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 either
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)
pathogenic (Huntington’s disease)
non pathogenic (forensics)
class
Size of repeat
Repeat block
Major chromosomal location
minisatellite
9-64 bp
0.1 – 20kb
Telomeres
microsatellites
1-13 bp
< 150 bp
Dispersed
Figure 11.8

8. Huntington’s disease -a microsatellite triplet repeat in a coding region
Figure 18.12: HMG3

9. 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

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

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

12. 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

13. 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

14. 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

15. 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

16. 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)

17. DNA fingerprinting
Originally described using minisatellite probes consisting of tandem repeats of the myoglobin locus (Nature, 1985, 316: 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

19. DNA fingerprinting
superceded by single locus probes (SLP) – just 2 bands per probe
Now superceded by SL-PCR
Use of allelic ladder markers
Advantages
Increased sensitivity
Small sample quantities sufficient
Uses microsatellites instead of minisatellites

20. 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

21. DNA fingerprinting
1995 – 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’

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

23. 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

24. 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’

25. 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

26. Mitochondrial DNA
Highest variation in control region (800bp)

27. Y chromosome
Haploid
Paternal inheritance
Binary polymorphisms

28. 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

29. Bioarchaeology, Anthropology, Evolution, and Human Migration
Applications of HGP
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

30. Microbial Genomics Applications of HGP 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

31. Risk Assessment Applications of HGP
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