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 in 3 ways

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?

Oct 2004, Vol 5 pg739

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?

(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

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

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