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System speeds up the pace of sequencing Schematic of New England Biolab's Genome Priming System Transposons are nothing new to molecular biologists--they have been used since the early 1970s for creating mutations, as well as for moving sequences from place to place in vivo. In the Genome Priming System (GPS™), New England BioLabs (NEB) has developed a novel, in vitro application of transposons for the production of sequencing templates. GPS replaces primer walking, nested deletions, and random subcloning, all of which can be tedious and time consuming, through the random insertion of a sequencing primer site--bearing transposon into a target DNA. Transposition can be a complicated process, requiring as many as four proteins and often resulting in complex products. But recently, Nancy Craig and Anne Stellwagen1,2 from Johns Hopkins University isolated a mutant that will transpose in vitro through a simple procedure to yield simple products. NEB's senior research scientist, Lise Raleigh, credits this discovery as the enabling technology for the development of the GPS kit. The mutant inserts randomly at high frequencies, generating 103-105 product molecules per reaction. Wild-type transposons, on the other hand, yield complex products, insert inefficiently, have strong site preferences, or combine some of these. GPS is a three-step process. First the donor transposon is incubated with the target DNA, such as a plasmid or cosmid, for the assembly process. The transposition reaction is then initiated by the addition of Mg2+. Finally, host bacteria are transformed--the entire process requires only 90 minutes. Because of the lack of site preference and "target immunity," which prohibits a second transposon insertion once a transposition event has occurred, this process generates a population of target molecules, each with a different insertion, that can be sequenced directly without subcloning or screening. Robert Munson, professor of Pediatrics at Ohio State University, who has been testing the GPS kit in his laboratory, has found that it works very well, and feels that it will be the method of choice for some sequencing projects. In conjunction with an automated sequencer and the now-standard software tools for stripping off primer and transposon sequences, a 5-10 kb plasmid can be sequenced completely in a few days, according to Munson. And while the coverage required might be greater than with other approaches, he feels the timesaving is worth the expense of doing sequencing. The donor transposons in GPS have other useful features. Two versions are provided with each kit: one that carries kanamycin resistance and one with chloramphenicol resistance, providing some flexibility to the user in choosing a target plasmid. In addition, the donors contain unique rare-cutting restriction enzyme sites to ease the process of restriction mapping, should it be required. Finally, the positions of the primer sites on the donor transposon are optimized for sequence acquisition--far enough from the ends that the first target base can be read, while not so far as to generate unwanted data. GPS works well with plasmids and cosmids, according to NEB, or any template that can be easily grown in moderate quantities. And the company is working to improve the system's efficacy with hard-to-get templates, like BACs. But for many sequencing projects, GPS changes the time-limiting step from template construction to sequencing. A.E. Stellwagen and N.L. Craig, "Gain-of-function mutations in TnsC, an ATP-dependent transposition protein that activates the bacterial transposon Tn7", Genetics 145,573-85, 1997. A.E. Stellwagen and N. L. Craig, "Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition", EMBO Journal 16:6823-34, 1997. The author, Laura De Francesco, can be contacted at ldefrancesco@the-scientist.com.For more information, please contact NEB at (800) 632-7799 or visit the company web site at www.neb.com.

Run, Don't Walk

Feb 14, 1999

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