1. Applications of HGP
Genetic testing
Forensics
Figure 11.3

2. 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
Used for mutation screening of disease genes e.g. HD, CFTR, DMD
Figure 11.3

3. Genetic testing can be done in 3 ways
Directly
Gene tracking
Population screening
Figure 11.3

4. DIRECT GENETIC TESTING
Based on either
MUTATION DETECTION: screening for KNOWN polymorphisms in DNA
MUTATION SCANNING: screening for UNKNOWN polymorphisms in DNA
Figure 11.6

5. SNPs by RFLP-PCR MUTATION DETECTION
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
Figure 11.7
Fig Hartwell

6. SNPs by ASOs MUTATION DETECTION
Figure 11.8
Very short specific probes (<21 bp) which hybridize to one allele or other
Such probes are allele-specific oligonucleotides (ASOs)
Fig. 11.8

7. Variation in length of DNA sequence (repetitive DNA)
MUTATION DETECTION
Variation in length of DNA sequence (repetitive DNA)
Huntington’s disease -a microsatellite triplet repeat in a coding region
Figure 11.8
Figure 18.12: HMG3

8. SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS
MUTATION SCANNING
SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS
RISKY
SENSITIVE
SPECIFIC
CFTR mutation frequency
F %
G551D 2.6 %
G542X 1.5%
PRE REQUISITES
Gene loci
Size
Frequency of known mutations
Figure 11.10

9. METHODS MUTATION SCANNING sequencing Direct sequencing Southern blots
dHPLC
Microarrays
Figure 11.10

10. MUTATION SCANNING Using dHPLC Exon 6 of DMD gene
normal
Figure 11.10
affected
Fig18.4: HMG3 by Strachan & Read

11. Using multiplex ARMS test
MUTATION SCANNING
Using multiplex ARMS test
Screening for 29 mutations of the CFTR gene
Figure 11.10
Fig18.10: HMG3 by Strachan & Read

12. 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
Figure 11.10

13. POPULATION SCREENING
Screening programs for well characterised traits must be both
SENSITIVE
ACCURATE
e.g. PKU tests /Guthrie (PAH activity)
ARMS test (CFTR mutations)
Figure 11.10

14. 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
Figure 11.15

15. Early markers DNA fingerprinting
Karl Landsteiner’s ABO blood typing
DNA fingerprinting
Originally described by Sir Alec Jeffereys (1985) (Nature, 1985, 316: Jeffereys et al)
Discovery of hypervariable loci
‘Differential lysis’ technique in parallel
First conviction using DNA fingerprinting was Colin Pitchfork in 1986
ABO proves exclusion
Proof of inclusion much more difficult

16. Simple sequence repeats (SSRs)
Repetitive sequences…
Simple sequence repeats (SSRs)
Microsatellites
1-13 bp repeats e.g. (A)n (AC)n
Minisatellites
bp repeats
3% of genome (dinucleotides - 0.5%)
HUMFES/FPS (ATTT)8-14

17. 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
ABO proves exclusion
Proof of inclusion much more difficult

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
Multiplex PCR: single tube PCR reaction amplifying multiple loci

20. 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)

21. Oct 2004, Vol 5 pg739

22. Current methods 1) Autosomal STR typing Needs ~300bp amplicons
SGMPlus database (UK) contains 5 multiplex loci
US FBI CODIS contain 13 STR loci
An electropherogram of the second-generation multiplex ‘SGM Plus’ profile from a male, including X- and Y-specific amelogenin products of 106 and 112 bp, respectively. Most short tandem repeats (STRs) are heterozygous and the alleles are evenly balanced. Numbers beneath STR peaks indicate allele sizes in repeat units. The STR profile is displayed in the green, blue and yellow channels of a four-colour fluorescent system, with the red channel
being used for a size marker (not shown).
electropherogram is a plot of fluorescence units over time.

23. Some STR electropherograms
Electropherogram of a second-generation multiplex ‘SGM Plus’ profile from a male
An electropherogram of the second-generation multiplex ‘SGM Plus’ profile from a male, including X- and Y-specific amelogenin products of 106 and 112 bp, respectively. Most short tandem repeats (STRs) are heterozygous and the alleles are evenly balanced. Numbers beneath STR peaks indicate allele sizes in repeat units. The STR profile is displayed in the green, blue and yellow channels of a four-colour fluorescent system, with the red channel
being used for a size marker (not shown).
electropherogram is a plot of fluorescence units over time.
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)

24. 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

25. Highest variation in control region (800bp)
Current methods
3) Mitochondrial DNA typing
Mutation rate ~1/33 generations
Heteroplasmy (original and mutated forms co-exist)
More stable for forensic analysis
Multicopy
16.5 kbp
Maternally inherited
Highest variation in control region (800bp)

26. Current methods 4) Y-chromosome typing Haploid
Recombination-deficient (mutations only)
Paternal inheritance
Binary polymorphisms

27. 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’

28. 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’
The evidential weight of a match between crime stain profile and suspect is quantified by the match probability (Pm);
the chance of two unrelated people sharing a profile. For independently inherited loci,Pm is calculated by multiplying
the individual allele frequencies in the profile in question (the ‘product rule’): the greater the number of loci, and the
greater the heterozygosity of each locus, the lower the value of Pm.However, there are a number of situations in which
Pm can be substantially increased:
• if the profile is partial because of degradation, reducing the number of informative loci;
• if a suspect and a perpetrator share many alleles by descent (for example, are brothers);
• if a suspect and a perpetrator originate from the same subpopulation.
POPULATION STRUCTURE can cause frequencies of alleles (and hence profiles) to vary between subpopulations — an issue
that caused great controversy in the application of SLP profiling90. The debate was resolved by applying guidelines to
ensure match probabilities quoted in court were conservative (that is, favourable to the defendant). Similar conservatism
is now applied to STR profiles91.

31. (A) PATERNITY TEST
(B) RAPE CASE
Figure 11.15

32. 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
Figure 11.15
Fig Hartwell

34. References Hum Mol Gen 3 by Strachan and Read Chapter 18
Hartwell et al – Chapter 11; pages
DNA profiling in forensics by Peter Gill et al