Solid-phase PCR with hybridization and time-resolved fluorometry for detection of HLA-B27.
BACKGROUND
Preactivated solid surfaces provide new possibilities for multiple consecutive reactions in a microtiter plate format. In this study, a combination of PCR and subsequent hybridization in the same microtiter well was applied for the detection of HLA-B27 alleles.


METHODS
A multiplex solid-phase PCR to amplify the HLA-B27 alleles together with beta-actin as an amplification control gene was performed on the NucleoLink (Nunc) surface. PCR was followed by hybridization and detection with time-resolved fluorescence. For the covalent capture of the PCR primers onto the solid support via a 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride-mediated reaction, different 5'-end modifications of oligonucleotides were tested [amination, phosphorylation, and a poly(dT)10 linker].


RESULTS
For covalent immobilization of the primers, amination of the 5' end combined with use of the poly(dT)10 linker was superior. At least 19.5% of the primer added per well was attached via a stable bond. When the standard time-resolved, fluorescence-based HLA-B27 detection system was compared with the newly developed method in a sample series of 82 genomic DNAs and the corresponding dried-blood spots, all results were in full agreement.


CONCLUSIONS
The new solid-phase PCR approach can be applied for multiple-target DNA detection. PCR followed by hybridization can be accomplished in a few hours using precoated strips and dried-blood spot PCR templates.
JDF Center for Prevention of Type 1 Diabetes in Finland, and Department of Virology, University of Turku, Turku, Finland. minna.sjoroos@utu.fi
M
M
Sjöroos
J
J
Ilonen
T
T
Lövgren
D020121:5' Untranslated Regions; D000199:Actins / Q000235:genetics; D001800:Blood Specimen Collection; D017931:DNA Primers / Q000737:chemistry; D015796:HLA-B27 Antigen / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence
D020121:5' Untranslated Regions; D000199:Actins; D017931:DNA Primers; D015796:HLA-B27 Antigen