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Rapid, Direct Sequencing of Bacterial Artificial Chromosome Clones Using TransposomesÔ--Epicentre Technologies Corporation, 1202 Ann Street, Madison, WI  53713-2410; 608-258-3080

Dr. Jerome Joseph Jendrisak, Principal Investigator

Dr. Gary A. Dahl, Business Official

DOE Grant No. DE-FG02-00ER83003

Amount:  $100,000

 

Due to their ability to maintain large (100-200 kb) inserts in a stable form, Bacterial Artificial Chromosomes (BACs) have found acceptance in large-scale sequencing applications. Typically, DNA from each low-copy-number BAC clone is purified to remove host genomic DNA and then subcloned to make smaller “shatter clones” for sequencing and subsequent assembly based on sequence overlaps.  If there were a simple, low-cost way to directly sequence BAC clones, without the need to extensively purify BAC DNA or make subclones, the time and cost of large-scale sequencing projects could be greatly reduced.  New methods were recently developed which permit efficient and highly random insertion of an artificial transposon (with bi-directional sequencing primer binding sites) into DNA in living cells (Goryshin, et al., Nature Biotechnology 18: 97-100, 2000).  This method will be used to generate insertion clones in BAC DNA by developing a specially-designed artificial transposon and a bacterial host in which chromosomal insertions will be lethal, thus permitting controlled generation of high-copy-number BAC insertion clone templates for use in direct sequencing following a simple alkaline lysis extraction of host DNA.  After proving the concept in small scale as part of a Phase I research program, implementation of the technology in existing and upcoming large-scale genomic sequencing projects will be pursued in Phase II.  We will construct a new artificial transposon containing a selectable marker, a conditional origin of replication, and bi-directional sequencing primer binding sites.  A new E. coli host cell will be constructed which will conditionally express up to 200 BAC molecules per cell but which, upon selection, is killed if the transposon inserts into the host chromosome. Then, a Transposome™ (a complex between hyperactive Tn5 transposase and an artificial transposon; see Goryshin, et al., Nature Biotechnology 18: 97-100, 2000) will be electroporated into living cells to obtain efficient and highly random transposon insertions into BAC clones.  Independent random insertion clones for sequencing will be selected simply by plating on medium containing an antibiotic to which resistance is encoded by the transposon, and BAC sequencing templates will be obtained by a simple alkaline lysis DNA prep.

 

Commercial Applications and Other Benefits as described by the awardee:  If successful, the methods developed will simplify and speed up genome sequencing operations, thereby greatly reducing the costs and time required to complete the Human Genome Project and future large-scale sequencing programs.  Also, because the proposed methods eliminate the duplication of methods, the assembly of the sequences may be easier and more accurate, so that the amount of sequencing redundancy needed to obtain an accurate, complete sequence could potentially be reduced.