1 제 5 장 동물 분자육종을 위한 DNA 표지인자의 이용 전통적인 통계적 보정...

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제 5 장 동물 분자육종을 위한 DNA 표지인자의 이용

전통적인 통계적 보정 방법에 의한 동물육종

DNA 상의 특성 및 유전자 조작에 의한 동물육종

동물육종 방법의 변화

( 교재 133 참조 )

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Molecular Methods for Identification of Genotypes Basic Concept

• The number of nucleic acid or amino acid differences between two organisms is proportional to the time since they

diverged from a common ancestor.

TIME

MOLECULAR

DIFFERENCES

1 AAGGCTA

2 AAGGGTA

3 AAGGGTG

Example Rate of Evolution = 1bp per 100 years

1 2 3

100years

200 years

DNA 의 다형현상

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MARKERS IN BIOLOGY

Phenotypic markers = Naked eye markers

P = E+G

Flower colors, shape of pods, etc..

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• Differences can be detected: – Visually – morphological traits

P1 F1 P2

B. Vandenberg

Polymorphisms

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Molecular markersMolecular markersR

eso l

utio

nR

eso l

utio

n po

wer

pow

er

RFLP, Restriction Fragment Length Polymorphisms (high)

SSCP, Single Strand Conformation Polymorphisms (very low)

RAPD RAPD ((medium) (random amplified polymorphic DNA)(random amplified polymorphic DNA)

AFLPAFLP(Amplified Fragment Length Polymorphism)(Amplified Fragment Length Polymorphism)

Multi-locus fingerprintsMulti-locus fingerprints

Microsatellites (SSRs)Microsatellites (SSRs)

Sequencing (SNPs)Sequencing (SNPs)

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DNA 의 다형현상(DNA Polymorphism=differences)

개체간의 DNA 의 차이를 DNA polymorphism 이라 함DNA 염기서열을 분석하여 개체간 특성 파악

제한효소 절편다형(Restriction Fragment Length Polymorphisms, RFLP)

단이가닥 입체다형(Single Strand Conformation Polymorphisms, SSCP)

임의증폭다형 (Random Amplified Polymorphic DNAs, RAPD)

초위성체에 의한 다형관찰법(Simple Sequence Length Polymorphism, SSLP)

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• Molecular differences can be caused by:– INSERTIONS

• Johnny is a boy• Johnny is a bad boy

– DELETIONS• The cow jumped over the moon• The cow over the moon

– MUTATIONS• Bean• Been

• These may cause phenotypic differences

왜 DNA 의 다형현상은 일어나는가 ?

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A type of DNA polymorphism

....CACACACACACA....

chromosome

PCR

No. of CAs varies (alleles)

DNA fragments

Electrophoresis1 2 3 4 5

large

small

T-T-G-A-C-T-A-C-C-C-A-G-A-T-C I I I I I I I I I I I I I I I

A-A-C-T-G-A-T-G-G-G-T-C-T-A-G

T-T-G-A-C-T-A-A-C-C-A-G-A-T-C I I I I I I I I I I I I I I I

A-A-C-T-G-A-T-T-G-G-T-C-T-A-GE. coli isolate A

E. coli isolate B

DNA 의 다형현상

SNP(single nucleotide polymorphism)

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• Restriction Fragment Length Polymorphism– 1980 Botstein et al.

• polymorphisms due to changes in restriction sites or in DNA between sites

제한효소 절편다형(Restriction Fragment Length Polymorphisms, RFLP)

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• Restriction fragment length polymorphism

• Co-dominant• Requires:

– single copy DNA probe– Restriction enzyme– Southern blotting– DNA polymorphism

RFLP

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Polymorphism-Parent 1 : one band-Parent 2 : a smaller band-Offspring 1 : heterozygote

= both bands-Offspring 2 : homozygote

parent 1

Polymorphism

Parent 1 : one band

-Parent 2 : no band

-Offspring 1 : homozygote parent 1

-Offspring 2 : ????

