Applications of HGPGenetic testing Forensics

testing for a pathogenic mutation in a certain gene in an individual that indicate a persons risk of developing or transmitting a disease Used for mutation screening of disease genes e.g. HD, CFTR, DMD

Genetic testing

Directly Gene trackingPopulation screening

Genetic testing can be done either

DIRECT GENETIC TESTINGBased on either MUTATION DETECTION: screening for KNOWN polymorphisms in DNA

MUTATION SCANNING: screening for UNKNOWN polymorphisms in DNA

SNPs by RFLP-PCRMust have sequence on either side of polymorphismAmplify fragmentExpose to restriction enzymeGel electrophoresise.g., sickle-cell genotyping with a PCR based protocolFig. 11.7 - HartwellMUTATION DETECTION

SNPs by ASOsVery short specific probes (

Variation in length of DNA sequence (repetitive DNA)pathogenic (Huntingtons disease)non pathogenic (forensics)MUTATION DETECTION

Huntingtons disease -a microsatellite triplet repeat in a coding regionFigure 18.12: HMG3

SCREENING TARGET LOCI FOR UNKNOWN MUTATIONSRISKYSENSITIVESPECIFIC

PRE REQUISITESGene lociSizeFrequency of known mutations

MUTATION SCANNINGCFTR mutation frequency

F50879.9%G551D2.6 %G542X1.5%

METHODSMUTATION SCANNINGDirect sequencingSouthern blotsdHPLCMicroarraysetcsequencing

Using dHPLCMUTATION SCANNINGExon 6 of DMD genenormalaffectedFig18.4: HMG3 by Strachan & Read

Using multiplex ARMS testMUTATION SCANNINGScreening for 29 mutations of the CFTR geneFig18.10: HMG3 by Strachan & Read

GENE TRACKINGAnalysis of linked markers in families for the inheritance of a high risk chromosome from heterozygous parents.The process has 3 steps1)find a closely linked marker for which the parents are heterozygous2)work out which chromosome carries the disease allele3) work out which chromosome the individual has inheritedUsed when map location of disease locus is known but not the actual disease gene

POPULATION SCREENINGScreening programs for well characterised traits must be bothSENSITIVEACCURATE

e.g. PKU tests /Guthrie (PAH activity) ARMS test (CFTR mutations)

ForensicsIdentify crime suspects / exonerate innocentIdentify victimsEstablish family relationshipsIdentify endangered speciesDetect pollutantsMatch organ donor with recipientDetermine seed / livestock pedigreeAuthenticate consummables

Extract DNAAnalyse specific regions using probes look for matches between 2 samples at many loci (multilocus)Scan ~ 10 DNA regions that show locus variability> 5 matchesCreate DNA profile (DNA fingerprint)

How does forensic ID work?

DNA fingerprintingOriginally 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) identifiedCore sequence GGAGGTGGGCAGGA2 of these used (33.15 and 33.6)Together, upto 36 independently inherited bands detected

DNA fingerprintingsuperceded by single locus probes (SLP) just 2 bands per probeNow superceded by SL-PCRUse of allelic ladder markers

AdvantagesIncreased sensitivitySmall sample quantities sufficientUses microsatellites instead of minisatellites

Simple sequence repeats (SSRs)Microsatellites 1-13 bp repeats e.g. (A)n (AC)nMinisatellites14 - 500 bp repeats3% of genome (dinucleotides - 0.5%)

Repetitive sequencesHUMFES/FPS (ATTT)8-14

DNA fingerprinting1995 National Criminal Intelligence Database (Forensic science service)700,000 samples storedStrength of evidence based on likelihood ratio (LR)LR = C / C

PROSECUTORS FALLACYThe 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 parentageE.g., DNA fingerprints confirmed Dolly the sheep was cloned from an adult udder cellDonor udder (U), cell culture from udder (C), Dollys blood cell DNA (D), and control sheep 1-12Fig. 11.15 - Hartwell

Is DNA effective in identification?Only if used intelligently!!Only regions showing the most variability must be usedMust cover large regions

Look for matches beyond a reasonable doubt

Mitochondrial DNAMulticopy16.5 kbp Maternally inheritedSequenced in 1981 (Nature,1981, 290:457-65)Mutation rate ~1/33 generationsHeteroplasmy (original and mutated forms co-exist)More stable for forensic analysis

Mitochondrial DNAHighest variation in control region (800bp)

Y chromosomeHaploid Paternal inheritanceBinary polymorphisms

ReferencesHum Mol Gen 3 by Strachan and Read Chapter 18Hartwell et al Chapter 11; pages 376-387

DNA profiling in forensics by Peter Gill et alwww.els.net

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 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 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 & carcinogensreduce the likelihood of heritable mutations Applications of HGP

Figure 11.3Figure 11.3Figure 11.3Figure 11.6Figure 11.7Figure 11.8Figure 11.8Figure 11.10Figure 11.10Figure 11.10Figure 11.10Figure 11.10Figure 11.10Figure 11.15Figure 11.15Figure 11.15