Presented by Amanda Walker, Crystal Snow, Erin Andersen: February 16, 2010

Transcription

1 Alec J. Jeffreys, Victoria Wilson: Department of Genetics, University of Leicester. Swee Lay Thein: Molecular Haematology Unit, Nuffield Dept. of Clinical Medicine at John Radcliffe Hospital Nature: Volume 314, March 1985 Sir Alec Jeffreys Presented by Amanda Walker, Crystal Snow, Erin Andersen: February 16, : Born at Oxford, England. Jeffreys enjoyed biology and biochemistry leading him to study biochemistry. He applied new techniques to the study of genetics. 1972: Graduated Merton College : European Molecular Biology Organization Research Fellow and University of Amsterdam Subsequent to that he went to the Department of Genetics at University of Leicester. Victoria Wilson: Frequent collaborator with Jeffreys A glossary of useful terms. Introduction DNA polymorphisms have modernized genetic analysis, revolutionzed genetic study from an ecological perspective. Important use in: Antenatal diagnosis, mapping human linkage groups, indirect localization of mitotic non-disjunction and recombination in inherited cancer. Single-copy human DNA probes were used to detect Restriction Fragment Length Polymorphisms (RFLPs). Treat sample with a restriction endonuclease recognizing specific short sequences. Resulting fragments are separated electrophoretically, mobility correlates to size. Southern blot procedure to transfer to a membrane. Hybridization with labeled ssdna probe determines length of sequences complementary to the probe. RFLP: Detected when on basis of variation in fragment length. Each fragment length is an allele --> potential for genetic analysis. Mutations affecting a single base-pair (SNPs) often create or destroy specific restriction endonuclease cleavage site. As a tool for genetic analysis RFLPs are limited, primarily by their dimorphism: A site either exists or does not. Consequently heterozygosity as a maximum can never exceed 50%, often falls far below that. The presence of such polymorphisms are too low for these enzymes to accurately detect hypervariability. Genetic analysis could be enhanced and simplfied by probes for hypervariable segments of the human genome, showing multiallelic variation and correspondingly high heterozygosity, regions that would come to be termed minisatellites. In these regions, allelic differences in the number of units of tandem repeats of a short sequence give rise to polymorphism. Unequal division or replication errors generate the repeats. The length of the region can be detected by restriction endonucleases which do not cleave the unit (so a probe can be used), providing an inherited genetic marker. First such region isolated by Wyman and White. Structural basis of the allelic variation was at the time unknown. Other highly variable regions found by Jeffreys et al near: human insulin gene, alpha-related globin genes A short minisatellite in the human myoglobin gene found, related to the others via transposition. Hypothesize that a probe based on the 33-bp repeat may be able to detect other minisatellites related by transposition. Probe for variable human DNA

2 Pure repeat probe human myoglobin minisatellite single 33-bp element puc 13 pav33.7 cleaved with BamHI, EcoRI 767-bp DNA insert comprised almost entirely of 23 repeats of 33-bp sequence Hybridization repeated with human DNA digested with HinfI and HaeIII Multiple fragments were detected. Polymorphism variation corresponds to minisatellite length. Isolation of minisatellites Human genomic library of kb Sau 3A partials. 40 hybridizing plaques from 3 x 10^5 recombinants. Random selection of 8: Purified --> Southern Blot Analysis --> Hybridization to one area of the phage genome. Presence of a minisatellite in each with clone-specific flanking DNA. Lambda 33.6 = 37-bp repeat composed of 12bp trimer (Significant when trying to estimate number of repeat units in an allele). Highly polymorphic minisatellites P-32 labelled ssdna probes from M13 (phage) subclones of each purified minisatellite. Hybridization of the single-copy probes hybridized to 6 individuals at random and at high stringency to panel of DNA from 14

