Ro_Lecture14

Ro_Lecture14 - RFLP analysis RFLP= Restriction fragment...

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1 RFLP analysis RFLP= Restriction fragment length polymorphism Refers to variation in restriction sites between individuals in a population These are extremely useful and valuable for geneticists (and lawyers) On average two individuals (humans) vary at 1 in 1000 bp The human genome is 3x10 9 bp This 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
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2 RFLP Lets generate a restriction map for a region of human X- chromosome 5kb 3kb The restriction map in the same region of the X chromosome of a second individual may appear as 8kb Normal GAATTC Mutant GAGTTC
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3 RFLP The internal EcoRI site is missing in the second individual For X1 the sequence at this site is GAATTC CTTAAG This is the sequence recognized by EcoRI The equivalent site in the X2 individual is mutated GAGTTC CTCAAG Now 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 region These polymorphisms usually do not have any phenotypic consequences Silent 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 protein Structure/function.
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4 RFLP RFLP are identified by southern blots In the region of the human X chromosome, two forms of the X-chromosome are Segregating in the population. B R R R B R 4 5 3 6 3.5 B R R B R 4 8 6 3.5
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5 RFLP Digesting with BamHI and performing Southern blots with the above probe produces the following results for X1/X1, X1/X2 and X2/X2 individuals: B R R R B R 4 5 3 6 3.5 B R R B R 4 8 6 3.5 X1 X2 There is no variation with respect to the BamHI sites, all individuals produce the same banding patterns on Southern blots If we used probe2 for southern blots with a BamHI digest what would be the Results for X1/X1, X1/X2 and X2/X2 individuals? If we used probe2 for southern blots with a EcoRI digest what would be the results for X1/X1, X1/X2 and X2/X2 individuals?
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6 RFLP RFLP’s are found by trial and error and they require an appropriate probe and enzyme They are very valuable because they can be used just like any other genetic marker to map genes They are employed in recombination analysis (mapping) in the same way as conventional Allelic variants are employed The 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 gene The phenotype in this case is a Southern blot rather than white eye/red eye Lets review standard mapping: To map any two genes with respect to one another, they must be heterozygous at both loci.
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Mapping Gene W and B are responsible for wing and bristle development Centromere Telomere W B To find the map distance between these two genes we need allelic variants at each locus W=wings B=Bristles w= No wings
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Ro_Lecture14 - RFLP analysis RFLP= Restriction fragment...

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