Presentation on theme: "RFLP analysis RFLP= Restriction fragment length polymorphism"— Presentation transcript:
1 RFLP analysis RFLP= Restriction fragment length polymorphism Refers to variation in restriction sites between individuals in a populationThese are extremely useful and valuable for geneticists (and lawyers)On average two individuals (humans) vary at 1 in 1000 bpThe human genome is 3x109 bpThis means that they will differ in more than 3 million bp.By chance these changes will create or destroy the recognition sites for Restriction enzymes
2 RFLPLets generate a restriction map for a region of human X-chromosome5kb3kbThe restriction map in the same region of the X chromosome of a second individual may appear as8kbNormal GAATTCMutant GAGTTC
3 RFLP The internal EcoRI site is missing in the second individual For X1 the sequence at this site is GAATTCCTTAAGThis is the sequence recognized by EcoRIThe equivalent site in the X2 individual is mutatedGAGTTCCTCAAGNow if we examine a large number of humans at this site we may find that 25% possess the EcoRI site and 75% lack this site.We can say that a restriction fragment length polymorphism exits in this regionThese polymorphisms usually do not have any phenotypic consequencesSilent mutations that do not alter the protein sequence because of redundancy in Codon usage, localization to introns or non-genic regions or do not affect proteinStructure/function.
4 RFLP RFLP are identified by southern blots In the region of the human X chromosome, two forms of theX-chromosome are Segregating in the population.X1BRRRBR45363.521Digest DNA withEcoRI and probe withprobe1What do we get?X2BRRBR4863.521
5 RFLPDigesting with BamHI and performing Southern blots with the above probe produces the following results:X1BR45363.5X2BR4863.521There is no variation with respect to the BamHI sites, allindividuals produce the same banding patterns on Southern blots
6 RFLP in individualsIf we used probe1 for southern blots with a BamHI digest what would be the Results for X1/X1, X1/X2 and X2/X2 individuals?18If we used probe1 for southern blots with a EcoRI digest what would be the results for X1/X1, X1/X2 and X2/X2 individuals?5 & 38, 5 & 38
7 RFLP RFLP’s are found by trial and error and they require an Appropriate probe AND appropriate enzymeThey are very valuable because they can be used just like any other genetic marker to map genesThey are employed in recombination analysis (mapping) in the same way as conventional morphological allele markers are employedThe presence of a specific restriction site at a specific locus on one chromosome and its absence at a specific locus on another chromosome can be viewed as two allelic forms of a geneThe phenotype in this case is a Southern blot rather than white eye/red eye
8 Using RFLPs to map human disease genes Which RFLP pattern segregates with the diseased individualsTop or bottomUsing DNA probes for different RFLPs you screen individuals for a RFLP pattern that shows co-inheritance with the diseaseConclusion: the actual mutation resides at or near the RFLP
9 Mapping Lets review standard mapping: To map any two genes with respect to one another, they must be heterozygous at both loci.Gene W and B are responsible for wing and bristle developmentWBCentromereTelomereTo find the map distance between these two genes we need allelicvariants at each locusW=wings B=Bristlesw= No wings b= no bristlesTo measure genetic distance between these two genes, the double heterozygote is crossed to the double homozygote
11 B b Mapping Both the normal and mutant alleles of gene B (B and b) are sequenced and we findWBCentromereTelomereB23Eb5GAATTCAAATTCBy chance, this mutation disrupts the amino acid sequence andalso a EcoRI site!If DNA is isolated from B/B, B/b and b/b individuals, cutwith EcoRI and probed in A Southern blot, the pattern that wewill obtain will beB/B BristleB/b Bristleb/b No bristle
12 Mapping Therefore in the previous cross (WB/wb x wb/wb), the genotype at the B locus can be distinguished either by the presence andabsence of bristles or Southern blotsWB/wb x wb/wbFemale MaleWings No wingsBristles No BristlesSouthern blot: Southern blot:5 and 2 kb band 5 kb bandThere are some phenotypes for specific genes that are verypainful to measureHaving a RFLP makes the problem easierJust like Genes, RFLPs mark specific positions on chromosomes and can be for mapping.