3 British caucasian HinfI DNA digests. Are any of the regions detected in the 8 phage clones polymorphic? Fragment length variation arises due to differences in repeat copy number. There is considerable variation in the level of repeat sequence homogeneity. The most polymorphic minisatellites exhibit the least divergence. Unequal exchange generates polymorphism in repeat number while diffusing sequence variants across mulitple repeat units. Estimating the Rate of Unequal Exchange to Maintain the Observed Number of Different Neutral Alleles na = number of neutral alleles in the sample population Ne = effective population size beginning with a monomorphic minisatellite of 30 repeats na determined by theta theta = 4 Ne u A Chi (X) Sequence in minisatellites? The question of how these dispersed, repeating regions throughout the genome relate arises. Initial theory: A common ancestral sequence gave rise to them by transposition. The repeat in myoglobin has 5'/3' terminal direct repeats. The cloned minisatellites exhibit vary in length and consensus sequnece. Dot-Matrix Comparison of Con. Sequences/Myoglobin 33-bp repeat probe reveals a unique bp core. Nearly invariant sequence of GGGCAGGAXG. Implications of a Common Core x: A generalized recombination signal in E. coli x and the common core: comparable in length/g-content. Current (1985) Models of x Function in Bacteria recbc encodes endonuclease V, an enzyme active in bacterial recombination. Endo. V unwinds and nicks, generating a ssdna projection needed for a Holliday junction.

4 (2250: Linkage Recombination and Mapping) DNA repair from the nick + ligation to ssdna projection = short tandem duplications. All with x sequence to generate unequal exchange --> Amplify into a minisatellite. Similarity of sequences in the human genome: Potential hotspots for recombination. Possible Model at Minisatellites: x-like region, binds recbc Nicking --> ss projection Assimilate into homologous duplex, Holliday junction precursor Or DNA repair synthesis Ligation--> segregation tandem duplicate with the chi sequence. Pedigree analysis Analysis of an Asian-Indian pedigree of Gujerati origin. 54 individuals over 4 generations Southern blot/ gel electrophoresis analysis confirms polymorphic variation is sufficient to distinguish a single sibship of a firstcousin marriage. Probe for hypervariable regions Repeat length half, the same, or double the length of the 33-bp probe from the human myoglobin gene. presence of core sequence in each repeat In-phase alignment of cross-hybridizable core sequence in each heteroduplex. If so, a probe of only tandem-repeated core sequence will detect. Human DNA fragments detected by pav33.7. Additional minisatellites, incapable of forming stable heteroduplexes. Minisatellite from lambda 33.15, 29 near-identical repeats of near-perfect 16-bp sequence, as probe for additional minisatellites. The probe detects a complex profile for hybridizing fragments in human DNA digested with HinfI Only the largest of these fragments resolve fully. Polymorphism is to the extent that hybridization profiles generate a unique, specific DNA 'fingerprint' A New Mutant Allele

5 Detection of extreme variability of the HinfI DNA fragments suggests mutation rate is very high AND can be directly measured. In hypervariable fragments, u= individuals surveyed: 240 clearly resolved offspring bands linked to one parent or the other except for one in individual 17. This gives a rate of which is relatively close to the mutation rate of the population --> Mutation rate is high and measureable. Conclusions Can design probes for hypervariable regions from human DNA The core may promote minisatellite formation. DNA fingerprints: Study marker loss in tumors: Detect inbreeding where offspring affliction may be due to homozygosity for autosomal recessive trait. Particular version of core in myoglobin gene used in initial hybridization impacts on the polymorphic loci detected. Detecting new length-related alleles of minisatellites may help analysis of rate/mechanisms of unequal exchange. Applications 1985: The first practical application of this technology was in resolution of an immigration disupute. 1986: First forensic application, in the solving of a rape/murder case at Enderby, England. DNA fingerprinting has subsequently come into use to both free and imprison suspects. Technological improvements like DNA amplification have brought worldwide acceptance in courts. DNA databases such as CODIS Other important applications of the method: Testing for bone marrow implants. Studying breeding in endangered species/aiding in breeding programs in zoos. Studying ancient man/the history of modern man. The technology was used to confirm the death of Nazi war criminal Josef Mengele. His alleged death in 1979 was verified in 1985 and the file on him was closed. An interview with Sir Jeffreys