Transcript powerpoint file

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: [email protected]
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?
Genotyping
genotype vs. phenotype
ABO blood group
locus:
ABO blood group
phenotype
A
B
AB
genotype
AA
BB
AB
AO
BO
alleles: A, B, O.
O
OO
Why genotyping ?
• Positional cloning
• research in molecular genetics
– parental origin of the defect
– haplotype analysis or linkage
disequilibrium
• 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
Why genotyping ?
• Positional cloning
• research in molecular genetics
– parental origin of the defect
– haplotype analysis or linkage
disequilibrium
• 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/lmsv2/chromema
p.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.
C290
organized into 15 panels
PCR REACTIONS
2000 / marker
DESCRIPTION
295 marker pairs
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 genotyping 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
reagent, internal ladder, high throughput operation)
• Hardwares
– 2 ABI PRISM 377XL
– (thermocyclers) (2 ABI PRISM 877)
– (Mac for post-electrophoresis analysis to maximize
the 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/cgibin/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.