Presentation on theme: "제5장 동물 분자육종을 위한 DNA 표지인자의 이용"— Presentation transcript:
1 제5장 동물 분자육종을 위한 DNA 표지인자의 이용 동물육종 방법의 변화전통적인 통계적 보정 방법에 의한 동물육종DNA상의 특성 및 유전자 조작에 의한 동물육종(교재 133 참조)
2 Molecular Methods for Identification of Genotypes Basic Concept DNA의 다형현상The number of nucleic acid or amino acid differences between two organisms is proportional to the time since they diverged from a common ancestor.1 AAGGCTA2 AAGGGTA3 AAGGGTGExample Rate of Evolution = 1bp per 100 yearsMOLECULARDIFFERENCES100years200 yearsTIME
3 P = E+G MARKERS IN BIOLOGY Phenotypic markers = Naked eye markers Flower colors, shape of pods, etc..P = E+G
4 Polymorphisms Differences can be detected: Visually – morphological traitsB. VandenbergP1F1P2
5 Molecular markers Sequencing (SNPs) Microsatellites (SSRs) Multi-locus fingerprintsAFLP (Amplified Fragment Length Polymorphism)Resolution powerRAPD (medium) (random amplified polymorphic DNA)RFLP, Restriction Fragment Length Polymorphisms (high)SSCP, Single Strand Conformation Polymorphisms (very low)
7 Molecular differences can be caused by: 왜 DNA의 다형현상은 일어나는가?Molecular differences can be caused by:INSERTIONSJohnny is a boyJohnny is a bad boyDELETIONSThe cow jumped over the moonThe cow over the moonMUTATIONSBeanBeenThese may cause phenotypic differences
8 DNA의 다형현상 E. coli isolate A E. coli isolate B 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 AE. coli isolate BSNP(single nucleotide polymorphism)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
10 Restriction Fragment Length Polymorphism 제한효소 절편다형(Restriction Fragment Length Polymorphisms, RFLP)Restriction Fragment Length Polymorphism1980 Botstein et al.polymorphisms due to changes in restriction sites or in DNA between sites
11 RFLP Restriction fragment length polymorphism Co-dominant Requires: single copy DNA probeRestriction enzymeSouthern blottingDNA polymorphism
12 Co-dominant marker Dominant marker Polymorphism -Parent 1 : one band -Parent 2 : a smaller band-Offspring 1 : heterozygote = both bands-Offspring 2 : homozygote parent 1P 2P 1O 2O 1Gel configurationDominant markerPolymorphismParent 1 : one band-Parent 2 : no band-Offspring 1 : homozygote parent 1-Offspring 2 : ????P 2Gel configurationP 1O 1O 2
14 RFLP linkage analysis AA Aa aa RFLPs provide useful markers for all of the human chromosomesdisease genes can be mapped by searching for linkage between anRFLP and the disease phenotypeRFLPprobedisease geneAnormal geneathe A/a polymorphism is linked to the disease-causing geneAA Aa aaAA homozygous for ‘A’Aa heterozygousaa homozygous for ‘a’This figure shows the linkage between an RFLP and a disease gene (the disease gene in most cases would be the result of a rare mutation). The larger 'A' fragment in this case is "linked" to the downstream disease gene. It should be emphasized that the RFLP in this case may be at quite a distance from the disease gene and may not have any relationship with the disease gene, except for the fact that it lies on the same chromosome. There may, in fact, be other genes lying between the RFLP and the disease gene (they, too, may not have any relevance to the disease phenotype). Most RFLPs are of this type - they are at a distance. However, it is also possible for RFLPs to lie close to or within disease genes, and they may actually be the result of the disease-causing mutation. In the example shown, Southern blotting followed by hybridization with the RFLP probe found that the 'A' allele is linked to the disease gene and that the 'a' allele is linked to the normal gene. Thus, an 'AA' individual in this family would be homozygous for the disease and an 'aa' individual would be homozygous for the normal state. If the disease was inherited in an autosomal recessive fashion, a heterozygous 'Aa' individual would be a non-affected carrier. If the disease was inherited in an autosomal dominant fashion, the heterozygous individual would be affected by the disease.In a dominant disease, AA and Aa wouldbe affectedIn a recessive disease, AA would beaffected and Aa would be a carrier
15 X affected child test subject mother father Aa Aa aa aa A a RFLP probe normal geneAXrecombination?disease geneathe test subject is determined to have thesame genotype as its sibling and thereforecan be predicted to get the diseasethe prediction must be qualified,however, because of thepossibility of recombinationbetween the polymorphicmarker and the disease genethe frequency of recombinationand therefore the reliabilityof the marker is dependenton the distance between thetwo sitesaffected childtest subjectmotherfatherAa Aa aa aaIn this family, it has been established that allele 'a' is linked to the disease gene. This was confirmed by showing that one of the affected children is homozygous for the 'a' allele. The test subject is then tested and found to also be homozygous for the 'a' allele, predicting that it will also be affected by the disease. This prediction must be qualified, however, because of the possibility of recombination between the RFLP locus and the disease gene locus (shown by the X). The frequency of recombination between loci is generally related to the distance between them. Therefore, the closer the loci are together, the more reliable the prediction.