14 MappingThe same southern blot method can be employed for the (W) wingLocus with a different restriction enzyme (BamHI) if anRFLP exists at this locus !!You make the DNA, digest half with EcoRI and probe with bristleprobeDigest the other half with BamHI and probe with the wing probe.W8GTATCCBBw44GGATCCBBB
15 Mapping To find the map distance between genes, multiple alleles are required.We can determine the distance between W and B by the classicalMethod because multiple alleles exist at each locus (W & w, B & b)You find a new gene C. There are no variants of this gene thatalter the phenotype of the fly, that you can observe. Say we don’teven know the function of this gene. You can’t even predict itsphenotype.However the researcher identified an RFLP variant in this gene.CentromereTelomereWBCR
16 MappingCc8E26With this RFLP, the C gene can be mapped with respect toother genes:Genotype/phenotype relationships for the W and C genesWW and Ww = Red eyesww = white eyesCC = 8kb bandC/c = 8, 6, 2 kb bandscc = 6, 2 kb bandsTo determine map distance between R and C, the following crossis performedW C w cw c w c
17 Mapping W B C R w c(6,2) W C(8) w c(6,2) w c(6,2) Male gamete (wc) Female gamete
18 MappingPrior to RFLP analysis, only a few classical markers existed in humansNow over 7000 RFLPs have been mapped in the human genome.Newly inherited disorders are now mapped by determining whether they are linked to previously identified RFLPs
19 Genetic polymorphismGenetic Polymorphism: A difference in DNA sequence amongindividuals, groups, or populations.Genetic Mutation: A change in the nucleotide sequence of aDNA molecule.Genetic mutations are a subset of genetic polymorphism.Genetic VariationSingle nucleotidePolymorphism(point mutation)Repeat heterogeneity
20 SNP A Single Nucleotide Polymorphism is a source variance in a genome. A SNP ("snip") is a single base change in DNA.SNPs are the most simple form and most common source ofgenetic polymorphism in the human genome (90% of all human DNA polymorphisms).There are two types of nucleotide base substitutions resulting in SNPs:Transition: substitution between purines (A, G) or between pyrimidines (C, T). Constitute two thirds of all SNPs.Transversion: substitution between a purine and a pyrimidine.While a single base can change to all of the other three bases, most SNPs have only one allele.
21 SNPs- Single Nucleotide Polymorphisms ACGGCTAAATGGCTAAInstead of using restriction enzymes, these are found by directsequencingThey are extremely useful for mappingMarkersClassical Mendelian ~200RFLPsSNPs 1.4x106SNPs occur every bp along the 3 billion long human genomeMany SNPs have no effect on cell function
22 SNPs Humans are genetically >99 per cent identical: it is the tiny percentage that is differentMuch of our genetic variation is caused by single-nucleotidedifferences in our DNA : these are called single nucleotidepolymorphisms, or SNPs.As a result, each of us has a unique genotype that typically differs in about three million nucleotides from every other person.SNPs occur about once every base pairs in the genome, and the frequency of a particular polymorphism tends to remain stable in the population.Because only about 3 to 5 percent of a person's DNA sequence codes for the production of proteins, most SNPs are found outside of "coding sequences".
23 How did SNPs arise? F2a----ACGGACTGAC----CCTTACGTTG----TACTACGCAT---- |F1 ----ACTGACTGAC----CCTTACGTTG----TACTACGCAT----P ----ACTGACTGAC----CCTTACGTTG----TACTACGCAT----F1 ----ACTGACTGAC----CCTTACGTTG----TACTAGGCAT----| |F2b----ACTGACTGAC----CCATACGTTG----TACTAGGCAT----Compare the two F2 progenyHaplotype1 (F2a) = SNP allele1----ACGGACTGAC----CCTTACGTTG----TACTACGCAT----Haplotype2 (F2b) = SNP allele2----ACTGACTGAC----CCATACGTTG----TACTAGGCAT----
24 SNPs, RFLPs, point mutations GAATTCGAATTCGAATTCGAATTCGAATTCGAATTCGAGTTCGAATTCGAATTCGACTTCRFLPSNPRFLPPt mutSNPPt mutSNPSNP
25 Coding Region SNPsTypes of coding region SNPsSynonymous: the substitution causes no amino acid change tothe protein it produces. This is also called a silent mutation.Non-Synonymous: the substitution results in an alteration ofthe encoded amino acid. A missense mutation changes theprotein by causing a change of codon. A nonsense mutationresults in a misplaced termination.One half of all coding sequence SNPs result innon-synonymous codon changes.
