基因體研究技術概論 Oct. 19, 1999

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

Most of the sides shown in this class are provided in this final version of outline. References or simple descriptions are given for those that are not available in

the Powerpoint format.

After this class you should be able to answer the following questions,

• 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?

• What does a core lab for genotyping provide (ideally)?– 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.

Genotyping

genotype vs. phenotype

ABO blood group

locus: ABO blood group

phenotype A B AB O

genotype AA BB AB OO

AO BO

alleles: A, B, O.

Why genotyping ?

• Positional cloning• research in molecular genetics

– parental origin of the defect– haplotype analysis or linkage disequilibriu

m• genetic counseling• forensics

Positional cloning

• Nature Genetics 1992; 1:3-6.• Nature Genetics 1993; 3:277-279.

Pedigree with genotyping

•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,

LOD score >3 => linkage is accepted;

LOD score <-2 => linkage is excluded,

LOD score between -2 and 3 means inconclusive.

Why genotyping ?

• Positional cloning• research in molecular genetics

– parental origin of the defect– haplotype analysis or linkage disequilibriu

m• genetic counseling• forensics

Polymorphic Markers (1)

• 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)

RFLP

• Restriction fragment length polymorphism

VNTR

• Variable number of tandem repeats

Microsatellite

• Short tandem repeats• Repeats of two, three or four nucleotides, for

example, (CA)n, (CAG)n, (GATA)n.• Evenly distributed in the human genome

A sequencing gel showing two (CA)n repeats

A slide showing that microsatellite markers are run on regular sequencing gels

A slide showing how dinucleotide repeats look like after autoradiography. The example given is D22S941.

In this gel seven alleles of D22S941 were observed. Only three out of sixteen individuals were homozygous.

Informativeness of a marker

• 3 alleles– assume equal frequency

=> 1/3 may be homozygous• 7 alleles

=> 1/7 may be homozygous• 10 alleles

=> 1/10 may be homozygous

Weissenback markers (Nature 1992; 359;794-801)

Why a core lab for genotyping?

You can definitely do the whole process in your own lab, but run on a autosequencer can

• 1. Increase the throughput by using multiple fluorescent dyes in a lane.

• 2. Genotyping software helps in genotyping and double checking.

• 3. Easily incorporate pedigree and clinical information to build a database and export in forms compatible for further analysis.

A slide showing the result of gel electrophoresis with four panels of fluorescent genotyping markers of the

PE ABI PRISM linkage mapping set 2.

Two slides showing the chromosome map of the PE ABI PRISM linkage mapping set 2 (now called MD10 for medium density or 10 cM). You can visit their web site at http://www.pebio.com/ab/apply/dr/lms

v2/chromemap.html.

A slide showing four panels, 13, 14, 15, and 16 of the PE ABI PRISM linkage mapping set 2 collectively

provide markers for human chromosomes 9, 10, and 11.

Weber’s marker

• low resolution– 169 markers in 20 panels, 25 cM spacing.– Average heterozygocity 0.78.– 94% are tri- and tetranucleotide repeats.

• high resolution– 387 markers in 44 panels, 10 cM spacing.– Average heterozygocity 0.76.– 89% are tri- and tetranucleotide repeats.

Human microsatellite sets for fluorescence-based genome mapping

The complete set is an expanded version of that described by Reed et al. (Nature Genetics 1994, 7, 390-395), which has been modified slightly so that the markers can be more easily multiplexed on ABI machines. It consists of 290 marker pairs labeled with either FAM, HEX or TET. Sets are multiplexed in groups of 20 individual markers on average, for rapid and efficient analysis. The resolution of the set is approximately 9cM (although we are constantly improving our set), with up to 2000 PCR reactions per pair. Subsets and individual chromosomes are also available: please apply for more information. CATALOGUE NO. PCR REACTIONS DESCRIPTION C290 2000 / marker 295 marker pairs organized into 15 panels

Microsatellite markers: Chromosomes 1 - 4 Chromosomes 5 - 8 Chromosomes 9 - 12 Chromosomes 13 - 16 Chromosomes 17 - 20 Chromosomes 21, 22, X

Experiment procedures

• 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.

Why a core lab for genotyping?

You can definitely do the whole process in your own lab, but run on a automated sequencer can

• 1. Increase the throughput by using multiple fluorescent dyes in a lane.

• 2. Genotyping software helps in genotyping and double checking.

• 3. Easily incorporate pedigree and clinical information to build a database and export in forms compatible for further analysis.

Two slides demonstrate the PE ABI PRISM GeneScan software, in one slide the lanes are aligned by scan, and in the other all the lanes are aligned by size, i.

e. all the internal size markers are lined up.

Three slides showing how the Genotyper software helps you with following analysis by importing analysis results from GeneScan, labeling peaks (doing genot

yping for you), and exporting the genotype results.

In addition to GeneScan and Genotyper, Genopedigree and GeneBase softwares provide links for further

analyses.

Applications of genotyping

• delineation of genetic traits--linkage analysis, association studies, …etc.– What do you need in addition to genotyping?

• Scale of analysis--whole genome vs. chromosome or even region specific

• genetic epidemiology• cancer genetics

– loss of heterozygocity

Two slides showing the theory and an exmple of LOH analysis in HCC using chromosome 22 markers.

Resources

• Man power: hopefully four full-time technicians• Reagents (DNA extraction, marker selection, PCR reag

ent, internal ladder, high throughput operation)• Hardwares

– 2 ABI PRISM 377XL– (thermocyclers) (2 ABI PRISM 877)– (Mac for post-electrophoresis analysis to maximize t

he throughput)• Softwares: supported by bioinformatics

Procedures for genetic study

• control/patient recruitment • phenotype analysis• pedigree analysis• DNA extraction• PCR setup• sample pooling• gel electrophoresis• genotype output• statistical analysis

Single nucleotide polymorphisms (SNP)

• Third Generation genetic map.• Power: ~2.5 SNPs equal to the power of one STR.• 2227 mapped as of May 1998, total >3000.• Map on the web: http://carbon.wi.mit.edu:8000/cg

i-bin/SNP/human/SNP_map.

Traditional ways of detection SNPs

• ASO (allele specific oligo), detects specific SNPs.• SSCP (single strand conformation polymorphism)• DGGE (denaturing gradient gel electrophoresis)• CDGE (constant denaturing gel electrophoresis)• heteroduplex analysis

Detect SNP using the WAVE system

• 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

SNP by microarray

• 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.

The human genome and various techniques for genome research

• 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

General references:

• 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.