ReviewThe real-time polymerase chain reaction
Section snippets
PCR amplification
PCR is performed on a DNA template, which can be single or double-stranded. Also needed are two oligonucleotide primers that flank the DNA sequence to be amplified, dNTPs, which are the four nucleotide triphosphates, a heat-stable polymerase, and magnesium ions in the buffer. The reaction is performed by temperature cycling. High temperature is applied to separate (melt) the strands of the double helical DNA, then temperature is lowered to let primers anneal to the template, and finally the
Gene expression measurements
Before a gene expression measurement can be performed by real-time PCR, the mRNA in the sample must be copied to cDNA by reverse transcription (RT) (Fig. 8). The RT step is critical for sensitive and accurate quantification, since the amounts of cDNAs produced must correctly reflect the input amounts of the mRNAs. Comparing the technical reproducibilities of the RT reaction and the PCR it was found that the RT reaction contributes with most of the variation to the experimental determination of
Acknowledgement
This work was supported in part by project no. AVOZ50520514 awarded by the Academy of Sciences of the Czech Republic.
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