P 2P 1 O 2O 1

Gel configuration

Co-dominant marker

P 2

Gel configurationP 1 O 1 O 2

Dominant marker

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Inheritance of RFLPs자손에게 유전

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RFLPprobe

RFLP linkage analysis• RFLPs provide useful markers for all of the human chromosomes• disease genes can be mapped by searching for linkage between an

RFLP and the disease phenotype

disease geneA

anormal gene

AA Aa aa AA homozygous for ‘A’Aa heterozygousaa homozygous for ‘a’

• the A/a polymorphism is linked to the disease-causing gene

• In a dominant disease, AA and Aa wouldbe affected

• In a recessive disease, AA would beaffected and Aa would be a carrier

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Aa Aa aa aa

disease gene

A

a

normal gene

mo

the

r

aff

ec

ted

ch

ild

tes

t s

ub

jec

t

• the test subject is determined to have thesame genotype as its sibling and thereforecan be predicted to get the disease

• the prediction must be qualified,however, because of thepossibility of recombinationbetween the polymorphicmarker and the disease gene

• the frequency of recombinationand therefore the reliabilityof the marker is dependenton the distance between thetwo sites

Xrecombination?

RFLPprobe

fath

er

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Genotype Determination using RFLP's and a Gene Probe

A DNA probe that hybridizes to the 5' end of the human beta globin gene (shown in blue on the diagram below) was used to identify RFLP pieces from members of a family in which sickle-cell hemoglobin (HbS) was segregating. The normal HB allele (HbA) is cut at three places by a particular restriction enzyme (positions shown with red arrows). The HbS mutation destroys the internal restriction site so the HbS gene is cut in only two places. Thus, the probe hybridizes to a 1.15 kb DNA fragment from HbA DNA and hybridizes to a 1.35 kb fragment from HbS DNA.

http://www.people.virginia.edu/~rjh9u/restrmst.html

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P1

P2

Restriction sites

e.g. EcoR1 recognizes GAATTC and cuts between G and A

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23 KB9.4 KB6.7 KB

4.3 KB

2.3 KB2.0 KB

0.6 KB

DNA taken from three individual animals, cut with a restriction enzyme and separated by gel electrophoresis...

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homozygote

heterozygotes

Autoradiograph of Southern blot produced from previous genomic DNA restiction digest, probed with a muscle actin gene probe...

DNA polymorphism: 3 different “genotypes”

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RFLP• DNA cut with restriction enzyme, separate

d by size on an agarose gel, hybridized to a filter

• To see bands, probe with a labeled fragment of DNA– probes = genomic clones, cDNAs, ESTs– Labeled with 32P or fluorescence

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RFLP techniques

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P1

P2

P1 P2 F1 F2Transfer to a membrane

Probe with DNA fragment

Southern Hybridization

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P1

P2

P1 P2 F1 F2

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P1 P2

자식들부모

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RAPD• Random Amplified Polymorphic DNA

– Williams et al. 1990• Amplify fragments of DNA using a SINGLE,

RANDOM oligonucleotide primer (usually 10mer)

• Run out product on agarose gel, stain with ethidium bromide and visualize using UV

• Polymorphisms due to differences in and between primer annealing sites

임의증폭다형 (Random Amplified Polymorphic DNAs, RAPD)

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RAPD

• Domimant markers• Reproducibility

problems

• Amplifies anonymous stretches of DNA using arbitrary primers

• Fast and easy method for detecting polymorphisms

Random Amplified Polymorphic DNA (RAPD)

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Random Amplified Polymorphic DNA (RAPD)

Strain 1Columbia

Strain 2Landsberg

Strain 1Columbia CCGTACGTAGCAAGT-.....NNNNNNNNN___...ACTTGCGGCGTA

C LCC/L

CC/LL L

PCR products on agarose gel

parental DNA

mapping population

CCGTACGTAGCAAGG-.....NNNNNNNNN___...ACTTGCGGCGTA

GCCGTAGCAAGT

GCCGTAGCAAGT

5’

5’

5’

5’5’

3’

GCCGTAGCAAGT5’

