Transcript Microsatellites

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
• Used for mutation screening of disease
genes e.g. HD, CFTR, DMD
Genetic testing can be done either
• 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 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
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)
• 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
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
etc
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
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
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)
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 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
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
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’
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 coexist)
• 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
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
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