16 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.
17 Restriction sites e.g. EcoR1 recognizes GAATTC and cuts between G and AP1P2
18 DNA taken from three individual animals, cut with a restriction enzyme and separated by gel electrophoresis...23 KB9.4 KB6.7 KB4.3 KB2.3 KB2.0 KB0.6 KB
19 Autoradiograph of Southern blot produced from previous genomic DNA restiction digest, probed with a muscle actin gene probe...homozygoteheterozygotesDNA polymorphism: 3 different “genotypes”
20 RFLPDNA cut with restriction enzyme, separated by size on an agarose gel, hybridized to a filterTo see bands, probe with a labeled fragment of DNAprobes = genomic clones, cDNAs, ESTsLabeled with 32P or fluorescence
25 RAPD Random Amplified Polymorphic DNA 임의증폭다형(Random Amplified Polymorphic DNAs, RAPD)RAPDRandom Amplified Polymorphic DNAWilliams et al. 1990Amplify fragments of DNA using a SINGLE, RANDOM oligonucleotide primer (usually 10mer)Run out product on agarose gel, stain with ethidium bromide and visualize using UVPolymorphisms due to differences in and between primer annealing sites
26 Random Amplified Polymorphic DNA (RAPD) Amplifies anonymous stretches of DNA using arbitrary primersFast and easy method for detecting polymorphismsDomimant markersReproducibility problems
27 Random Amplified Polymorphic DNA (RAPD) PCR products on agarose gelCC/LCC/LCLLLStrain 1ColumbiaStrain 2LandsbergparentalDNAmapping population순수 혹은 잡종분별5’PrimerStrain 1ColumbiaGCCGTAGCAAGT5’3’CCGTACGTAGCAAGT-.....NNNNNNNNN___...ACTTGCGGCGTAGCCGTAGCAAGT5’Primer5’Strain 2LandsbergGCCGTAGCAAGT5’5’CCGTACGTAGCAAGG-.....NNNNNNNNN___...ACTTGCGGCGTA
28 RAPD Polymorphisms among landraces of sorghum Sequences of 10-merRAPD primersName SequenceOP 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’MRAPD gel configuration
29 Microsatellites (SSR) 초위성체에 의한 다형관찰법(Simple Sequence Length Polymorphism, SSLP)Microsatellites (SSR)polymorphisms in the number of di-, tri-, tetra- or penta-nucleotide repeatsscattered throughout the genomeOften arise due to ‘stuttering’ during DNA synthesis or uneven pairing and crossing-overprimers designed based on flanking DNA sequencesRequires large amounts of sequencing to develop - $$$
30 R S S R AGTGCATGAGCGCGCGCGCGCGTCTCTATGTC Parent 1 - resistantAGTGCATGAGCGCGCGCGCGCGTCTCTATGTCParent 2 - susceptibleAGTGCATGAGCGCGCGCGCGCGCGCGTCTCTATGTCRSSR
31 Microsatellites (SSR) PCR products run out on agarose gels (if not too close in size) or more often, on polyacrylamide gels (if only a few bases separate the 2 genotypesVisualized by using labeled primers (radioactive or fluorescent) or with ethidium bromide or silver staining
32 SSR repeats and primers GGT(5)SequenceGCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTCATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGATTCCGGTCGTCAGAATCAGAATCAGAATCGATATGGTGGCAGTGGTGGTGGTGGTGGTGGTTTTGGTGGTGGTGAATCTAAGGCGGATGGAGTGGATAATTGGGCGGTTGGTAAGAAACCTCTTCCTGTTAGATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCTGCTGTGGAACCA…..GAGGGCTGATGAGGTGGATAATCTTATGGCGGTTCTCGTG
33 SSR polymorphisms AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG P1 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCGP2P1P2Gel configuration
34 Genographer image of microsatellite sORB30 in a B. napus population bp207169sORB30P1P2Agriculture andAgri-Food Canada
35 Simple Sequence Length Polymorphism (SSLP) ReversePrimerReversePrimerForwardPrimerForwardPrimerACGT GA (GA)44 GA CCTGACGT GA (GA)78 GA CCTGStrain 1ColumbiaStrain 2LandsbergPCRStrain 1ColumbiaStrain 2Landsberg
42 PCR hypervariable fragment of gene. 단이가닥 입체다형(Single Strand Conformation Polymorphisms, SSCP)(교재 )Theory: the conformation of single-stranded DNA is dependent on its primary sequencePurpose: 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 gelPCR products with different sequences will run a different speeds on the gel.
