【正文】 tic, and biomedical research implications, SNP maps are helping to identify thousands of additional markers along the genome, thus simplifying navigation of the much larger genome map generated by researchers in the HGP SNP資料庫簡介與運用 YUHSHAN JOU, . 周玉山 博士 DIVISION OF MOLECULAR AND GENOMIC MEDICINE NATIONAL HEALTH RESEARCH INSTITUTES Example order of bases in a section of DNA on a chromosome: Some people have a different base at a given location What is a SNP? ...C C A T T G A C... ...C C G T T G A C... … G G T A A C T G... … G G C A A C T G... DNA Polymorphism Discovery Panels for Human Genome Variation Genome Res 8(12):1229, 1998 Characteristics of SNP in Databases Science 291:1331 (2022) SNP Databases: (Dec. 2022) ? Celera+PFP : 2,104,820 entries ? TSC : 585,811 entries ? Kwok (Wash U): 438,032 entries Dec. 2022 Classification of SNP by location ? Coding region: Synonymous: mutation does not change amino acid. Nonsynonymous: mutation change amino acid seq. ie rare mutations that cause medelian diseases with allele frequency below 1%. ? Noncoding region: 5’ and 3’ UTR’s Introns Space Features of Single Nucleotide Polymorphisms (SNPs) Markers ? the variant sequence type has a frequency of at least 1% in the population. ? high frequency of SNPs in human genome: estimated ~1 SNP/Kb. ? SNP :biallelic markers with 2 mon nucleotide substitution alleles (% of total SNPs is triallelic markers in TSC data). ? SNPs has lower mutation rate than do repeat sequences, but not as informative as microsatellite markers. ? detection methods for SNPs are potentially more suitable for geic screening in automated and largescale. ? the SNPs are likely be responsible for the functional change of the diseases: cSNPs. ? Allele frequencies of SNPs are tend to be very populationspecific. SNPs Genotyping ? Genome scans for Linkage Analysis: 1,000 to 5,000 markers. ? Candidate Gene (LD mapping): 100 to 1,000 markers per cM. ? Genome scans for Association Studies: 100,000 markers ? ? Requires PCR amplification per variant position. ? Currently resource intensive to find/characterize SNPs : 60,000 100,000, allele frequencies, map location and order. ? Comprehensive map may not be available until the genome is sequenced. ? Regional and gene specific SNPs will probably e first. ? Highdensity SNP mapping: require more efficient technologies and putational capability to analyze or recognize patterns from hundreds of thousands of SNPs. Mapping Disease Genes ? Look for geic linkage of disease to marker ? Microsatellite markers are too widely spaced to get to the individual gene level. ? There are mon SNPs in every gene. chromosome genes microsatellite SNPs disease gene Genotyping SNP markers for association studies If average size of linkage disequilibrium (LD) is 30~50 Kb. ? 50 Kb per SNP marker ? 60,000 SNP markers analyzed per association study. ? at least 1,000 individual patient samples in a cohort. A total of 60,000,000 SNP genotyping data per study and try to associate with available phenotypes. Cost: 60,000,=1,200,000 US dollars NRG 2:931 (2022) Assays for Known SNPs ? DNA sequencing: capillaries electrophoresis ? Microarray: oligonucleotide arrays ? DHPLC: ? Mismatch Repair Enzymes: T4 Endo VII, Cleavase etc ? Templatedirected Dyeterminator Incorporation assay with Fluorescence Polarization detection: ddNTP* ? MALDITOF: on silicon chips or various fragment sizes ? KiicPCR: sample pooling? ? More technologies are under developed. Purpose: confirmation of SNPs in various races Technologies: Highthroughput and Automation Strategies: haplotyping, Pool markers or Pool genomic DNA Mass Spectrometry for Analysis and Scoring Use mass spec to score which base(s) add Multiplex 5 with known primer masses Pool 50 to 500 samples Haff and Smirnov, Genome Res. 7 (1997): 378 Sequenom MALDITOF assays for SNPs MALDITOF assays for SNPs (cont.) Selected list of SNP databases ? ? ? ? ? ? ?