Genetic Mapping Techniques PDF

 History of gene mapping  In 1911, by Thomas Hunt Morgan, gene for eye-color was located on the X chromosome of fruit fly.  Shortly after that, E.B. Wilson attributed the sex-linked genes responsible for color-blindness and hemophilia in human beings to be located on the X-chromosome,...

 History of gene mapping  In 1911, by Thomas Hunt Morgan, gene for eye-color was located on the X chromosome of fruit fly.  Shortly after that, E.B. Wilson attributed the sex-linked genes responsible for color-blindness and hemophilia in human beings to be located on the X-chromosome,  similar to the many X-linked factors being described by the Morgan group in flies.  History of gene mapping cont…  It wasn't until 1968 that an autosomal assignment of linkage was made. History of gene mapping cont…  Gene Mapping  Techniques  in situ hybridization  somatic cell hybridization  dosage effect  chromosome aberration  linkage analysis  Strategies  Gene mapping  "Gene mapping" refers to the mapping of genes to specific locations on chromosomes.  It is a critical step in the understanding of genetic diseases.  There are two types of gene mapping:  Genetic Mapping - using linkage analysis to determine the relative position between two genes on a chromosome.  Physical Mapping - using all available techniques or information to determine the absolute position of a gene on a chromosome.  Gene mapping cont…  Requires informative markers – polymorphic and a population with known relationships  Best if measured between “close” markers.  Unit of distance in genetic maps = centiMorgans, cM  1 cM = 1% chance of recombination between markers Physical mapping  Relies upon observable experimental  Outcomes  hybridization  amplification  May or may not have a distance measure.  gene mapping  The ultimate goal of gene mapping is to clone genes, especially disease genes. Once a gene is cloned, we can determine its DNA sequence and study its protein product.  For example: cystic fibrosis (CF).  In 1985, the gene was mapped to chromosome 7q31-q32 by linkage analysis.  Four years later, it was cloned by Francis Collins and his co- workers. We now know that the disease is caused by the defect of a chloride channel – the protein product of this disease gene.  Gene maps  Genetic map  Physical map  Transcription map  Sequence map Technique 1 --- Gene mapping by In situ Hybridization  The method which involves hybridizing labeled DNA (or RNA) probes directly to metaphase chromosomes. In situ Hybridization cont… In situ Hybridization cont… FISH FISH  Specialties  Simple, convenient.  Only for known genes or known DNA segments  Cannot obtain high-resolution maps on metaphase chromosomes or pre-metaphase chromosomes Technique 2 --- Gene mapping by somatic cell hybridization  The method which to fuse somatic cells from different species.  An experimentally based segregation technique used to map genes to human chromosomes. Somatic cell hybridization (cell fusion)  Cells from two different species (e.g. humans and rodents) are artificially fused together.  These culture lines are developed by mixing human and mouse cells in the presence of the Sendai virus.  The virus facilitates the fusing of the two cell types to form a hybrid cell. Chromosome loss of hybrid cells  For a reason that is not entirely known, most, but not all, human chromosomes are randomly lost from the hybrid cell lines.  Usually a few human chromosomes are retained.  Because the human and mouse chromosomes can be distinguished by chromosome staining techniques, it can be determined which human chromosomes are retained with a specific cell line. (https://www.youtube.com/watch?v=MqubuIdR8NY) Exposure to virus or to chemical agent leads to cell fusion A cell with two nuclei forms (heterokaryon) The nuclear membranes then fuse Each cell then randomly loses some human chromosomes Colonies arise from these cells… Somatic cell hybridization  Specialties:  Simple, convenient.  Only for analyzing the known genes or their products；  Cannot map genes accurately；  Because of the homologies between human genes and rodent genes, this method cannot succeed in mapping some of the genes. Technique 3 --- Gene mapping by gene dosage using patient cells  The method which is used to detect dosage differences in either gene products or gene sequences themselves between patients’ cell lines containing different numbers of copies of a particular gene. dosage effect  The gene dosage strategy was originally used to assign genes to chromosome 21 by detecting levels of enzyme activity in cell lines from patients with Down syndrome that were 1.5-fold higher than levels in cell lines from chromosomally normal persons. Dosage effect  Specialties:  Simple and convenient；  For the known genes or their protein products；  The cell lines involving numerical chromosomal aberrations are infrequent naturally. Technique 4 --- Gene mapping by chromosomal aberration  To detect directly chromosomal aberration involving genes which may lead to particular disease. chromosomal aberration cont… Example: 1: DMD（Duchenne muscular dystrophy） X－A translocation DMD, X-linked recessive inheritance, is very rare in female. Karyotype analysis of several affected female indicated common X-A translocation. Although these translocations involved different autosomes, their broken points on X chromosomes were commonly located on Xp21. This indicated that the broken points were inner of the gene for DMD. ② Microdeletion in X chromosome In one well-studied example, a boy with no known family history of any genetic disease presented with 4 ordinarily distinct X-linked conditions: DMD, chronic glaucomatous disease (CGD), retinitis pigmentosa (RP), and a rare red blood cell phenotype. Careful cytogenetic analysis revealed a small but detectable deletion in band Xp21.2.  chromosomal aberration cont…  Specialties:  Simple, convenient.  For mapping the unknown genes；  Most of the individuals with (structural) chromosomal aberrations are eliminated naturally. Technique 5 --- Gene mapping by linkage analysis  Linkage analysis is a method of mapping genes that uses family studies to determine whether two genes show linkage when passed on from one generation to the next. Linkage analysis  Mapping by genetic linkage analysis differs from mapping by physical methods because physical mapping relies on having a laboratory method to localize a gene by FISH or by somatic cell hybridization.  In contrast, linkage analysis is a tremendously important and powerful approach in medical genetics because it is the only method that allows mapping of genes, including disease genes, that are detectable only as phenotypic traits. Linkage analysis cont… Linkage analysis cont… How to know the linkage between any locus and the faulty gene?  Genome scanning  The 10 cM (centiMorgan) limit LOD score: are two loci linked?  The statistical term that is generally calculated to evaluate the significance of linkage results is called a LOD score (or Z).  The LOD score compares the likelihood of obtaining the test data if the two loci are indeed linked, to the likelihood of observing the same data purely by chance.  Likelihood is similar to probability and is very dependent on the distance between the two test loci.  LOD=log10  likelihood of data if loci linked at a particular position and likelihood of data if loci unlinked ( =0.5)  LOD score ≥+3, definitely linked  LOD score≤-2±, exclusion of linkage  LOD score＝±1, linked uncertainly Summary for common methods Strategies -Mapping a Known Gene Strategies - Mapping a disease gene