1. Bacterial Artificial Chromosome Libraries BY JOE HAYES UNIVERSITY OF NEW MEXICO DEPARTMENT OF BIOLOGY BIO446

2. Say What Dude??

3. What are BACs and BAC Libraries?  Bacterial Artificial chromosome libraries are an effective tool for doing genomic research that consist of using a DNA construct, based on F-plasmid genes, as a vector for molecular cloning and cellular transformations.  BACs are modified bacterial chromosomes (circular& lack telomeres)  The BAC act like a vector to carry DNA fragments(large) into bacterial cells where they can be copied  Bacterial cells can be easily cultured and stored, storing the entire genome in the process  Episome vs Plasmid!

4. Why use BAC libraries? DNA sequencing can be an expensive and tedious process. Early sequences were only of small genomic organisms( Phages and plasmids)Why? Well….. Reliability and plasmid cloning fragment size limitations(~5000Bp) For GBp long genomes, such as snails, this would take 200,000plasmids for only one gig(still not enough for proper sequencing due to the probability of fragment repeats)!!! BACs are capable of up taking base pair fragments of 100-500Kbp ~60000 BACs/1GBp

5. Methodology

6. Recap  So to recapitulate….  We take our genome DNA of interest  We fragment it with restriction enzymes to select for desired sizes  We use the DNA construct/Bacterial Artificial Chromosome to uptake DNA(Large scale genomic cloning)  Take BACs and insert them into bacteria(E coli)  Spread plate to culture  Selection for correct inserts using various chemicals, and phenotypic expressions

7. Continuing on  Southern blotting can then be used to ID clone with the insert of interest  Computer recognition of particular spot on filter paper, labeled and archived  This can then be used for easy recognition and retrieval of desired colonies

8. Other applications?  Sequence a gene region  ID and sequence desired genes  Sequence BAC ends Allows for the detection of variation in polymorphisms, deletions, insertions, inversions etc. through sequencing of the ends of the inserted DNA fragments in BAC libraries Structural variance in disease tissue Evolutionary studies Physical mapping

9. FISH (Fluorescence in-situ hybridization)

10. Quick FISH Video  https://www.youtube.com/watch?v=2Z29usot2Dg https://www.youtube.com/watch?v=2Z29usot2Dg  https://www.youtube.com/watch?v=w5l5SmKvS1o https://www.youtube.com/watch?v=w5l5SmKvS1o

11. Eventual Replacement  Exponential increases in technological advancement and ideas have lead way to the replacement of BAC libraries almost entirely  NGS (next generation sequencing)  Requires less DNA  Also more sensetive  More economical$$$

12. FIN  Thanks  Colleagues  UNM department of Biology

13. References  https://en.wikipedia.org/wiki/Genomic_library https://en.wikipedia.org/wiki/Genomic_library  https://en.wikipedia.org/wiki/DNA_construct https://en.wikipedia.org/wiki/DNA_construct  https://en.wikipedia.org/wiki/Bacterial_artificial_chromosome https://en.wikipedia.org/wiki/Bacterial_artificial_chromosome  https://en.wikipedia.org/wiki/End-sequence_profiling https://en.wikipedia.org/wiki/End-sequence_profiling  http://filmgarb.com/wp-content/uploads/film-the_big_lebowski-1998- the_dude-jeff_bridges-tops-pendleton_shawl_cardigan.jpg  C.M. Adema, M.-Z. Luo, B. Hanelt, L.A. Hertel, J.J. Marshall, S.-M. Zhang, et al., A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni, (2006)  H. Wu, Z. Xia, Achievement, Challenge and Future Perspective of BAC Library Technology with a Focus on its Application to Soybean Genomics Researches.2015