BioSci D145 Winter 2014: Osoegawa Presentation

1. BioSci D145 Winter 2014: Osoegawa Presentation By: Argenis Ruvalcaba & Andrew Banuelos

2. Big Picture The researchers worked with mice (murine genome) because they provide the ability to identify, manipulate, and understand genes related to humans. Comparative analysis between mouse and human simplifies the understanding of processes affected in human diseases. They examined the usefulness of BAC and PAC libraries for genome research by constructing the libraries and performing functional analysis.

3. Background Genomic library: a collection of the total genomic DNA from a single organism Clone: copy of DNA (genetic material) Contig: a set of overlapping DNA segments that together represent a consensus region of DNA (below) BAC = Bacterial Artificial Chromosome PAC = P1-derived Chromosome (from bacteriophage P1) YAC = Yeast Artificial Chromosome

4. BACs and PACs BAC: an engineered DNA molecule used to clone DNA sequences in bacterial cells. As the bacterial cells grow and divide, they amplify the BAC DNA, which can then be isolated and used to sequence DNA. PAC: similar cloning vector, but from the bacterial P1-plasmid. Advantages: Stability, replication as plasmids, stringent copy control, allows you to copy large DNA sequences, can live in bacterial cultures. sNational Human Genome Research Institute

5. Methods Pulsed-field Electrophoresis (sizing) and Agarose Gel Electrophoresis (separate DNA) LB Media and colony were used to grow PAC and BAC clones and SOC medium was used to induce the expression of the antibiotic resistant gene. Constructed a large insert PAC library in a bacterial/mammalian shuttle for functional gene studies. Constructed BAC libraries for genome mapping and sequencing.

6. Methods cont. Recombinant clones were arrayed by picking colonies into 384-well plates containing LB with glycerol and antibiotics. PAC and BAC DNA insert size analysis was performed. Libraries were screened with 22 probes and sequencing was performed by the use of a Perkin Elmer Applied Biosystems 377 DNA Sequencer (depicted at right).

7. Results

8. Constructing Mouse PAC & BAC Libraries • 1 PAC (top) and 2 BAC (2 bottom) libraries were prepared from different strains (DNA sources) • RPCI-21 PAC library was prepared by 6 ligations of MboI partially digested DNA and the BamHI-cut pPAC4 vector. • RPCI-22 was constructed from 2 ligations. • RCPI-23 was prepared using 9 ligations.

9. Size distribution for genomic inserts • Size distribution for the RPCI-21 • A total of 434 clones from RPCI-22 • Library provides a 11.4 fold mouse genome representation for RPCI-21.

10. Size distribution for genomic inserts • Size distribution for the RPCI-22 • A total of 282 clones from RPCI-22 • Library provides a 10.9 fold mouse genome representation for RPCI-22.

11. Size distribution for genomic inserts • Size distribution for the RPCI-23 • A total of 285 clones from RPCI-23 • Library provides a 11.2 fold mouse genome representation for RPCI-23.

12. Screening the Libraries • 22 markers were amplified from 129-strain genomic DNA. • Markers were used for hybridization-based screening of the 3 libraries. • A large number of of positive clones identified in RPCI-21 library using three probes: AMY1A, GAPD, and LDHA. • The 19 other probes were used to look at the genome representation in all of the libraries.

13. Southern Hybridization • Detected contig sequences • Used to confirm positive clones and clone integrity • Used to evaluate the genomic representation of the library being mapped • Important to the library screening process

14. Screening Libraries • In total 28 Markers were used to find genomic redundancies in the genomic inserts. • Genomic redundancies represent the amount of genes found at a specific locus, depending on its fold representation.

15. Clone Chimerism & Rearrangements • BAC and PAC end sequences were used to design hybridization probes for screening all of the overlapping clones. • Clone and marker analysis was performed for the CAT, IGFBP1, and MLR markers. • Inconsistent markers can identify clone rearrangements or deletions. • Only 1 chimeric clone and 6 deletions were observed from the 113 total PAC and BAC colonies. • 71 of 113 clones were nonchimeric since both markers were mapped to the confirmed contigs.

16. BAC-PAC contigs • To the left is a picture of one of three the 400-kb high resolution end probe-based BAC-PAC contigs • The dots at the end of the 1-221A10 indicate unusual representation of contig markers. This is indicative of a chimeric clone found.

17. Clone heterogeneity • There 3 small rearrangements were detected in the contigs as heterogeneity between duplicate subcolonies. • Inconsistent bands in fingerprint of a clone that is not present in other clones is a sign of a rearrangement • 4 out of 113 clones analyzed showed inconsistent patterns. • 3 out of 113 clones showed clonal heterogeneity.

18. Summary • Goal = BAC libraries with low levels of cloning artifacts, high clone stability, and an unbiased representation of the entire genome. • BAC and PAC libraries are useful resources for mouse genome mapping, sequencing, and functional analysis. • BAC and PAC chimeric clones occur very infrequently at levels about 1% and they are stable. • Mapped markers are derived from BAC & PAC since FISH assay does not work well with low chimeric cloning levels. • Thus the usefulness of BAC libraries for genomic research is supported.

19. Thank you for your time Questions?