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Applications of HGP
Genetic testing
Forensics
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
PURPOSE
 Medical management
 Forensics
 Research
Genetic testing can be done in 3
ways
• Directly
• Gene tracking
• Population screening
DIRECT GENETIC TESTING
Based on either
a) MUTATION DETECTION: screening for
KNOWN polymorphisms in DNA
b) MUTATION SCANNING: screening for
UNKNOWN polymorphisms in DNA
MUTATION DETECTION
SNPs
by ASOs
• Very short specific probes (<21 bp) which hybridize to one allele or other
• Such probes are allele-specific oligonucleotides (ASOs)
Fig. 11.8
MUTATION DETECTION
Variation in length of DNA sequence
(repetitive DNA)
Huntington’s disease -a microsatellite
triplet repeat in a coding region
Figure 18.12: HMG3
MUTATION SCANNING
SCREENING TARGET LOCI FOR UNKNOWN
MUTATIONS
 RISKY
 SENSITIVE
 SPECIFIC
PRE REQUISITES
Gene loci
Size
Frequency of known mutations
CFTR mutation
frequency
F508
G551D
G542X
79.9%
2.6 %
1.5%
MUTATION SCANNING
METHODS
 Direct sequencing
 Southern blots
 dHPLC
 Microarrays
sequencing
MUTATION SCANNING
Using dHPLC
Exon 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 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
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)
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
Early markers
• Karl Landsteiner’s 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
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
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
…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
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)
Oct 2004, Vol 5 pg739
Current methods
1) Autosomal STR typing
– Needs ~300bp amplicons
– SGMPlus database (UK) contains 5 multiplex loci
– US FBI CODIS contain 13 STR loci
Some STR electropherograms
Electropherogram of a second-generation multiplex ‘SGM Plus’ profile from a male
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)
Current methods
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
3) Mitochondrial DNA typing
• Mutation rate ~1/33 generations
• Multicopy
• Heteroplasmy (original and mutated
• 16.5 kbp
forms co-exist)
• Maternally inherited
• More stable for forensic analysis
Highest variation in control region (800bp)
Current methods
4) Y-chromosome typing
• Haploid
• Recombinationdeficient (mutations
only)
• Paternal inheritance
• Binary polymorphisms
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’
Is DNA effective in casework?
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’
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
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