43 (Single Strand Conformation Polymorphisms, SSCP) 단이가닥 입체다형(Single Strand Conformation Polymorphisms, SSCP)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 mutantconformation may electrophorese differently tothe wild type conformation.
46 NT kid blood (Jinsoon)Recipient bloodDonor cell linePCR- SSCP analysis of the second exon of the goat MHC class ll DRB genePCR 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.
47 SNPs (Single Nucleotide Polymorphisms) Hybridization using fluorescent dyesSNPs on a DNA strandAny two unrelated individuals differ by one base pair every1,000 or so, referred to as SNPs.Many SNPs have no effect on cell function and thereforecan be used as molecular markers.
48 (Single Nucleotide Polymorphism) SNP(Single Nucleotide Polymorphism)SNPs, the most common form of genetic polymorphism causing diversities among different individuals.SNPs are estimated to occur every bp (3,000,000 to 6,000,000 SNPs)To facilitate large scale genetic association studiesApproximately 1,000,000 human SNP’s currently mappedUseful in pharmacogenomics, advanced disease screening studies, etc…
49 SNP Analysis SNP Discovery: Direct sequencing and comparative analysis Quality ValuesCEQuence InvestigatorSNP Scoring: Rapid identification of known SNPs by microsequencing or primer extensionCEQ or SNPstream AnalysisPrimer Extension chemistry
50 DNA polymorphism 분석에 의한 동물육종 개체에 의한 유전자형을 손쉽고 정확하게 파악하여 개체에 대한 육종가 추정대동물에서 착상전 수정란의 성감별유전병 등 열성형질을 제거하기 위한 종축집단 구축육종 프로그램내의 혼선을 방지하기 위한 개체나 집단간의 혈연관계 확인형질관련 유전자의 분리를 위한 도구로서 유전자 지도 작성시의 이용
51 Marker Assisted Selection Breeding for specific traits in plants and animals is expensiveand time consumingThe progeny often need to reach maturity before a determination of the success of the cross can be madeThe greater the complexity of the trait, the more time and effortneeded to achieve a desirable result.
52 Marker Assisted Selection(MAS) The goal to MAS is to reduce the time needed to determine if the progeny have traitThe second goal is to reduce costs associated withscreening for traitsIf you can detect the distinguishing trait at the DNA levelyou can identify positive selection very early.
53 Developing a MarkerBest marker is DNA sequence responsible for phenotype i.e. geneIf you know the gene responsible and has been isolated, compare sequence of wild-type and mutant DNADevelop specific primers to gene that will distinguish the two forms
54 Developing a Marker If gene is unknown, screen contrasting populations Use populations rather than individualsNeed to “blend” genetic differences between individual other than trait of interest
55 Developing MarkersCross individual differing in trait you wish to develop a markerCollect progeny and self or polycross the progenyCollect and select the F2 generation for the trait you are interested inSelect individuals in the F2 showing each trait
56 Developing Markers Extract DNA from selected F2s Pool equal amounts of DNA from each individual into two samples - one for each traitScreen pooled or “bulked” DNA with what method of marker method you wish to useMethod is called “Bulked Segregant Analysis”
57 Marker DevelopmentOther methods to develop population for markers exist but are more expensive and slower to developNear Isogenic Lines, Recombinant Inbreeds, Single Seed DecentWhat is the advantage to markers in breeding?
59 Capillary Electrophoresis: The Basic Design Dideoxy-terminator labeledfragments are heated in plate to denature.Current is applied across the capillary, and charged fragments are drawn into capillary.Fragments migrate according to size through capillary.As fragments pass detector window, diode lasers excite dye terminators.Dye terminators fluoresce.Filter wheel masks spurious signal.Filtered signal excites photomultiplier tube (pmt).Software interprets signal and calls bases321
60 CEQ Applications: Fragment Analysis 2) DNA Sequencing STR/SSR/Microsatellite AnalysisAmplified Fragment Length Polymorphism (AFLP)Random Fragment Length Polymorphism (RFLP)Loss of Heterozygosity (LOH)MI (Micro satellite Instability)SNP GenotypingGene ExpressionOther sizing, peak ratio/quantitation applications2) DNA SequencingShort 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)
61 Benefits of Optimization with CE: Can modify methodsFor high signal = inject lessFor low signal = inject moreFor short template = decrease separation timeFor longer template = increase separation timeRe-run samples at many different methods(with limitations!!!!)
