Novel PCR technology to analyze multiple genomic regions on a DNA microarray

Abstract

DNA microarrays are fabricated by robotic machines that arrange DNA spots in micro-scale onto the solid substrate. Each DNA spot contains very low amount of DNA probes which are covalently attached to the solid surface with several distinct chemistries. Since DNA probes are spotted at hundreds or thousands of sites from 10 to 500 microns size, it is possible to detect and measure thousands of genetic information simultaneously. Therefore, DNA microarray analysis has become the most widely used technology for the high-throughput analysis of nucleic acids including gene expression profiling, comparative genomic hybridization, single nucleotide polymorphism detection, and pathogen detection. Target nucleic acids to be analyzed are amplified by PCR amplification and hybridize to the specific capture probes by forming hydrogen bonds between complementary nucleic acids pairs. Particularly, when various genomic regions are analyzed simultaneously, multiplex PCR method is generally used to amplify several target nucleic acids in a single tube by using many primer pairs. However, an increase in the number of genetic regions for simultaneous amplification by multiplex PCR leads to the generation of non-specific amplification products due to the cross-reaction among primers. For the purpose of overcoming this problem, we have developed a novel PCR technology for the amplification of multiple genomic regions simultaneously which can be analyzed by DNA microarrays. In the first strategy described in chapter 2, we have developed a novel multiplexed single-nucleotide polymorphisms (SNPs) genotyping method based on amplification of separated ligation-dependent probe (ASLP). In the ASLP technique, allele-specific ligation reaction was first performed between annealed allele-specific oligonucleotide (ASO) probe and locus-specific oligonucleotide (LSO) probe containing the unique regions of homology to target genomic DNA. The ASO probe covering up to SNP site is modified with un...