• 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
DIRECT GENETIC TESTINGDIRECT 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)MUTATION DETECTION
Figure 18.12: HMG3
Huntington’s disease -a microsatellite triplet repeat in a coding region
SCREENING TARGET LOCI FOR UNKNOWN SCREENING TARGET LOCI FOR UNKNOWN MUTATIONSMUTATIONS
RISKY SENSITIVE SPECIFIC
PRE REQUISITES
Gene loci
Size
Frequency of known mutations
MUTATION SCANNINGMUTATION SCANNING
CFTR mutation frequency
F50879.9%
G551D 2.6 %
G542X 1.5%
METHODS
Direct sequencing Southern blots dHPLC Microarrays
sequencing
MUTATION SCANNINGMUTATION SCANNING
Using dHPLCExon 6 of DMD gene
normal
affected
Fig18.4: HMG3 by Strachan & Read
MUTATION SCANNINGMUTATION SCANNING
Using multiplex ARMS test
Screening for 29 mutations of the CFTR gene
Fig18.10: HMG3 by Strachan & Read
MUTATION SCANNINGMUTATION SCANNING
GENE TRACKINGGENE 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 SCREENINGPOPULATION SCREENING
Screening programs for well characterised traits must be both
SENSITIVE
ACCURATE
e.g. PKU tests /Guthrie (PAH activity)
ARMS test (CFTR mutations)
ForensicsForensics
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
Early markersEarly markers
• Karl Landsteiner’s ABO blood typing
DNA fingerprintingDNA fingerprinting
Originally described by Sir Alec Jeffereys (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
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
1985 technique using hybridisation of Multi 1985 technique using hybridisation of Multi locus probes (MLP)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
……now superceded by PCR-based methodsnow superceded by PCR-based methods
Discovery of STR (short tandem repeats)Use of STR multiplex PCRAutosomal SNP typing, Y-chromosome / mtDNA markers
AdvantagesIncreased sensitivitySmall sample quantities sufficientUses microsatellites, instead of minisatellites
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?How does forensic ID work?
1) Autosomal STR typing1) Autosomal STR typing– Needs ~300bp amplicons– SGMPlus database (UK) contains 5 multiplex loci– US FBI CODIS contain 13 STR loci
Current methodsCurrent methods
Some STR electropherogramsSome 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
2) Autosomal SNP typing2) 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 methodsCurrent methods
Current methodsCurrent methods
• Multicopy
• 16.5 kbp
• Maternally inherited
Highest variation in control region (800bp)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 typing3) Mitochondrial DNA typing
Current methodsCurrent methods4) Y-chromosome typing4) Y-chromosome typing
• Haploid
• Recombination-deficient (mutations only)
• Paternal inheritance
• Binary polymorphisms
Is DNA effective in casework?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’
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
‘Prosecutor’s 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?Is DNA effective in casework?
• 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