63 CEQTM One Gel, One Array, One Software Perform sequencing and fragment sizing applications without changing gels, capillaries, or plates!Gel cartridge- preloaded syringe, linearized polyacrylamide (LPA), nontoxicCapillary array- patented fixed coating eliminates electrosmotic flow (EOF)
64 CEQTM Well-Red Dyes- Designed for CE Minimal spectral overlapsuperior resolutionInfrared dyeslow intrinsic background with no interference from biological componentsSimilar sizes and mobilitiesmove optimally in a capillary arrayMinimal Spectral overlap means greater resolution. Think of it like swimming in murky waters. The more dirt the more interference in your eyes, the harder it is to navigate where you are going. This is essentially what the spectral overlap represents. The less overlap the better the resolution the better the data analysis.Also, Well-Red dyes read in the near infra red region. This eliminates the large biological interference that naturally occurs using he dyes red in the Blue/green region…like Big Dye chemistry. All naturally occurring organisms are red in that region meaning there is far more intrinsic background. This cuts down on resolution and limits the ability of the instrument. Because the Well-Red dye have such low background, they can detect things like heterzygocity which require much greater sensitivity and resolution.
69 Amplified Fragment Length Polymorphisms AFLP (and RFLP) reveal the DNA fragment length polymorphisms due to mutation at restriction sites or any insertion or deletion between two restriction sites.Why Use AFLP (or RFLP)?Rapid analysis of unknown genome without sequence knowledgeIdentification of different speciesRelationship studyGenome wide genotypingHigh discriminatory power and relatively easy to perform.
70 AFLP Theory AFLP: Amplified Fragment Length Polymorphism Mutations at restriction enzyme cutting sites result in fragment length polymorphismLigation of adapters to genomic restriction fragmentsSelective PCR amplification with adapter-specific primersAdapters and amplification w/ selective primers are the keys in AFLP
71 AFLP -How It Works: Fragment length polymorphism due to: Mutation at restriction sitesInsertions or deletions between restriction sitesMutation adjacent to restriction sites and complementary to the selective primer extension
73 (Single Nucleotide Polymorphism) SNP(Single Nucleotide Polymorphism)SNPs, the most common form of genetic polymorphism causing diversities among different individuals.SNPs are estimated to occur every bp (3,000,000 to 6,000,000 SNPs)To facilitate large scale genetic association studiesApproximately 1,000,000 human SNP’s currently mappedUseful in pharmacogenomics, advanced disease screening studies, etc…
74 SNP Analysis SNP Discovery: Direct sequencing and comparative analysis Quality ValuesCEQuence InvestigatorSNP Scoring: Rapid identification of known SNPs by microsequencing or primer extensionCEQ or SNPstream AnalysisPrimer Extension chemistry
75 Life Cycle of a SNP CEQ 8000 and 8800: 1 – 4,600 SNP’s SNPstream: 4,600 – 800,000 SNP’s
76 Primer Extension Technology: The Genotyping Gold StandardfunctionDetectPurpose: Reinforce simplicity of the fundamental chemistry and set stage for tag-array description – SIMPLE AND HIGHLY ACCURATE.First, the primer complimentary to the target sequence adjacent to the SNP site, hybridizes to the pre-amplified DNA. This is the first level of specificity inherent in the reaction and enhances the previous specificity from the PCR amplification primers.The extension primer is next extended enzymatically with a labeled terminating nucleotide at the target SNP site. The terminating nature of the nucleotides limits extension to only one base. The combination of the fidelity of the polymerase and its sensitivity to 3’ mismatches produces the second level of SNP-IT specificity and ensures a highly accurate genotyping result.The labeled nucleotide on the extended SNP-IT primer is then detected. The readout method is dependent only on the type of labeled terminator used – in SNP Stream it is fluorescent with a ccd camera. Detect:FluorescenceAnneal:Simple Primer ExtensionExtend:Accurate “lock and key” enzyme
79 Visualize Manage genetic study data including genotypic, clinical, and phenotypic information and pedigrees.Create and view customizable, interactive pedigrees.Explore genotypic and phenotypic patterns with advancedvisualization tools.Perform population statistical analyses including alleleand genotype frequencies, Hardy-Weinberg equilibrium,and chi-squared analysis.