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Week 11: Mapping

Week 11: Mapping. November 8, 2001 Todd Scheetz. Introduction. What is mapping? determining the location of elements within a genome, with respect to identifiable landmarks. Types of mapping… genetic mapping physical mapping restriction mapping cytogenetic mapping

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Week 11: Mapping

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  1. Week 11: Mapping November 8, 2001 Todd Scheetz

  2. Introduction • What is mapping? • determining the location of elements within a genome, with respect to identifiable landmarks. • Types of mapping… • genetic mapping • physical mapping • restriction mapping • cytogenetic mapping • somatic cell mapping • radiation hybrid mapping • comparative mapping

  3. Introduction • Genetic mapping • Utilize recombination events to estimate distance between genetic markers. • RFLP • STRP • SNP • Look at a population and estimate the recombination fraction •  = # recombinants / # total

  4. Introduction • Physical mapping • Relies upon observable experimental outcomes • hybridization • amplification • May or may not have a distance measure.

  5. Genetic Mapping Requires informative markers -- polymorphic and a population with known relationships Best if a measured between “close” markers. Unit of distance in genetic maps = centimorgans, cM 1 cM = 1% chance of recombination between markers

  6. Genetic Mapping A2 B2 A2 B2 A2 B2 A1 B1 A1 B2 A1 B1 A2 B1 A1 B1 A2 B2 A1 B1 A1 B1 A1 B1 A2 B2 A1 B1 A1 B1 A1 B1 A1 B2 A1 B1 A2 B2 A1 B1 A1 B1 A1 B1 A2 B1 A1 B1 A2 B2 A1 B1 NR NR R NR NR R NR  = # recombinant / # total = 2/7 = 0.286

  7. Genetic Mapping Theta calculation with inbred population… det+ det bn bn+ x bn det+ bn+ det det+ det bn bn x bn+ det bn det det+ det det det+ bn bn+ bn bn+ bn det bn det bn det bn det  = # recombinant # total = 5/1000 = 0.005 banded detached banded, detached wild-type 483 512 2 3

  8. Genetic Mapping • : theoretical maximum of 50% • Best if a measured between “close” markers. • Unit of distance in genetic maps = centimorgans, cM • d = - 0.5 ln(1 - 2) • d = 0.25 ln[(1 + 2)/(1 - 2)] • 1 cM = 1% chance of recombination between markers

  9. Genetic Mapping

  10. Restriction Mapping • Background on restriction enzymes • cut DNA at specific sites • Ex. EcoRI cuts at GAATTC • sites are often palindromic • GAATTC • CTTAAG • may leave blunt ends or overlaps GGCC GG CC CCGG CC GG GAATTC G AATTC CTTAAG CTTAA G

  11. Restriction Mapping Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA. EcoRI HindIII BamHI PstII HindIII BamHI PstII

  12. Restriction Mapping BamHI +PstI BglII +BamHI BglII +PstI BglII BamHI PstI 5.2 4.2 3.6 3.5 3.3 2.6 1.7 1.7 1.4 1.4 1.2 1.2 1.2 1.0 1.0 0.9 0.7 0.5 0.3 0.3 0.3 BglII BamHI PstI BglII PstI 0.3 0.7 2.6 0.9 0.5 1.2

  13. Restriction Mapping • Creating a restriction map from a double digest experiment is NP-complete. • No polynomial-time solution. • As the number of fragments increase, the complexity increases as A!B!. • if the two single-enzyme reactions generate • 6 and 8 times respectively, • 29,030,400 potential permutations to evaluate A A! 1 1 2 2 3 6 4 24 5 120 6 720 7 5040 8 40,320

  14. Restriction Mapping • Multiple valid solutions possible. • Reflections • Equivalence • A = {1,3,3,12} B = {1,2,3,3,4,6} • A^B = {1,1,1,1,2,2,2,3,6} 3 12 3 1 4320 map configurations, but only 208 distinct solutions. A 6 3 3 1 2 4 B 3 1 2 1 1 1 2 2 6 A^B 1 3 12 3 A’ 3 3 1 2 4 6 B’ 1 1 1 3 1 2 2 6 2 A^B’

  15. Cytogenetic Mapping Cytogenetic mapping refers to observing a map location in reference to a chromosomal banding pattern.

