Biomedical Engineering Reference
In-Depth Information
Fig. 1. Flowchart for the real-competitive polymerase chain reaction (rcPCR)
approach for DNA quantification. The DNA or cDNA (reverse-transcribed from RNA)
sample is spiked with a competing DNA oligonucleotide standard (60-80 bases) with
an artificial single-base mutation in the middle of the sequence. These two DNA
sequences are co-amplified with PCR in the same reaction. Excess dNTPs are removed
by shrimp alkaline phosphatase (not shown). A primer extension reaction is carried
out with an extension primer annealing to the region immediately next to the mutation
site. Depending on the mutation introduced and the ddNTP/dNTP mixture used, either
one or two bases are added to the extension primer. The extension products are detected
and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spec-
trometry.
