title: The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA.
abstract: The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.
authors.affiliation: Pharmacia Genetic Engineering Inc., La Jolla, CA 92037.
authors.forename: P
authors.initials: P
authors.lastname: Dahlén
authors.forename: A
authors.initials: A
authors.lastname: Iitiä
authors.forename: V M
authors.initials: VM
authors.lastname: Mukkala
authors.forename: P
authors.initials: P
authors.lastname: Hurskainen
authors.forename: M
authors.initials: M
authors.lastname: Kwiatkowski
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D004279:DNA, Viral / Q000032:analysis*; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D005063:Europium; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D019809:Streptavidin
chemical_list: D001426:Bacterial Proteins; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D005063:Europium; D001710:Biotin; D019809:Streptavidin; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases