Published on

identification of human disease gene

Published in: Education
1 Like
  • Be the first to comment

No Downloads
Total views
On SlideShare
From Embeds
Number of Embeds
Embeds 0
No embeds

No notes for slide


  1. 1. Presented BY: Pradeep JaswaniM.S.c MHG III semesterJiwaji University Gwalior (M.P)
  2. 2. ContentsIntroductionPrinciples & strategies in identifying disease genePosition independent strategies for identifying disease gene Identifying disease gene though knowing the protein product Identifying disease gene though an animal model Identification of a diseases gene using position independent DNA sequence knowledgePositional cloning Single stand conformation polymorphism analysis Hetero duplex analysisCandidate gene approach
  3. 3. INTRODUCTION“Identifying genetic determinants of human phenotypes”.The approach described are equally applicable to identifying determinants of diseases or of normal variations such as red hair or red- green color blindness.
  4. 4. PRINCIPLES & STRATEGIES IN IDENTIFYING DISEASE GENE• There are many different ways of arriving at the final identification, but all path converge on a candidate gene.• One way or another, a candidate gene is identified; the researcher then test the hypothesis that these gene is disease gene by screening it for mutation
  5. 5. • Candidate genes may be identified without reference to their chromosomal location but more commonly, first a candidate chromosomal region is pinpointed, and then candidate genes are identified from within that region.• Positional information reduces the list of possible candidates from all 30000 or so on human genes to may be 10-30genes in a candidate region.
  6. 6. • Over and over again, when a disease gene is finally identified, it remains a complete mystery why mutation should cause that particular disease• For e.g. why should loss of function of the FMR1 protein, involved in transporting RNA from nucleus to cytoplasm cause, cause mental retardation (Fragile-X syndrome)
  7. 7. POSITION INDEPENDENT STRATEGIES FOR IDENTIFYING DISEASE GENEHistorically, the first diseases genes were identified by position independent methods, simply because no relevant mapping information existed an no techniques were available to generate it.Under those circumstances the candidate must be suggested by the knowledge of the gene product: β-globin for sickle cell disease, phenylalanine hydroxylase for PKU.
  8. 8. E1 B1 N D1 GeneticA1 Y C1 Collect SuccessfullyMapped Candidate mapping: chromosomal families located?candidate genome region? for Nhomolog? search mapping Y D2 E2 C2 Clone the Think again N B2 Chromosoma chromoso about mode of Check l deletions or mal inheritance,A3 heterogeneity database translocation breakpointsCloned s for genes D3 E3 Think againcandidate Identify new B3 abouthomolog? human candidate B3 genes gene & go to Possible A3 B2 D3 D4 candidate Work out full N gene sequence & E5 Y N structure Pathogenic B4 mutations C5 D5 Has it been found? Collect Look for fully Y Y unrelated mutations Model characterized patients organism Database searching Laboratory work success Clinical Input
  9. 9. IDENTIFYING DISEASE GENE THOUGH KNOWING THEPROTEIN PRODUCTModern proteomic technique allow even very tiny quantities of protein to be identified or partially sequenced by mass spectrophotometery.As only one of the nucleotide in the mixture will corresponding to the authentic sequence, it is important to keep the number of different oligonucleotides low so as to increase the chance of identifying correct target.
  10. 10. The number of possible permutations should be reduced by ligating the target cDNA to a vector and using one vector-specific primer and one degenerate protein specific-primerHost cells containing clones with the desired gene should produce the protein or at least parts of the protein, and could be indentified using colony filters from the library with an appropriate antibody.A more rapid alternative is to use partially degenerate oligonucleotides as PCR primers
  11. 11. IDENTIFYING DISEASE GENE THOUGH AN ANIMAL MODEL• Many human diseases genes have been identified with the help of animal models-but nearly always this has been after checking positional informationsA mouse mutant and a phenotypically similar human diseases are mapped to chromosomal location that are corresponds.If the mouse gene is cloned its human homolog became a natural candidate.
  12. 12. • Alternatively a diseases gene may be identified in the mouse and then the human homolog isolated; this can be mapped by fluorescence in situ Hybridization, and becomes a candidate gene for any relevant diseases mapping to that location.• This is how the MIFT gene was identified as a cause of type2 waardenburg syndrome (Hughes et al 1994).
  13. 13. IDENTIFICATION OF A DISEASES GENE USING POSITION INDEPENDENT DNA SEQUENCE KNOWLEDGE• Positional independent candidates are also generated by expression array experiment in which mRNA samples from patient and controls are compared to produce a list of genes whose expression is alter in the disease.• An interesting application of positional independent DNA sequence knowledge is attempt to clone genes containing novel trinucleotide repeats.
  14. 14. • The repeat expansion detection method of schalling et al 1993 permits detection of expanded repeats in unfractionated genomic DNA of affected patient and method have been developed for cloning expanded repeats detected (koob et al 1998).
  15. 15. Positional cloningA diseases is identified knowing nothing except its appropriate chromosomal location.The first successful application was identification of the gene for X-linked chronic granulomatous disease (Royer- Pokora et al 1985)The successful conclusion of these work in 1986 marked the start of triumphant new era for human molecular genetics.One after another, the genes underlying important disorder such as cystic fibrosis, Huntingtons diseases.
  16. 16. Define the candidate region Obtain clones of all the DNA of the region Identify all the genes in the region Prioritize them for mutation screening Test candidate genes for mutation in affected peopleFig: Logic of positional cloning
  17. 17. A NUMBER OF POSITIONAL CLONING METHODS ARE USED AS FOLLOWS:-Single strand conformation polymorphism (SSCP)Denaturing gradient gel electrophoresis (DGGE)Heteroduplex analysisChemical mismatch cleavage protein truncation test (PTT)
  19. 19. CANDIDATE GENE APPROACH• A functional/candidate gene cloning project starts with either the known protein that is responsible for an inherited disorder or a protein that is considered a likely candidate based on the symptoms and biochemistry of the disease.• The amino acid sequence of the protein is used to deduce the possible cloning sequence of the corresponding gene.
  20. 20. Fig: candidate gene approach Known (or candidate) protein Deduce nucleic acid sequence Examine human genome database Retrieve bacterialLocalize Characterize gene artificialchromosome Identify exons chromosome (BAC) structureregion clones Develop Devise DNA mutation Mutation diagnostic detection assays phenotype studies tests
  21. 21. REFERENCES HUMAN MOLECULAR GENETICS “Tom strachan & Read” AN INTRODUCTION TO HUMAN MOLECULAR GENETICS “Jack J.Pasternek” agnostic_Direct_Methods