Multiple sample amplification and genotyping integrated on a single electronic microarray.
We report a novel method that allows simultaneous in situ amplification and then genotyping of single nucleotide polymorphism (SNP) for multiple samples on a single electronic microarray. The locus coding for one of the common inherited thrombosis risk factors, Factor V Leiden (FVL), was chosen as a model system for SNP analysis. This method combines strand displacement amplification (SDA) with electrophoretic movement and concentration of DNA on electronic microarrays to provide a single platform for DNA amplification and analysis. The method includes: electronic anchoring of allele-specific SDA amplifiable primers (APs) and a nonamplifiable primer (NAP) to different electrodes, electronic hybridization of genomic DNA from different samples to those primers, in situ amplification of target DNA, and genotyping of FVL. Compared to previous anchored SDA methods, the addition of a NAP improves detection signals by at least 20-fold. The sensitivity of this method is dependent on the amplification time. Using this method, nine different genomic DNA samples with known FVL genotypes were amplified and correctly genotyped on a single electronic microarray without any contamination between samples. The present method could streamline development of nucleic acid-based assays in applications of molecular diagnostic, point-of-care testing, and forensic detection, which often require the capability to analyze multiple samples efficiently.
Nanogen Inc., San Diego, CA 92121, USA. yhuang@nanogen.com
Ying
Y
Huang
Jack
J
Shirajian
Astrid
A
Schroder
Zuxu
Z
Yao
Tamara
T
Summers
Dalibor
D
Hodko
Ron
R
Sosnowski
D001483:Base Sequence; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D005838:Genotype; D036103:Nanotechnology; D020411:Oligonucleotide Array Sequence Analysis
D017931:DNA Primers; D004247:DNA