FoLT PCR: a simple PCR protocol for amplifying DNA directly from whole blood.
FoLT (formamide low temperature) PCR is a protocol for amplifying DNA directly from whole blood without any preparative steps. Up to 10% (vol/vol) whole blood can be added directly into the tube containing the PCR mixture. There is no need for transfers, centrifugations, pre-boiling or any preparative step. It involves the use of formamide (18% vol/vol) as well as reduced incubation temperatures (cycles of 85 degrees, 40 degrees, 60 degrees C). The type of anticoagulant used was critical: sodium heparin or EDTA being superior to lithium or fluoride heparin. Our studies indicate that FoLT PCR probably works by reducing the amount of protein coagulation and allowing more DNA template to be accessible for amplification. The sensitivity of FoLT PCR is such that a single copy gene from 5.5 nucleated cells in 1 microliter of whole blood can be detected.
Panaccio
M
M
Victorian Institute of Animal Sciences, Australia.
Georgesz
M
M
Lew
A M
AM
D000925:Anticoagulants; D001483:Base Sequence; D002021:Buffers; D004247:DNA / Q000097:blood* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004492:Edetic Acid; D005559:Formamides; D006493:Heparin; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
D000925:Anticoagulants; D002021:Buffers; D004269:DNA, Bacterial; D005559:Formamides; D007202:Indicators and Reagents; C031066:formamide; D006493:Heparin; D004247:DNA; D004492:Edetic Acid