• 教師: 鍾明怡 電話: 28712121 ext. 3265
• email: mychung@vghtpe.gov.tw
• Genotyping Core Laboratory

• Chapters 11-14 in “Human molecular genetics” by T.Strachan and AP Read.
• “Principles of medical genetics” by TD Gelehrter, and FS Collins.
• “Genetics in Medicine” by Thompson and Thompson.
• Some of the mentioned techniques can be found in biotech companies as Applied Biosystems (autosequening, genotyping, TaqMan, SNaPshot), BioRad (DGGE, CDGE, TGGE, SSCP), Transgenomics (dHPLC)

• What is genotyping?
• What are genotype and phenotype?
• Define some of the related genetic terms.
• What is genotyping for?
• Explain positional cloning and linkage analysis briefly.
• How is genotyping done in a lab?
• What is a “marker”?
• Why are microsatellite markers widely used these days?
• How do we perform “high throughput” genotyping in a core lab?
• What is SNP?
• How are SNPs detected traditionally?
• How are SNPs detected nowadays?
• Briefly describe the relative size of the components of the human genome and techniques applied to genome research.

genotype, phenotype, locus, alleles, polymorphic, dominant, recessive, co-dominant

Why genotyping ?

• Positional candidate cloning
• research in molecular genetics
• genetic counseling
• forensics

• Protein: ABO, HLA blood groups...etc..
• DNA sequences:
• RFLP (restriction fragment length polymorphism)
• VNTR (variable number of tandem repeats)
• microsatellite (STR, short tandem repeats)
• SNP (single nucleotide polymorphism)

• example: D22S264
• “D” means DNA
• “22” means chromosome 22.
• “S” means simple sequence, “Z” means repetitive on a single chromosome, and “F” means repetitive on multiple chromosomes.
• “264” means the 264th markers deposited, Independent of position
• Markers can be non-polymorphic, a.k.a. STS, “sequence tagged sites”, or expressed or not.

• Restriction fragment length polymorphism
• Methods of detection
• restriction enzyme digestion of genomic DNA + southern blot + hybridization with a specific probe
• restriction enzyme digestion of PCR-amplified DNA fragment.

• Variable number of tandem repeats
• Methods of detection
• restriction enzyme digestion of genomic DNA + southern blot + hybridization with a specific probe
• electrophoresis of PCR product on a gel (usually polyacrylamide).

• Short tandem repeats (STR)
• Repeats of two, three or four nucleotides, for example, (CA)n, (CAG)n, (GATA)n.
• Evenly distributed in the human genome

Why microsatellite markers?

• Informativeness of a marker
• Weissenback markers (Nature 1992; 359;794-801); even distribution in the genome
• allele size range
• fluorescent dyes and matrix
• autosequencer

• PCR setup for each marker
• multiplexing may work for some markers
• Pool PCR products of the same panel.
• Add loading dye with internal size standard.
• Gel electrophoresis.

• parental origin
• LOH
• microsatellite instability

• Third Generation genetic map.
• Power: ~2.5 SNPs equal to the power of one STR.
• In human, 1,592,751mapped as of Feb. 12, 2001, total 2,554,830 submitted.

• Home page: http://www.ncbi.nlm.nih.gov/SNP/index.html

SNPs: detection, confirmation, high throughput

• SSCP (single strand conformation polymorphism), UFD1L as an example
• DGGE (denaturing gradient gel electrophoresis)
• CDGE (constant denaturing gel electrophoresis)
• TGGE (temperature gradient gel electrophoresis)
• Heteroduplex analysis and dHPLC (denaturing high performance liquid chromatography)
• sequencing
• RFLP of PCR product
• ASO (allele specific oligo), detects specific SNPs by hybridization.
• Allelic discrimination by TaqMan probe
• Primer extension (SNaPshot)
• molecular beacon

• dHPLC = denaturing HPLC
• Fragment length: 150-450 bp (= 1.5 Kb)
• Four key aspects of mutation detection
• PCR primer design,
• PCR protocol,
• separation gradient,
• separation temperature
• DYS271and an unknown gene as examples.

• Affymetrix HuSNP genotyping chip.
• If you want to see the microarry chip, you can try to find it on the Research Genetics web site: http://www.resgen.com/.
• about 1500 SNP covering all 22 autosomes and the X chromosome.
• Primarily for linkage studies, also for LOH and association studies.
• Use only 0.5 micrograms of DNA.

• Variants of proteins.
• High density

• Nature Genetics 1992; 1:3-6.
• Nature Genetics 1993; 3:277-279.
• Linkage analysis
• Basic mechanism: meiotic recombination between chromosome homologues.
• unit: centiMorgan (cM), Morgan (M). Genetic distance is a function of recombination fraction. Two loci which show 1% recombination are defined as being 1 cM apart on a genetic map.
• LOD score to evaluate whether the recombination fraction you measured is statistically significant. Usually,

• Positional candidate cloning
• single gene
• multifactorial
• traits and susceptibility
• Research in molecular genetics
• parental origin of the defect
• haplotype analysis or linkage disequilibrium
• Genetic counseling
• Forensics

• chromosomes (estimated 64 Mb to 400 Mb)
• linkage analysis
• FISH (fluorescent in situ hybridization)
• PFGE (pulse field gel electrophoresis), regular agarose gel electrophoresis
• cloning vectors: YAC (yeast artificial chromosome), PAC, BAC, P1 phage, cosmid, plasmid
• Gene, gene complex