Primer

Primer

Strain 2Landsberg

순수 혹은 잡종분별

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RAPD Polymorphisms among landraces of sorghum

M

Sequences of 10-mer RAPD primers

Name Sequence

OP A08 5’ –GTGACGTAGG- 3’OP A15 5’ –TTCCGAACCC- 3’OP A 17 5’ –GACCGCTTGT- 3’OP A19 5’ –CAAACGTCGG- 3’OP D02 5’ –GGACCCAACC- 3’

RAPD gel configuration

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Microsatellites (SSR)

• polymorphisms in the number of di-, tri-, tetra- or penta-nucleotide repeats

• scattered throughout the genome• Often arise due to ‘stuttering’ during DNA synth

esis or uneven pairing and crossing-over• primers designed based on flanking DNA seque

nces– Requires large amounts of sequencing to develop -

$$$

초위성체에 의한 다형관찰법(Simple Sequence Length Polymorphism, SSLP)

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AGTGCATGAGCGCGCGCGCGCGCGCGTCTCTATGTC

AGTGCATGAGCGCGCGCGCGCGTCTCTATGTC

S RSRR S R S R SR S

Parent 1 - resistant

Parent 2 - susceptible

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Microsatellites (SSR)

• PCR products run out on agarose gels (if no

t too close in size) or more often, on polyacrylamide gels (if only a few bases separate the 2 genotypes

• Visualized by using labeled primers (radioactive or fluorescent) or with ethidium bromide or silver staining

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Sequence

GCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTC

ATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGATTCCGGTCGTCAGAATCAGAATCAGAATCGATATGGTGGCAGTGGTGGTGGTGGTGGTGGTTTTGGTGGTGGTGAATCTAAGGCGGATGGAGTGGATAATTGGGCGGTTGGTAAGAAACCTCTTCCTGTTAG

ATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCTGCTGTGGAACCA…..

Repeat

GGT(5)

SSR repeats and primers

GAGGGCTGATGAGGTGGATA

ATCTTATGGCGGTTCTCGTG

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AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGGP1

AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCGP2

P1 P2

SSR polymorphisms

Gel configuration

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Genographer image of microsatellite sORB30 in a B. napus population

P2P1

bp

207

169

sORB30

Agriculture and

Agri-Food Canada

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Simple Sequence Length Polymorphism (SSLP)

Strain 1Columbia

ACGT GA (GA)44 GA CCTG

ForwardPrimer

ReversePrimer

ACGT GA (GA)78 GA CCTG

Strain 2Landsberg

Strain 1Columbia

Strain 2Landsberg

PCR

ForwardPrimer

ReversePrimer

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범인 1 범인 2 희생자

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엄마 형제자매? 엄마 형제자매?

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ABI 377-96 microsatellite mapping ABI 377-96 microsatellite mapping gelgel

Agriculture and

Agri-Food Canada

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Theory: the conformation of single-stranded DNA is dependent on its primary sequence

Purpose: to electrophoretically separate PCR fragments that differ by a few bases.

• PCR hypervariable fragment of gene.• Denature into single-strands by heating.• Run on a polyacrylamide gel • PCR products with different sequences

will run a different speeds on the gel.


(교재 136-137)

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Single strand conformation polymorphismanalysis (SSCP), takes advantage of the secondarystructure (conformation) of single stranded DNA.

A mutation in a DNA strand may changethe conformation of that strand. The mutant

conformation may electrophorese differently tothe wild type conformation.


44

Denature

Renature rapidly

Wild type Mutant


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Wild type Mutant

Electrophorese

ss DNA

ds DNAWT M


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PCR- SSCP analysis of the second exon of the goat MHC class ll DRB gene

PCR products were denatured at 80°C for 5 min and immediately chilled on ice for 2min. The samples were run for 3.5hrs on 10% polyacrylamide gels in 1% TBE buffer at 100V and the bands were visualized by ethidium bromide under the UV light.

