Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.
In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B). Using a dilution series of purified PCR products, we consistently found a lower detection limit of 1.5 fmol for method A than the 0.15-fmol limit for method B. In the second assay we used the peroxidase-based enhanced chemiluminescence system in combination with a cooled charge-coupled device camera to quantify PCR fragments that were spotted on membranes. A linear logarithmic response was observed between the amount of light produced within a certain time interval and the number of DNA molecules. With an exposure time of 5 min, a detection limit of 0.15 fmol was found. Longer exposure times did not result in a higher sensitivity. We conclude that the assays are of sufficient sensitivity for application in quantitative PCR strategies. The nonradioactive technology facilitates implementation of these assays in routine settings.
Vlieger
A M
AM
Department of Cytochemistry and Cytometry, Sylvius Laboratory, Leiden University, The Netherlands.
Medenblik
A M
AM
van Gijlswijk
R P
RP
Tanke
H J
HJ
van der Ploeg
M
M
Gratama
J W
JW
Raap
A K
AK
D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D003124:Colorimetry; D004247:DNA / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D005240:Feasibility Studies; D005434:Flow Cytometry; D005914:Globins / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D019809:Streptavidin
D001426:Bacterial Proteins; D004279:DNA, Viral; D001710:Biotin; D005914:Globins; D004247:DNA; D019809:Streptavidin