Genotyping Technology And Applications For Cancer

Cancer is one of the major killers currently threatening human life.Individualized treatment is the most promising method to cure cancer,according to an in-depth study of cancer.Cancer is a gene disease.Genotyping of the individual cancer is require to achieve individualized treatment.Molecular genetic markers for genotyping include restriction fragment length polymorphisms(RFLPs),microsatellite loci(STRs),single nucleotide polymorphisms(SNPs)and copy number variations(CNVs).Because of the complexity of cancer,CNVs based profiling of genomic changes has become an important means of cancer genotyping.Array-based comparative genomic hybridization technology,which combines the benefits of comparative genomic hybridization(CGH)and microarray chips,have the advantages of high-throughput and being amendable to automation,thus making it a major means of CNVs research.Polymerase chain reaction(PCR)is an effective method for in vitro DNA amplification.Amplifying probes by PCR and finding more effective conditions of array production are basic steps for the preparation of whole-genome comparative genome hybridization array,which is the main task of the thesis.The thesis work consists four parts as follows:1.Probe amplification:Over seventeen millions of unique oligo probes were designed to cover the whole genome.These probes were synthesized by in situ surface synthesis into twelve probe libraries.A combination set of primer pairs were used to separate these probes into 23033 probe bins,each of which contains 700 individual oligos covering about 128 kb genomic region.We used three rounds of PCR to amplify the probe bins to a quantity sufficient for array production.The conditions for each step of PCR were optimized.2.Probe preparation:Amplified PCR products were concatenated using specially designed primers,precipitated,and then silanized for covalent coupling.3.Test Array preparation:We selected a small group of probes to make test arrays to find the optimal condition of array preparation and array hybridization.We then finalized the protocols for scale-up probe preparation and array production.4.Cancer aCGH arrays:Through literature searches we identified 140 regions that showed recurrent copy number alteration across 11 types of cancer.We selected 3267 probe bins to provide full coverage of these regions.3500 probes scatter evenly across the genome were selected as reference.We used a set of endometrial cancer to validate the arrays.