Quantitation of rare DNAs by PCR.
This unit presents a protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence from 1 to 20,000 molecules per sample. In addition, it helps assess the presence of contaminating sequences, the bane of this kind of procedure. The DNA of interest is prepared and its concentration is determined. A known amount of this DNA is then mixed with two sets of oligonucleotide primers, one set specific for the DNA of interest (e.g., a virus) and the other set specific for an internal control (e.g., a single-copy gene encoded by the host organism). The sequences between the primers are amplified, electrophoresed on a gel, transferred to a filter, and probed with oligonucleotides specific for each amplified product. The amounts of the amplified products from the DNA of interest can then be quantitated by comparison to the internal control. For simplicity, the protocol is written in terms of quantitating viral DNA molecules relative to host cellular sequences; however, it can be adapted readily for other applications.
Coen
Donald M
DM
Harvard Medical School, Boston, Massachusetts.
D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D017931:DNA Primers; D004279:DNA, Viral