Published online 9 August 2005
Methods Online |
Headloop suppression PCR and its application to selective amplification of methylated DNA sequences
CSIRO Molecular and Health Technologies PO Box 184, North Ryde NSW 1670, Australia 1The Garvan Institute for Medical Research 384 Victoria Street, Darlinghurst NSW 2010, Australia
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Received May 19, 2005. Revised July 12, 2005. Accepted July 14, 2005.
Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5' extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure—this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5' extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 105-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.