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Applications of HGP

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.

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Applications of HGP

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  1. Applications of HGP Genetic testing Forensics

  2. 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

  3. Genetic testing can be done in 3 ways • Directly • Gene tracking • Population screening

  4. DIRECT GENETIC TESTING Based on either • MUTATION DETECTION: screening for KNOWN polymorphisms in DNA • MUTATION SCANNING: screening for UNKNOWN polymorphisms in DNA

  5. MUTATION DETECTION SNPsby 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

  6. MUTATION DETECTION SNPsby ASOs • Very short specific probes (<21 bp) which hybridize to one allele or other • Such probes are allele-specific oligonucleotides (ASOs) Fig. 11.8

  7. Variation in length of DNA sequence (repetitive DNA) MUTATION DETECTION Huntington’s disease -a microsatellite triplet repeat in a coding region Figure 18.12: HMG3

  8. RISKY SENSITIVE SPECIFIC PRE REQUISITES Gene loci Size Frequency of known mutations MUTATION SCANNING SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS CFTR mutation frequency F508 79.9% G551D 2.6 % G542X 1.5%

  9. MUTATION SCANNING METHODS sequencing • Direct sequencing • Southern blots • dHPLC • Microarrays

  10. MUTATION SCANNING Using dHPLC Exon 6 of DMD gene normal affected Fig18.4: HMG3 by Strachan & Read

  11. MUTATION SCANNING Using multiplex ARMS test Screening for 29 mutations of the CFTR gene Fig18.10: HMG3 by Strachan & Read

  12. 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

  13. POPULATION SCREENING Screening programs for well characterised traits must be both SENSITIVE ACCURATE e.g. PKU tests /Guthrie (PAH activity) ARMS test (CFTR mutations)

  14. 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

  15. Early markers DNA fingerprinting • Karl Landsteiner’s ABO blood typing • 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

  16. 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

  17. 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

  18. …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

  19. 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)

  20. Oct 2004, Vol 5 pg739

  21. Current methods 1) Autosomal STR typing • Needs ~300bp amplicons • SGMPlus database (UK) contains 5 multiplex loci • US FBI CODIS contain 13 STR loci

  22. 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)

  23. 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

  24. 3) Mitochondrial DNA typing Current methods Mutation rate ~1/33 generations Heteroplasmy (original and mutated forms co-exist) More stable for forensic analysis • Multicopy • 16.5 kbp • Maternally inherited Highest variation in control region (800bp)

  25. 4) Y-chromosome typing Current methods • Haploid • Recombination-deficient (mutations only) • Paternal inheritance • Binary polymorphisms

  26. 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’

  27. 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’

  28. (A) PATERNITY TEST (B) RAPE CASE

  29. 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

  30. 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 Gillet al www.els.net

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