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 A2B2A2B2 A2B2A2B2 A2B2A2B2 A2B2A2B2 A2B2A2B2 A2B2A2B2 A2B2A2B2 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B1A1B1 A1B2A1B2 A2B1A2B1 A1B2A1B2 A2B1A2B1 NR RR  = # recombinant / # total = 2/7 = 0.286

7. Genetic Mapping Theta calculation with inbred population… bn det+ bn bn+ det bn+ bn det+ detbn+ bn det bn bn+ det bn det+ detbn det bn bn+ det+ detbn bandeddetachedbanded, detached wild-type 483512 23 x x  = # recombinant # total = 5/1000 = 0.005

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. HindIIIBamHI PstII BamHI HindIII EcoRI

12. Restriction Mapping BglIIBamHIPstI BglII +BamHI BglII +PstI BamHI +PstI 4.2 5.2 3.6 3.5 3.3 2.6 1.7 1.4 1.2 1.0 1.2 0.7 0.9 0.5 0.3 BglII BamHIPstIBglIIPstI 0.30.7 2.60.90.51.2

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 AA!12 36 424 5120 6720 75040 840,320

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} A B A^B A’ B’ A^B’ 1 11 1 1 1 1 1 1 111 33 33 3 33 33 3 2 22 2 22 2 2 12 4 4 6 6 6 6 4320 map configurations, but only 208 distinct solutions.

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

16. Cytogenetic Mapping 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 Probe1 Probe2 Probe3 12345 Chromosome 01 11 11111 000 000 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. 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_ALB0100110102010001100100100000102210010.. RN_HEM0101110102000100101100200010100110010..

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 + T H (R A + R B - 2R A R B ) 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 Mapping Sources of Information sequence mapping BLAST potential orthologs colocalization sequence mapping Putative orthologs and syntenic segments

29. Comparative Mapping Sources 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 Mapping Examples RNO18 MMU18 300 600 900 1200 0 100 200 300 400

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