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Bassem M. Shoucri Bio Sci D145 January 9, 2014

Bassem M. Shoucri Bio Sci D145 January 9, 2014. Why do we want to map genomes?. Clinical applications! Identify genes causing diseases Treatment? Compare genomes of different species Understand overall genome structure Understand relationship between genes and regulatory elements

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Bassem M. Shoucri Bio Sci D145 January 9, 2014

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  1. Bassem M. Shoucri Bio Sci D145 January 9, 2014

  2. Why do we want to map genomes? • Clinical applications! • Identify genes causing diseases • Treatment? • Compare genomes of different species • Understand overall genome structure • Understand relationship between genes and regulatory elements • Map vs. sequence

  3. Classic linkage mapping • “distance” on map is actually recombination frequency (cM)

  4. During meiosis • Recombination (meiosis I) • Segregation (meiosis II)

  5. HAPPY Mapping: HAPloid equivalents of DNA and the PolYmerase chain reaction • Mapping method that emulates recombination and segregation in vitro • Why? • Eliminate need for in vivo experiments • Control over frequency of breakage • Claim more feasible than existing methods

  6. Meiosis Recombination HAPPY Segregation

  7. Maps need markers • “a marker can be any single-copy sequence that can be amplified using the PCR to give copies that can be identified” • Examples: • Sequence-tagged sites (STS) • Variable number tandem repeats (VNTR) • Single nucleotide polymorphisms (SNP) • Restriction fragment length polymorphism (RFLP)

  8. Approach Isolate genomic DNA Fragment randomly Dilute haploid equivalents Assess linkage, assign LOD Create map

  9. Methods • Mapped 7 markers at the DMD locus (1.24 Mbp) on the X chromosome • Lymphocytes from female blood  captured in agarose beads (3 ug DNA/mL packed beads) • Break DNA by γ-irradiation or shearing

  10. Methods cont’ • Load DNA onto gel for Pulse-field gel electrophoresis (PFGE) • Extract DNA of appropriate size • 1.75, 2.75 Mbp from γ-irradiated samples • 0.05 – 0.4 Mbp from sheared samples

  11. Mapping panel • 2-phase or nested PCR • Reduce nonspecific products • First external primers (9-EXT) then internal primers (9-INT) • Also carried out whole genome amplification (via random 15-mers) and 2-phase PCR

  12. Assessing linkage • LOD score: probability the markers are linked. Logarithm of the ODds> 3 (1000:1) is evidence for linkage • Θ, recombination fraction: probability there is breakage between markers. Maximum Θ of 1 = complete breakage

  13. LOD,Θ for 1.75 Mbp map

  14. Compare LOD, Θ with known inter-marker distances! • Large fragments allow for reliable mapping over greater distances • Small fragments allow for greater resolution

  15. HAPPY Maps! • Brackets indicate uncertain order • Sheared fragments (not shown) can map small distances (D31-D32) Known map 1.75 Mbp 2.75 Mbp WG 1.75 Mbp

  16. Discussion, Conclusions • in vitro approach to linkage mapping using a clever analog of meiosis • Pros • Control frequency of breakage • Can use any marker • No cloning required • Cons • Must know something about sequence • Whole genome amplification is limiting

  17. “Up to date, only eight maps have been generated using the HAPPY approach and all of them were contributed by the inventors’ group [Dear and Cook].”

  18. Why so SAD? • Pros • Control frequency of breakage • Can use any marker • No cloning required • Cons • Must know something about sequence • X. tropicalis genome sequenced but NOT assembled • Whole genome amplification is limiting • Material, coverage, amplification bias • Resolve with Multiple displacement amplification (MDA, Φ29 polymerase)  SNPs

  19. Frogs?? • Study of vertabrate embryogenesis • X. tropicalis • Only diploid Xenopus • 10 chromosome pairs • Sequenced, not assembled

  20. Sequencing will only get you so far… • Contig is a continuous stretch of gDNA in which the sequence is known with high confidence

  21. Assembling the genome • 90% X. tropicalisgenome are contigs (1.33Gbp), 10% gaps • Genome is complex with many scattered repeats • Interspersed gaps interfere with understanding of complete genome

  22. Methods are essentially the same EXCEPT • SNPs as markers • MDA for genome wide amp. Jiang et al. Int J Biol Sci. 2009.

  23. MDA • Primers are bound • Φ29 DNA Pol elongates, strands are displaced • Displaced strands can also serve as primer templates • LOTS of material is generated Lovmar et al. Hum Mutat. 2009.

  24. HAPPY Mapping of X. Tropicalisusing SNPs within ultra-conserved elements

  25. Further reading… • Jiang, Rokhsar, Harland. Int J Biol Sci. 2009; 5(6): 621. • Acknowledgment • Dr. Amanda Janesick

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