26 Intergenic SNPsResearchers have found that most SNPs are not responsible for a disease state because they are intergenic SNPsInstead, they serve as biological markers for pinpointing a disease on the human genome map, because they are usually located near a gene found to be associated with a certain disease.Scientists have long known that diseases caused by single genes and inherited according to the laws of Mendel are actually rare.Most common diseases, like diabetes, are caused by multiple genes. Finding all of these genes is a difficult task.Recently, there has been focus on the idea that all of the genes involved can be traced by using SNPs.By comparing the SNP patterns in affected and non-affected individuals—patients with diabetes and healthy controls, for example—scientists can catalog the specific DNA variations that underlie susceptibility for diabetes
27 PCRIf a region of DNA has already been cloned and sequenced, thesequence information can be used to isolate and amplify that sequence from other individuals in a population.Individuals with mutations in p53 are at risk for colon cancerTo determine if an individual had such a mutation, prior to PCR one would have to clone the gene from the individual of interest (construct a genomic library, screen the library, isolate the clone and sequence the gene).With PCR, the gene can be isolated directly from DNA isolated from that individual.No lengthy cloning procedureOnly small amounts of genomic DNA required30 rounds of amplification can give you >109 copies of a gene
28 PCR and RFLPWT CCTGAGGAGGGACTCCTCMSTIIMut CCTGTGGAGGGACACCTCPCR amplify DNA from normal and sickle cell patientDigest with MstIIWTMut500400300200100
29 Genotype and Haplotype In the most basic sense, a haplotype is a “haploid genotype”.Haplotype: particular pattern of sequential SNPs (or alleles) found on a single chromosome in a single individual.The DNA sequence of any two people is 99 percent identical.Sets of nearby SNPs on the same chromosome are inherited in blocks.Blocks may contain a large number of SNPs, but a few SNPs areenough to uniquely identify the haplotypes in a block.The HapMap is a map of these specific SNPs that identify the haplotypes are called tag SNPs.This will make genome scan approaches to finding regions with genes that affect diseases much more efficient and comprehensive.Haplotyping: involves grouping individuals by haplotypes, or particular patterns of sequential SNPs, on a single chromosome.There are thought to be a small number of haplotype patterns for each chromosome.Microarrays, PCR and sequencing are used to accomplish haplotyping.
30 SNP mapping is used to narrow down the known physical location of mutations to a single gene. The human genome sequence provided us with the list ofmany of the parts to make a human.The HapMap provides us with indicators which we can focus on in looking for genes involved in common disease.By using HapMap data to compare the SNP patterns of peopleaffected by a disease with those of unaffected people, researcherscan survey the whole genome and identify genetic contributions tocommon diseases more efficiently than has been possible withoutthis genome-wide map of variation: the HapMap Project hassimplified the search for gene variants.Oligonucleotide chips contain thousands of short DNA sequences immobilised at different positions. Such chips can be used to discriminate between alternative bases at the site of a SNP.Chips allow many SNPs to be analyzed in parallel.Short DNA sequences on the chip represent all possible variations at a polymorphic site;A labeled DNA will only stick if there is an exact match. The base is identified by the location of the fluorescent signal.
32 Mapping recessive disease genes with DNA markers DNA markers are mapped evenly across the genome. The markers are polymorphic- they look slightly different in different individuals.We can tell looking at a particular individual which grandparent contributed a certain part of its DNA.If we knew that grandparent carried the disease, we could say that part of the DNA might be responsible for the disease.1234567894 different alleles at each locusPosition1 can be A or C or G or TPosition2 can be A or C or G or TPosition3 ………………..Grandparent1234ChromosomeA-A-A-A-A-A-A-A-AC-C-C-C-C-C-C-C-CG-G-G-G-G-G-G-G-GT-T-T-T-T-T-T-T-T
34 Haplotyping with microarrays AlleleAAlleleBSNPSNPDesign 20mer oligonucleotide probes complementary to thePolymorphismsThe probes are arrayed on a slideEach spot corresponds to a polymorphismIsolate DNALabel DNA and hybridize to arrayLabeled Chromosomal 20mer probeHybridizationsignalNo signalThere are ~3 thousand different probes per microarray
35 Genetic polymorphismGenetic Polymorphism: A difference in DNA sequence amongindividuals, groups, or populations.Genetic mutations are a kind of genetic polymorphism.Genetic VariationSingle nucleotidePolymorphism(point mutation)Repeat heterogeneity
36 RepeatsVariation between people- small DNA change – a single nucleotide polymorphism [SNP] – in a target site,RFLPs and point mutations are proof of variation at the DNA level.Satellite sequences: a short sequence of DNA repeated many times.Chr1InterspersedChr2tandem
37 Mini Satellite Repeats and Blots Mini Satellite sequences: a short sequence (20-100bp long) of DNA repeated many times (alleles vary in length from 0.5 to 20 kb)EEEE256Chr1Chr2EEEE3140.5tandem531Repeat probe
38 Repeat expansionTandem repeats expand and contract during recombination.Mistakes in pairing leads to changes in tandem repeat numbersThese can be detected by Southern blottingIndividual 1EE2Individual 2EE3Ind1Ind25There are on average between 2 and 10 alleles (repeats) per mini-sat locus31
40 DNA finger printingVariation between people- small DNA change – a single nucleotidepolymorphism [SNP] – in a target site,RFLPs and SNPs are proof of variation at the DNA level,Satellite sequences: a short sequence of DNA repeated many times.Micro satellite are 2-4 bp repeats in tandem repeats times in a rowMini satellite are bp repeats in tandem (0.5 to 20kb long)Class size No of loci methodSNP 1 bp 100 million PCR/microarrayMicro ~200bp 200,000 PCRMini kb 30,000 southern blot