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Transcript
Applications of HGP Genetic testing Forensics Genetic testing • testing for a pathogenic mutation in a certain gene in an individual that indicate a person’s risk of developing or transmitting a disease • Used for mutation screening of disease genes e.g. HD, CFTR, DMD Genetic testing can be done in 3 ways • Directly • Gene tracking • Population screening DIRECT GENETIC TESTING Based on either a) MUTATION DETECTION: screening for KNOWN polymorphisms in DNA b) MUTATION SCANNING: screening for UNKNOWN polymorphisms in DNA MUTATION DETECTION SNPs by RFLP-PCR • Must have sequence on either side of polymorphism – Amplify fragment – Expose to restriction enzyme – Gel electrophoresis • e.g., sickle-cell genotyping with a PCR based protocol Fig. 11.7 - Hartwell MUTATION DETECTION SNPs by ASOs • Very short specific probes (<21 bp) which hybridize to one allele or other • Such probes are allele-specific oligonucleotides (ASOs) Fig. 11.8 MUTATION DETECTION Variation in length of DNA sequence (repetitive DNA) Huntington’s disease -a microsatellite triplet repeat in a coding region Figure 18.12: HMG3 MUTATION SCANNING SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS RISKY SENSITIVE SPECIFIC PRE REQUISITES Gene loci Size Frequency of known mutations CFTR mutation frequency F508 G551D G542X 79.9% 2.6 % 1.5% MUTATION SCANNING METHODS Direct sequencing Southern blots dHPLC Microarrays sequencing MUTATION SCANNING Using dHPLC Exon 6 of DMD gene normal affected Fig18.4: HMG3 by Strachan & Read MUTATION SCANNING Using multiplex ARMS test Screening for 29 mutations of the CFTR gene Fig18.10: HMG3 by Strachan & Read GENE TRACKING Analysis of linked markers in families for the inheritance of a high risk chromosome from heterozygous parents. Used when map location of disease locus is known but not the actual disease gene The process has 3 steps 1) find a closely linked marker for which the parents are heterozygous 2) work out which chromosome carries the disease allele 3) work out which chromosome the individual has inherited POPULATION SCREENING Screening programs for well characterised traits must be both SENSITIVE ACCURATE e.g. PKU tests /Guthrie (PAH activity) ARMS test (CFTR mutations) Forensics Identify crime suspects / exonerate innocent Identify victims Establish family relationships Identify endangered species Detect pollutants Match organ donor with recipient Determine seed / livestock pedigree Authenticate consummables Early markers • Karl Landsteiner’s ABO blood typing DNA fingerprinting Originally described by Sir Alec Jeffereys (1985) (Nature, 1985, 316: 76-79- Jeffereys et al) Discovery of hypervariable loci ‘Differential lysis’ technique in parallel First conviction using DNA fingerprinting was Colin Pitchfork in 1986 Repetitive sequences… Simple sequence repeats (SSRs) Microsatellites 1-13 bp repeats e.g. (A)n (AC)n Minisatellites 14 - 500 bp repeats 3% of genome (dinucleotides - 0.5%) HUMFES/FPS (ATTT)8-14 1985 technique using hybridisation of Multi locus probes (MLP) Minisatellite probes consisting of tandem repeats of the myoglobin locus Number of multiple loci probes (MLP) identified Core sequence GGAGGTGGGCAGGA 2 of these used (33.15 and 33.6) hybridised to Southern Blots of restriction-digested genomic DNA Shared ‘core’ sequences at multiple loci creates hypervariable, multi-band patterns called DNA ‘fingerprints Together, upto 36 independently inherited bands detected 2 probes gave a match probability of <5 x 10-19 …now superceded by PCR-based methods Discovery of STR (short tandem repeats) Use of STR multiplex PCR Autosomal SNP typing, Y-chromosome / mtDNA markers Advantages Increased sensitivity Small sample quantities sufficient Uses microsatellites, instead of minisatellites How does forensic ID work? Extract DNA Analyse specific regions using probes look for matches between 2 samples at many loci (multilocus) Scan ~ 10 DNA regions that show locus variability > 5 matches Create DNA profile (DNA fingerprint) Oct 2004, Vol 5 pg739 Current methods 1) Autosomal STR typing – Needs ~300bp amplicons – SGMPlus database (UK) contains 5 multiplex loci – US FBI CODIS contain 13 STR loci Some STR electropherograms Electropherogram of a second-generation multiplex ‘SGM Plus’ profile from a male Electropherogram profile from a mixture Mixtures can only be identified if the alleles of the minor component are above the background ‘noise’ in an electropherogram (in practice a ratio of ~1:10) Current methods 2) Autosomal SNP typing – – – – Lower heterozygosities compared to STR (0.5) ~ 50 SNPs need to be typed for low Pm Difficult to resolve mixtures ~50bp template sizes enough Current methods 3) Mitochondrial DNA typing • Mutation rate ~1/33 generations • Multicopy • Heteroplasmy (original and mutated • 16.5 kbp forms co-exist) • Maternally inherited • More stable for forensic analysis Highest variation in control region (800bp) Current methods 4) Y-chromosome typing • Haploid • Recombinationdeficient (mutations only) • Paternal inheritance • Binary polymorphisms Is DNA effective in casework? Techniques must be robust and reproducible for sample variability Only if used intelligently!! Only regions showing the most variability can be used Must cover large regions Must be validated Look for matches ‘beyond a reasonable doubt’ Is DNA effective in casework? evidential weight of a match between crime stain profile and suspect is quantified by the match probability (Pm) Strength of evidence based on likelihood ratio (LR) LR = C / C ‘Prosecutor’s fallacy’ or ‘fallacy of the transposed conditional’ ‘The probability of the DNA evidence, if it came from the suspect, is 1 in 50 million’ (A) PATERNITY TEST (B) RAPE CASE • DNA fingerprints can identify individuals and determine parentage • E.g., DNA fingerprints confirmed Dolly the sheep was cloned from an adult udder cell • Donor udder (U), cell culture from udder (C), Dolly’s blood cell DNA (D), and control sheep 1-12 Fig. 11.15 - Hartwell References Hum Mol Gen 3 by Strachan and Read Chapter 18 Hartwell et al – Chapter 11; pages 376-387 DNA profiling in forensics by Peter Gill et al www.els.net