  16. CytogeneticMapping These methods allow a rough determination of location, but to not yield a direct measure of distance.

  17. Cytogenetic Mapping

  18. Somatic Hybrid Mapping • Somatic cell mapping can be used to map an element to a portion of a genome. • typically with chromosome resolution • Exploits the ability of rodent (hamster) cells to stably integrate genetic material from other species. • Cells from the target genome are fused with hamster cells. The resulting cells are then screened for cells (hybrids) that have retained one or more of the chromosomes from the target genome. • Ideally, a complete set of hybrids can be constructed such that each has retained a single chromosome from the target genome.

  19. Somatic Hybrid Mapping Chromosome 1 2 3 4 5 Probe1 0 1 0 0 0 Probe2 0 0 1 1 0 Probe3 1 1 1 1 1 Probe1 -- maps to chromosome 2 Probe2 -- maps to chromosomes 3 and 4 -- possible paralogs, pseudogene, or low-copy repeat Probe3 -- maps to all chromosomes -- possible high-copy repeat or ribosomal genes

  20. Somatic Hybrid Mapping A subset of the data used to map the Blood Coagulating Factor III to human chromosome 1.

  21. Somatic Hybrid Mapping Finer mapping (higher resolution) can be obtained if hybrids are present in the panel that contain partial chromosomes. (E.g., translocations) Such a strategy is expensive, because numerous hybrids have to be screened to identify hybrids containing the partially retained chromosomes. A more cost-effective and high-resolution alternative is Radiation Hybrid Mapping.

  22. Radiation Hybrid Mapping Radiation hybrid mapping is a method for high-resolution mapping. Exploits the ability of rodent cells (hamster cells) to stably incorporate genetic material from fused cells. Pro: Resolution is “tunable”, relatively cheap Con: Difficult to compare results from different groups

  23. Radiation Hybrid Mapping

  24. Radiation Hybrid Mapping • The data obtained from a radiation hybrid experiment is similar to that from a somatic cell hybrid. It is the retention data for the given locus for each hybrid. • This data is generally displayed as a vector of numbers or letters… • 1 or + for retention • 0 or - for non-retention • 2 or ? for ambiguous or unknown • Ex. • RN_ALB 0100110102010001100100100000102210010.. • RN_HEM 0101110102000100101100200010100110010..

  25. Radiation Hybrid Mapping • Analytical methods -- • Many ranging from minimizing the number of obligate breaks to sophisticated methods relying on maximum likelihood or maximum posterior probability methods. •  = A+B- + A-B+ • TH(RA + RB - 2RARB) • d = - ln (1 - ) • NOTE:   [0,1]

  26. Summary of Mapping Strategies

  27. Comparative Mapping Can be very useful in utilizing animal models of human disease, and also in exploring the causes of complex diseases. Comparing gene content, localization and ordering among multiple species.

  28. Comparative MappingSources of Information sequence sequence BLAST mapping mapping potential orthologs colocalization Putative orthologs and syntenic segments

  29. Comparative MappingSources of Information • GeneMap 99 (human) • 42,000 ESTS • 12,500 genes • Mouse RH consortium (mouse) • 14,000 ESTs • UIowa EST placements (rat) • 13,793 ESTs

  30. Current Status • Initial comparative map (Welcome Trust and Otsuka Lab) • about 500 previously identified orthologs • human-mouse-rat • University of Iowa comparative maps • 13,973 placed ESTs • 3057 significant mouse hits • 9109 significant human EST hits • 10,148 significant hits to GenBank’s nt database • 2479 rat ESTs in preliminary human-rat comparative map • 1671 rat ESTs in preliminary mouse-rat comparative map

  31. Comparative MappingExamples RNO18 MMU18 0 1200 100 900 200 600 300 300 400

  32. Comparative MappingExamples HSA7 RNO4 400 0 RNO12 { } { } { 100 500 RNO12 { } HSA11 { } 600 200 { RNO5 { } 300 700 } HSA7p } HSA4

  33. Resources Genome browsers http://genome.ucsc.edu/goldenPath/hgTracks.html http://www.ensembl.org http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search GeneMap99 http://www.ncbi.nlm.nih.gov/genemap99

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