The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA.
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.
Dahlén
P
P
Pharmacia Genetic Engineering Inc., La Jolla, CA 92037.
Iitiä
A
A
Mukkala
V M
VM
Hurskainen
P
P
Kwiatkowski
M
M
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
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