Download - Applications of HGP
Applications of HGP
Genetic testing
Forensics
• 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
Genetic testing
• Directly
• Gene tracking
• Population screening
Genetic testing can be done either
DIRECT GENETIC TESTING
Based on either
a) MUTATION DETECTION: screening for KNOWN polymorphisms in DNA
b) MUTATION SCANNING: screening for UNKNOWN polymorphisms in DNA
SNPs by 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
MUTATION DETECTION
• 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
Variation in length of DNA sequence (repetitive DNA)
• pathogenic (Huntington’s disease)
• non pathogenic (forensics)
MUTATION DETECTION
class Size of repeat
Repeat block
Major chromosomal
location
minisatellite 9-64 bp 0.1 – 20kb Telomeres
microsatellites 1-13 bp < 150 bp Dispersed
Huntington’s disease -a microsatellite triplet repeat in a coding region
Figure 18.12: HMG3
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%
G551D 2.6 %
G542X 1.5%
METHODSMUTATION SCANNING
Direct sequencingSouthern blotsdHPLCMicroarraysetc
sequencing
MUTATION SCANNING
Using dHPLCExon 6 of DMD gene
normal
affected
Fig18.4: HMG3 by Strachan & Read
MUTATION SCANNING
Using multiplex ARMS test
Screening for 29 mutations of the CFTR gene
Fig18.10: 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 inherited
Used when map location of disease locus is known but not the actual disease gene
POPULATION SCREENING
Screening programs for well characterised traits must be both
SENSITIVE
ACCURATE
e.g. PKU tests /Guthrie (PAH activity)
ARMS test (CFTR mutations)
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
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 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 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)n
Minisatellites14 - 500 bp repeats3% of genome (dinucleotides - 0.5%)
Repetitive sequences…
HUMFES/FPS (ATTT)8-14
DNA fingerprinting1995 – 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’
Oct 2004, Vol 5 pg739
(A) PATERNITY TEST
(B) RAPE CASE
• 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
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
• 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 & carcinogens
• reduce the likelihood of heritable mutations
Applications of HGP