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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. - PowerPoint PPT Presentation
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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
http://www.ornl.gov/hgmis/project/benefits.html
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"
DefinitionsDNA 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
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.
RFLPs
Fig. 11.7 – genetics/ Hartwell
- Amplify fragment
- Expose to restriction enzyme
- Gel electrophoresis
e.g., sickle-cell genotyping with a PCR based protocol
SSLPs Similar principles used in detection of RFLPs However, no change in restriction sitesChanges in length of repeats
SNPs (single nucleotide polymorphisms)
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
Fig. 11.8
Sites resulting from a single change in individual bp
Hybridized and labeled with ASO for allele 1
Hybridized and labeled with ASO for allele 2
Fig. 11.9 d, e
How to identify disease genes
• Identify pathology• Find families in which the disease is
segregating• Find ‘candidate gene’• Screen for mutations in segregating
families
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)
Position independent approach1) 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
2) Animal model approachcompares 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
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
B) Positional Cloning StepsStep 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
Step 2 - Identifying a candidate regionGenetic 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 linkedneed a lod of 3 to prove linkage and a lod of -2 against linkage
Halpotype maps
HapMap published in Oct27 2005 Nature
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
Step 4 – identifying candidate genes
Expression: Gene expression patterns can pinpoint candidate genes
Northern blot analysis reveals only one of candidate genes is expressed in lungs and pancreas
RNA expression by Northern blot or RT-PCR or microarrays
Look for misexpression (no expression, underexpression, overexpression)
CFTR gene
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
Step 4 – identifying candidate genes
Homology: look for homolog (paralog or ortholog)
Both mapped to 5q
Beals syndromefibrillin gene FBN2
Marfan syndrome fibrillin gene FBN1
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 locusHomologous to HuP2
Splotch mouse WS type1
Step 5 – confirm a candidate gene
Mutation screeningSequence differences
Missense mutations identified by sequencing coding region of candidate gene from normal and abnormal individualsTransgenic modelKnockout / knockin the mutant gene into
a model organismModification of phenotype
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