1. Applications of HGP Genetic testing Forensics

2. Testing for a pathogenic mutation in a certain gene in an individual that indicate a persons risk of developing or transmitting a disease PURPOSE Medical management Forensics Research Genetic testing

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

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

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

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

7. SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS RISKY SENSITIVE SPECIFIC PRE REQUISITES Gene loci Size Frequency of known mutations MUTATION SCANNING CFTR mutation frequency F50879.9% G551D2.6 % G542X1.5%

8. METHODS Direct sequencing Southern blots dHPLC Microarrays sequencing MUTATION SCANNING

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

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

11. GENE TRACKING Analysis of linked markers in families for the inheritance of a high risk chromosome from heterozygous parents. 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 Used when map location of disease locus is known but not the actual disease gene

12. POPULATION SCREENING Genetic Screening programs should meet the following criteria 1.The condition to be screened for should be serious. 2.The diagnostic methodology should be accurate and sensitive. 3.The condition must be sufficiently common to make the program economically feasible. 4.The individual identified as at risk must have some options, preferably either effective early treatment or prenatal diagnosis e.g. PKU tests /Guthrie (PAH activity) ARMS test (CFTR mutations)

13. 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

14. Early markers Karl Landsteiners ABO blood typing DNA fingerprinting Originally described by Sir Alec Jeffreys (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

15. 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%) Repetitive sequences… HUMFES/FPS (ATTT) 8-14

16. 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. 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) How does forensic ID work?

20. Oct 2004, Vol 5 pg739

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

22. Some STR electropherograms 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) Electropherogram of a second-generation multiplex SGM Plus profile from a male

23. 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

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

25. Current methods 4) Y-chromosome typing 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. 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 Prosecutors fallacy or fallacy of the transposed conditional The probability of the DNA evidence, if it came from the suspect, is 1 in 50 million Is DNA effective in casework?

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 Gill et al www.els.net