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 either

DIRECT 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)

• pathogenic (Huntington’s disease)

• non pathogenic (forensics)

MUTATION DETECTION

class Size of repeat

Repeat block

Major chromosomal

location

minisatellite 9-64 bp 0.1 – 20kb Telomeres

microsatellites 1-13 bp < 150 bp Dispersed

Huntington’s disease -a microsatellite triplet repeat in a coding region

Figure 18.12: HMG3

SCREENING TARGET LOCI FOR UNKNOWN MUTATIONS

RISKY SENSITIVE SPECIFIC

PRE REQUISITES

Gene loci

Size

Frequency of known mutations

MUTATION SCANNING

CFTR mutation frequency

F50879.9%

G551D 2.6 %

G542X 1.5%

METHODSMUTATION SCANNING

Direct sequencingSouthern blotsdHPLCMicroarraysetc

sequencing

MUTATION SCANNING

Using dHPLCExon 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 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 SCREENING

Screening programs for well characterised traits must be both

SENSITIVE

ACCURATE

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

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?

DNA fingerprinting

Originally described using minisatellite probes consisting of tandem repeats of the myoglobin locus (Nature, 1985, 316: 76-79- 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

DNA fingerprintingsuperceded by single locus probes (SLP) – just 2 bands per probeNow superceded by SL-PCRUse of allelic ladder markers

AdvantagesIncreased sensitivitySmall sample quantities sufficientUses microsatellites instead of minisatellites

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

DNA fingerprinting1995 – 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’

Oct 2004, Vol 5 pg739

(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

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’

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

Mitochondrial DNA

• Highest variation in control region (800bp)

Y chromosome

• Haploid

• Paternal inheritance

• Binary polymorphisms

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

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

Applications of HGP

Microbial Genomics

• 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

Applications of HGP

Risk Assessment

• 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

Applications of HGP