.
• A PCR consists of multiple cycles of annealing of the oligonucleotides, synthesis of a DNA chain, denaturation of the synthesized DNA.
• Theoretically, a geometric amplification of a fragment whose ends are defined by the 5' ends of the primers should occur, while the longer template sequence is amplified at most arithmetically.
• PCR is used widely in:
• Molecular cloning
• Pathogen detection
• Genetic engineering
• Mutagenesis
• Genetics, producing molecular markers

• The availability of thermostable polymerases and the design of thermal cycler machines made PCR widely accessible.
• Synthesis times can be manipulated to guarantee just the synthesis of shorter DNAs.
• To amplify RNA rather than DNA sequences, a cDNA copy of the RNA can be made by reverse transcription prior to PCR. The combination is called RT-PCR.
• A diverse variety of PCR's have been devised.
• PCR has been made quantitative by the use of fluorescence that can be read at each cycle. There are several strategies: Taqman, Molecular Beacons, dual oligo binding.
• PCR is extremely sensitive. Contamination is a constant worry. On line help is available.
• A "Shockwave" animation is available.