Recip

ient

blo

odDon

or cel

l lin

eNT k

id blo

od (J

insoo

n)

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SNPs (Single Nucleotide Polymorphisms)

•Any two unrelated individuals differ by one base pair every 1,000 or so, referred to as SNPs.•Many SNPs have no effect on cell function and therefore can be used as molecular markers.

Hybridization using fluorescent dyesSNPs on a DNA strand

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SNPs, the most common form of genetic polymorphism causing diversities among different individuals.

SNPs are estimated to occur every 500-1000 bp (3,000,000 to 6,000,000 SNPs)

To facilitate large scale genetic association studies Approximately 1,000,000 human SNP’s currently mapped Useful in pharmacogenomics, advanced disease

screening studies, etc…

SNP (Single Nucleotide Polymorphism)

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SNP Analysis

• SNP Discovery: Direct sequencing and comparative analysis– Quality Values– CEQuence Investigator

• SNP Scoring: Rapid identification of known SNPs by microsequencing or primer extension– CEQ or SNPstream Analysis– Primer Extension chemistry

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DNA polymorphism 분석에 의한 동물육종

개체에 의한 유전자형을 손쉽고 정확하게 파악하여 개체에 대한 육종가 추정

대동물에서 착상전 수정란의 성감별

유전병 등 열성형질을 제거하기 위한 종축집단 구축

육종 프로그램내의 혼선을 방지하기 위한 개체나 집단간의 혈연관계 확인

형질관련 유전자의 분리를 위한 도구로서 유전자 지도 작성시의 이용

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Marker Assisted Selection

• Breeding for specific traits in plants and animals is expensive

and time consuming

• The progeny often need to reach maturity before a determination of the success of the cross can be made

• The greater the complexity of the trait, the more time and effort

needed to achieve a desirable result.

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• The goal to MAS is to reduce the time needed to determine if the progeny have trait

• The second goal is to reduce costs associated with

screening for traits

• If you can detect the distinguishing trait at the DNA level

you can identify positive selection very early.

Marker Assisted Selection(MAS)

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Developing a Marker

• Best marker is DNA sequence responsible for phenotype i.e. gene

• If you know the gene responsible and has been isolated, compare sequence of wild-type and mutant DNA

• Develop specific primers to gene that will distinguish the two forms

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Developing a Marker

• If gene is unknown, screen contrasting populations

• Use populations rather than individuals• Need to “blend” genetic differences

between individual other than trait of interest

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Developing Markers

• Cross individual differing in trait you wish to develop a marker

• Collect progeny and self or polycross the progeny

• Collect and select the F2 generation for the trait you are interested in

• Select 5 - 10 individuals in the F2 showing each trait

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Developing Markers

• Extract DNA from selected F2s• Pool equal amounts of DNA from each

individual into two samples - one for each trait

• Screen pooled or “bulked” DNA with what method of marker method you wish to use

• Method is called “Bulked Segregant Analysis”

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Marker Development

• Other methods to develop population for markers exist but are more expensive and slower to develop

• Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent

• What is the advantage to markers in breeding?

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Capillary Electrophoresis: The Basic Design

1. Dideoxy-terminator labeled

fragments are heated in plate to denature.

2. Current is applied across the capillary, and charged fragments are drawn into capillary.

3. Fragments migrate according to size through capillary.

4. As fragments pass detector window, diode lasers excite dye terminators.

5. Dye terminators fluoresce.

6. Filter wheel masks spurious signal.

7. Filtered signal excites photomultiplier tube (pmt).

8. Software interprets signal and calls bases

33

22

11

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CEQ Applications:1) Fragment Analysis STR/SSR/Microsatellite Analysis Amplified Fragment Length Polymorphism (AFLP) Random Fragment Length Polymorphism (RFLP) Loss of Heterozygosity (LOH) MI (Micro satellite Instability) SNP Genotyping Gene Expression Other sizing, peak ratio/quantitation applications

2) DNA Sequencing Short PCR (75 – 300 bp in 25 minutes) Long Fast (700 – 900 bp in 80 minutes) Extended Read Lengths (increase reads by 20% - over 1000

bp)

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Benefits of Optimization with CE:

• Can modify methods– For high signal = inject less– For low signal = inject more– For short template = decrease separation

time– For longer template = increase separation

time– Re-run samples at many different methods(with limitations!!!!)

