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BAC Libraries

BAC Libraries. Biol 446; 9.28.18 Alan Buser. What is a BAC?. Bacterial Artificial Chromosome Small pieces of circular bacterial DNA used as cloning vectors Based on F plasmid, also engineered to contain specific sequences DNA Libraries (Genomics)

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BAC Libraries

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  1. BAC Libraries Biol 446; 9.28.18 Alan Buser

  2. What is a BAC? • Bacterial Artificial Chromosome • Small pieces of circular bacterial DNA used as cloning vectors • Based on F plasmid, also engineered to contain specific sequences • DNA Libraries (Genomics) • BACs can store ~200 kbp, genomes are much larger but full of cut sites • DNA sequences are stable for many generations • Sequencing a genome in parts (shotgun) is simpler than doing so all at once • Colonies can also be stored until needed

  3. Digest Genomic DNA Gel Electrophoresis Ligation to Vector

  4. Let’s Get Historical • Human Genome project: 1990-2003 • PCR existed at this point, but impractical for the entire genome • Cloning existed as well, but yeast chromosomes (YACs) were challenging • How to use large sequences? And we need stability and easy retrieval

  5. OK, Neat. How do we make one? • Obtain genomic DNA • Digest with restriction enzymes • Select appropriately sized DNA fragments and ligate into BACs • Transform BACs into bacteria

  6. Lyse cells and digest with restriction enzymes Place agarose ‘plugs’ into gel for electrophoresis Immobilize cells in agarose gel Get DNA back into solution and ligate to BACs Measure location of 200 kbp, cut appropriate piece of gel Cut out ladders (on edges) an visualize with UV light Pick colonies into 96 well plates so each well contains a single BAC clone Transform BACs into bacteria, grow on selective media Scientists choice: use for sequencing or freeze for storage

  7. Bac to Bac Sequencing Requires physical ‘map’ of chromosome before sequencing This was the public lab approach to human genome sequencing

  8. Pure Shotgun: the Celera Approach This approach works from the bottom up Reliant on public BACs as well as their own sequencing from individuals

  9. Let’s get Mathematical • We can now see that only a few clones will ultimately be useful for sequencing, so excess coverage is necessary • A reasonable number is 5x the desired sequence length, how many colonies do we need to make a BAC? • 3 billion bp in human genome, assume 200 kbp per BAC (likely a little lower in reality) (3,000,000,000 * 5) / 200,000 = 75,000 individual BACs

  10. References Adema, C., Knight, M., Lewis, F., and Loker, E.S. nd. Request for making a BAC library from Biomphalaria glabrata (gastropod mollusc), the prominent snail species contributing to transmission of human schistomiasis. Adema, C., Luo, M., Hanelt, B., Hertel, L., Marshall, J., Zhang, S., DeJong, R., Kim, H., Kudrna, D., Wing, R., Soderlund, C., Knight, M., Lewis, F., Caldeira, R., Jannotti-Passos, L., Carvalho, O. and Loker, E. (2006). A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni. Memórias do Instituto Oswaldo Cruz, 101(suppl 1), pp.167-177. Chial, H. (2018). Human Genome Project: Sequencing the Human Genome | Learn Science at Scitable. [online] Nature.com. Available at: https://www.nature.com/scitable/topicpage/dna-sequencing-technologies-key-to- the-human-828 [Accessed 28 Sep. 2018]. Ghai, R. (2018). Map of the novel hybrid BAC vector pUvBBAC. [online] Research Gate. Available at: https://www.researchgate.net/figure/Map-of-the-novel-hybrid-BAC-vector-pUvBBAC-This-derivative-of-the- second-generation-BAC_fig1_5628943 [Accessed 27 Sep. 2018]. Shizuya, H., Birren, B., Kim, U., Mancino, V., Slepak, T., Tachiiri, Y. and Simon, M. (1992). Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proceedings of the National Academy of Sciences, 89(18), pp.8794-8797. Trivedi, B. (2018). SEQUENCING THE GENOME. [online] Genomenewsnetwork.org. Available at: http://www.genomenewsnetwork.org/articles/06_00/sequence_primer.shtml [Accessed 28 Sep. 2018]. Venter, J. C., Smith, H. O., & Hood, L. (1996). A new strategy for genome sequencing. Nature,381(6581), 364-366. doi:10.1038/381364a0 Yourgenome.org. (2018). What are BAC libraries?. [online] Available at: https://www.yourgenome.org/facts/what-are-bac-libraries [Accessed 28 Sep. 2018]. Zhang, H. and Wu, C. (2001). BAC as tools for genome sequencing. Plant Physiology and Biochemistry, 39(3-4), pp.195- 209.

  11. BAC Image credits: https://www.amazon.com/Backstreet-Boys-BACKSTREET-BOYS/dp/B0000A0A4D https://deadline.com/2015/10/back-to-the-future-day-racks-up-dough-from-trilogy-re-release-1201590945/ https://www.researchgate.net/figure/Map-of-the-novel-hybrid-BAC-vector-pUvBBAC-This-derivative-of-the-second-generation-BAC_fig1_5628943

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