Applications of HGP Genetic testing Forensics Figure 11.3

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 Figure 11.3

Genetic testing can be done either Directly Gene tracking Population screening Figure 11.3

DIRECT GENETIC TESTING Based on either MUTATION DETECTION: screening for KNOWN polymorphisms in DNA MUTATION SCANNING: screening for UNKNOWN polymorphisms in DNA Figure 11.6

SNPs by RFLP-PCR MUTATION DETECTION 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 Figure 11.7 Fig. 11.7 - Hartwell

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

Variation in length of DNA sequence (repetitive DNA) MUTATION DETECTION Variation in length of DNA sequence (repetitive DNA) pathogenic (Huntington’s disease) non pathogenic (forensics) class Size of repeat Repeat block Major chromosomal location minisatellite 9-64 bp 0.1 – 20kb Telomeres microsatellites 1-13 bp < 150 bp Dispersed Figure 11.8

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

SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS MUTATION SCANNING SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS RISKY SENSITIVE SPECIFIC CFTR mutation frequency F508 79.9% G551D 2.6 % G542X 1.5% PRE REQUISITES Gene loci Size Frequency of known mutations Figure 11.10

METHODS MUTATION SCANNING sequencing Direct sequencing Southern blots dHPLC Microarrays etc Figure 11.10

MUTATION SCANNING Using dHPLC Exon 6 of DMD gene normal Figure 11.10 affected Fig18.4: HMG3 by Strachan & Read

Using multiplex ARMS test MUTATION SCANNING Using multiplex ARMS test Screening for 29 mutations of the CFTR gene Figure 11.10 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 Figure 11.10

POPULATION SCREENING Screening programs for well characterised traits must be both SENSITIVE ACCURATE e.g. PKU tests /Guthrie (PAH activity) ARMS test (CFTR mutations) Figure 11.10

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 Figure 11.15

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)

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

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

Simple sequence repeats (SSRs) 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

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’

(A) PATERNITY TEST (B) RAPE CASE Figure 11.15

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 Figure 11.15 Fig. 11.15 - Hartwell

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’

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

Mitochondrial DNA Highest variation in control region (800bp)

Y chromosome Haploid Paternal inheritance Binary polymorphisms

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

Bioarchaeology, Anthropology, Evolution, and Human Migration 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

Microbial Genomics Applications of HGP 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

Risk Assessment Applications of HGP 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