1. applications of genome sequencing projects
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applications of genome sequencing projects
1)   Molecular Medicine
2)  Energy sources and environmental applications
3)  Risk assessment
4)  Bioarchaeology, anthropology, evolution, and human migration
5)  DNA forensics
6) Agriculture, livestock breeding, and bioprocessing

2. Molecular medicine improved diagnosis of disease
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Molecular medicine
improved diagnosis of disease
eearlier detection of genetic predisposition to disease
rational drug design
gene therapy and control systems for drugs
ppharmacogenomics "custom drugs"

3. Definitions
DNA polymorphism: A DNA sequence that occurs in two or more variant forms
Alleles: any variations in genes at a particular location (locus)
Haplotype: combination of alleles at multiple, tightly-linked loci that are transmitted together over many generations
Anonymous locus : position on genome with no known function
DNA marker: polymorphic locus useful for mapping studies
RFLP Variation in the length of a restriction fragment detected by a particular probe due to nucleotide changes at a restriction site
SNP: two different nucleotides appear at the same position in genomic DNA from different individuals
DNA fingerprinting: Detection of genotype at a number of unlinked highly polymorphic loci using one probe
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

4. DNA markers/polymorphisms
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DNA markers/polymorphisms
RFLPs (restriction fragment length polymorphisms)
- Size changes in fragments due to the loss or gain of a restriction site
SSLPs (simple sequence length polymorphism) or microsatellite repeats. Copies of bi, tri or tetra nucleotide repeats of differing lengths e.g. 25 copies of a CA repeat can be detected using PCR analysis.
SNPs (single nucleotide polymorphisms)-Sites resulting from a single change in individual bp.

5. RFLPs Amplify fragment Expose to restriction enzyme
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RFLPs
Amplify fragment
Expose to restriction enzyme
Gel electrophoresis
e.g., sickle-cell genotyping with a PCR based protocol
Fig – genetics/ Hartwell

6. SSLPs Similar principles used in detection of RFLPs
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SSLPs
Similar principles used in detection of RFLPs
However, no change in restriction sites
Changes in length of repeats

7. SNPs (single nucleotide polymorphisms)
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SNPs (single nucleotide polymorphisms)
Sites resulting from a single change in individual bp
SNP detection using allele-specific oligonucleotides
(ASOs)
Very short probes (<21 bp) specific which hybridize to one allele or other
Such probes are called ASOs
Figure 11.8
Fig. 11.8

8. ASOs can determine genotype at any SNP locus
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ASOs can determine genotype at any SNP locus
Figure 11.9 a-c
Fig a-c

9. Hybridized and labeled with ASO for allele 1
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Hybridized and labeled with ASO for allele 1
Hybridized and labeled with ASO for allele 2
Figure 11.9 d,e
Fig d, e

10. How to identify disease genes
Identify pathology
Find families in which the disease is segregating
Find ‘candidate gene’
Screen for mutations in segregating families

11. How to map candidate genes
2 broad strategies have been used
A. Position independent approach (based on knowledge of gene function)
1)  biochemical approach
2) animal model approach
B. Position dependent approach (based on mapped position)

12. Position independent approach
1) Biochemical approach: when the disease protein is known E.g. Factor VIII haemophilia
Blood-clotting cascade in which vessel damage causes a cascade of inactive factors to be converted to active factors

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Blood tests determine if active form of each factor in the cascade is present
Figure c
Fig c

14. Techniques used to purify Factor VIII and clone the gene
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Techniques used to purify Factor VIII and clone the gene
Figure d
Fig d
Fig d

15. 2) Animal model approach
compares animal mutant models in a phenotypically similar human disease.
E.g. Identification of the SOX10 gene in human Waardenburg syndrome4 (WS4)
Dom (dominant megacolon) mutant mice shared phenotypic traits similar to human patient with WS4 (Hirschsprung disease, hearing loss, pigment abnormalities)
WS4 patients screened for SOX10 mutations
confirmed the role of this gene in WS4.
Dom mouse
Hirschsprung

16. B) Positional dependent approach
Positional cloning identifies a disease gene based on only approximate chromosomal location. It is used when nature of gene product / candidate genes is unknown.
Candidate genes can be identified by a combination of their map position and expression, function or homology

18. B) Positional Cloning Steps
Step 1 – Collect a large number of affected families as possible
Step 2 - Identify a candidate region based on genetic mapping (~ 10Mb or more)
Step 3 - Establish a transcript map, cataloguing all the genes in the region
Step 4- Identify potential candidate genes
Step 5 – confirm a candidate gene

19. Step 2 - Identifying a candidate region
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Genetic map of <1Mb
Genetic markers: RFLPs, SSLPs, SNPs
Lod scores:
log of the odds: ratio of the odds that 2 loci are linked or not linked
need a lod of 3 to prove linkage and a lod of -2 against linkage
Halpotype maps
LoD- log of the Odds: ratio of the odds that 2 loci are linked or not linked; lod of 3 is 95%
Kay davies green man vs green car analogy
HapMap published in Oct Nature

20. Step 3 – transcript map which defines all genes within the candidate region
Search browsers e.g. Ensembl
Computational analysis
Usually about 17 genes per 1000 kb fragment
Identify coding regions, conserved sequences between species, exon-like sequences by looking for codon usage, ORFs, and splice sites etc
Experimental checks – double check sequences, clones, alignments etc
Direct searches – cDNA library screen

21. Step 4 – identifying candidate genes
Expression: Gene expression patterns can pinpoint candidate genes
RNA expression by Northern blot or RT-PCR or microarrays
Look for misexpression (no expression, underexpression, overexpression)
CFTR gene
Northern blot analysis reveals only one of candidate genes is expressed in lungs and pancreas

22. Step 4 – identifying candidate genes
Function: Look for obvious function or most likely function based on sequence analysis
e.g. retinitis pigmentosa
Candidate gene RHO part of phototransduction pathway
Linkage analysis mapped disease gene on 3q (close to RHO)
Patient-specific mutations identified in a year

23. Step 4 – identifying candidate genes
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Homology: look for homolog (paralog or ortholog)
Congenital contractural arachnodactyly (Beals syndrome) is a rare autosomal dominant disorder caused by mutation in fibrillin 2 (FBN2) gene that is phenotypically similar to but less severe than Marfan syndrome
Marfan syndrome
fibrillin gene FBN1
Beals syndrome
fibrillin gene FBN2
Both mapped to 5q

24. Step 4 – identifying candidate genes
Animal models: look for homologous genes in animal models especially mouse
e.g. Waardenburg syndrome type 1
Linkage analysis localised WS1 to 2q
Splotch mouse mutant showed similar phenotype
Could sp and WS1 be orthologous genes?
Pax-3 mapped to sp locus
Homologous to HuP2
Splotch mouse
WS type1

25. Step 5 – confirm a candidate gene
Mutation screening
Sequence differences
Missense mutations identified by sequencing coding region of candidate gene from normal and abnormal individuals
Transgenic model
Knockout / knockin the mutant gene into a model organism
Modification of phenotype

26. Transgenic analysis can prove candidate gene is disease locus
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Transgenic analysis can prove candidate gene is disease locus
Figure 11.21
Fig

28. Optional Reading on Molecular medicine
HMG3 by T Strachan & AP Read : Chapter 14 AND/OR Genetics by Hartwell (2e) chapter 11
Optional Reading on Molecular medicine
Nature (May2004) Vol 429 Insight series
human genomics and medicine pp439 (editorial)
predicting disease using medicine by John Bell pp