Abstract:
Starting with minimal amounts of DNA, the polymerase chain reaction (PCR) allows to amplify specific DNA-fragments of 500 to 1000 bp length. The amplified DNA can then be demonstrated e.g. in an agarose-gel , or processed further with other methods. The basis of PCR consists of a heat resistant Taq-polymerase, which synthesizes from a single strand-DNA the corresponding counter-strand. The fact that the synthesis can only start from double strand-DNA is used to define the DNA sequence to be amplified by adding specific oligonucleotides (primer). The DNA-fragment is multiplied exponentially by running 25-30 cycles with 3 temperature levels. At 95°C, DNA denaturates to single strands. At 55-68°C, the oligonucleotides (primer) bind specifically to the single strand-DNA (annealing) and thus start the Taq-polymerase reaction. At 72°C, the counter strand is synthesized (extension).
With reverse transcription-PCR, specific RNA-sequences can be demonstrated. RNA is first converted with RT-PCR to complementary DNA and then amplified with "normal" PCR.
In quantitative real-time PCR, a nucleotide marked with fluorescein is released. Thus, the synthesized DNA can be reliably and exactly quantified.
Indication:
There are many uses of the PCR-method hematological diseases. Here some example: