Sequence-specific detection of PCR-amplified DNA by restriction enzyme release of hybrids.
We have developed a method to detect PCR amplicons that contain a restriction enzyme site. The method is called Polymerase Chain Reaction-Hybridization and Enzymatic Release Assay (PCR-HERA). The PCR product is hybridized to a synthetic oligonucleotide that is immobilized on a microtitre plate. Specific cleavage of the resulting hybrid by a restriction endonuclease liberates a fluorescein labeled arm of the immobilized DNA. We demonstrate the use of PCR-HERA to detect species-specific sequences of extra-chromosomal, ribosomal DNA, of Entamoeba histolytica. This highly specific assay does not require any washing steps and can be easily automated.
Estrada
G
G
Department of Molecular Recognition and Structural Biology, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
Colin
L
L
Gaytan
P
P
Alagón
A
A
Lizardi
P M
PM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004262:DNA Restriction Enzymes / Q000032:analysis*; D016054:DNA, Protozoan / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D004748:Entamoeba histolytica / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D004262:DNA Restriction Enzymes