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CEQ 8000 Series: CEQ 8000 Series: HardwareHardware

Fully Automated

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Perform sequencing and fragment sizing applications without changing gels,

capillaries, or plates!

CEQTM One Gel, One Array, One Software

Capillary array- patented fixed coating eliminates

electrosmotic flow (EOF)

Gel cartridge- preloaded syringe, linearized polyacrylamide (LPA),

nontoxic

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CEQTM Well-Red Dyes- Designed for CE

h

0

2000

4000

6000

8000

1 10 4

650 700 750 800 850

Wavelength (nm)

ddT ddG ddC ddA

Rela

tive

Fluo

resc

ence

•Minimal spectral overlap superior resolution

• Infrared dyes low intrinsic background with no

interference from biological components

• Similar sizes and mobilities

move optimally in a capillary array

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Heterozygosity: the status that the two copies of a gene or non-gene segment at a locus have different sequences

Loss of Heterozygosity (LOH)Cancer Research

Heterozygous

PCR CEQPCR CEQ

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Abnormalities (deletions) through mutagenesis

Loss of Heterozygosity (LOH) in Cancer

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AFLP (and RFLP) reveal the DNA fragment length polymorphisms due to mutation at restriction sites or

any insertion or deletion between two restriction sites.

Amplified Fragment Length Polymorphisms

Amplified Fragment Length Polymorphisms

Why Use AFLP (or RFLP)? Rapid analysis of unknown genome without

sequence knowledge

Identification of different species

Relationship study

Genome wide genotyping

High discriminatory power and relatively easy to perform.

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AFLP Theory

• AFLP: Amplified Fragment Length

Polymorphism

– Mutations at restriction enzyme cutting sites result in fragment length polymorphism

– Ligation of adapters to genomic restriction fragments

– Selective PCR amplification with adapter-specific primers

• Adapters and amplification w/ selective primers are the keys in AFLP

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AFLP -How It Works:

• Fragment length polymorphism due to:

– Mutation at restriction sites

– Insertions or deletions between restriction sites

– Mutation adjacent to restriction sites and complementary to the selective primer extension

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SNPs, the most common form of genetic polymorphism causing diversities among different individuals.

SNPs are estimated to occur every 500-1000 bp (3,000,000 to 6,000,000 SNPs)

To facilitate large scale genetic association studies Approximately 1,000,000 human SNP’s currently mapped Useful in pharmacogenomics, advanced disease

screening studies, etc…

SNP (Single Nucleotide Polymorphism)

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SNP Analysis

• SNP Discovery: Direct sequencing and comparative analysis– Quality Values– CEQuence Investigator

• SNP Scoring: Rapid identification of known SNPs by microsequencing or primer extension– CEQ or SNPstream Analysis– Primer Extension chemistry

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Life Cycle of a SNP

CEQ 8000 and 8800: 1 – 4,600 SNP’s

SNPstream: 4,600 – 800,000 SNP’s

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Primer Extension Technology:The Genotyping Gold Standard

Detect

functionAnneal:

Simple Primer Extension

Extend:

Accurate “lock and key” enzyme

Detect:

Fluorescence

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Primer Extension Technology

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Multiplexed SNP’s

on the CEQ 8800

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Manage genetic study data including genotypic, clinical,

and phenotypic information and pedigrees.

Create and view customizable, interactive pedigrees.

Explore genotypic and phenotypic patterns with advanced

visualization tools.

Perform population statistical analyses including allele

and genotype frequencies, Hardy-Weinberg equilibrium,

and chi-squared analysis.

1 제 5 장 동물 분자육종을 위한 DNA 표지인자의 이용 전통적인 통계적 보정...

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Transcript of 1 제 5 장 동물 분자육종을 위한 DNA 표지인자의 이용 전통적인 통계적 보정...