Applications of HGP

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 either • 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. MUTATION DETECTION Variation in length of DNA sequence (repetitive DNA) • pathogenic (Huntington’s disease) • non pathogenic (forensics)

8. Huntington’s disease -a microsatellite triplet repeat in a coding region Figure 18.12: HMG3

9. 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%

10. MUTATION SCANNING METHODS sequencing Direct sequencing Southern blots dHPLC Microarrays etc

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

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

13. 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

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

15. 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

16. 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)

17. DNA fingerprinting • Originally described using minisatellite probes consisting of tandem repeats of the myoglobin locus (Nature, 1985, 316: 76-79- Jeffereys et al) • Number of multiple loci probes (MLP) identified • Core sequence GGAGGTGGGCAGGA • 2 of these used (33.15 and 33.6) • Together, upto 36 independently inherited bands detected

19. DNA fingerprinting • superceded by single locus probes (SLP) – just 2 bands per probe • Now superceded by SL-PCR • Use of allelic ladder markers Advantages • Increased sensitivity • Small sample quantities sufficient • Uses microsatellites instead of minisatellites

20. 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

21. DNA fingerprinting • 1995 – National Criminal Intelligence Database (Forensic science service) • 700,000 samples stored  • Strength of evidence based on likelihood ratio (LR) • LR = C / C PROSECUTOR’S FALLACY ‘The probability of the DNA evidence, if it came from the suspect, is 1 in 50 million’

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

23. 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

24. Is DNA effective in identification? • Only if used intelligently!! • Only regions showing the most variability must be used • Must cover large regions Look for matches ‘beyond a reasonable doubt’

25. Mitochondrial DNA • Multicopy • 16.5 kbp • Maternally inherited • Sequenced in 1981 (Nature,1981, 290:457-65) • Mutation rate ~1/33 generations • Heteroplasmy (original and mutated forms co-exist) • More stable for forensic analysis

26. Mitochondrial DNA • Highest variation in control region (800bp)

27. Y chromosome • Haploid • Paternal inheritance • Binary polymorphisms

28. 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

29. Applications of HGP Bioarchaeology, Anthropology, Evolution, and Human Migration • study evolution through germline mutations in lineages • study migration of different population groups based on female genetic inheritance • study mutations on the Y chromosome to trace lineage and migration of males • compare breakpoints in the evolution of mutations with ages of populations and historical events

30. Applications of HGP Microbial Genomics • new energy sources (biofuels) • environmental monitoring to detect pollutants • protection from biological and chemical warfare • safe, efficient toxic waste cleanup • understanding disease vulnerabilities and revealing drug targets

31. Applications of HGP Risk Assessment • assess health damage and risks caused by radiation exposure, including low-dose exposures • assess health damage and risks caused by exposure to mutagens & carcinogens • reduce the likelihood of heritable mutations