Kim_et_al_1992_-_Fosmids

Kim_et_al_1992_-_Fosmids - NucleicAcids Research, Vol....

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Unformatted text preview: NucleicAcids Research, Vol. 20,No. 5 1083-1085 Stable propagation of cosmid sized human DNA inserts in an F factorbased vector Ung-Jin Kim, Hiroaki Shizuya, PieterJ.de Jong', Bruce Birrenand Melvin l.Simon* DivisionofBiology, 147-75,California Instituteof Technology, Pasadena, CA 91125 and 'Human Genome Center, L-452, Lawrence Livermore National Laboratory,Livermore, CA 94550, USA Received December 2,1991;Accepted January5,1992 ABSTRACT InstabilityofcomplexmammaliangenomicDNA inserts iscommonplace in cosmid libraries constructed in conventional multicopyvectors.To developameans toconstructstablelibraries,we have developed alow copynumbercosmidvector based onthe E.coll Ffactor replicon(Fosmid).We havetestedrelativestabilityof human DNA insertsinFosmidsandintwoconventional multicopy vectors (Lawrist 16 and Supercos) by comparing the frequency of changes in restriction patterns of the inserts after propagating randomly pickedhumangenomicclonesbasedonthese vectors. We found that the clones based on Fosmid vector undergo detectable changes at a greatly reduced frequency. We also observed that sequences that undergodrasticrearrangementsanddeletionsduring propagation in a conventional vector were stably propagated when recloned as Fosmids. The results indicate that Fosmid system may be useful for constructingstablelibrariesfrom complex genomes. INTRODUCTION Duetothepresenceofmultiplerepeatedelementsandmodified bases,mammalian genomicDNA clonedinE. colifrequently undergoesmodifications includingrearrangementsanddeletions. AlthoughtheinstabilityofhumanDNA insertsingenomiccosmid librariesisreported(1)andwidely acknowledgedinanecdotal fashion,quantitativeanalysis onthe instabilityofcomplexinserts in genomiclibrarieshasnotbeendocumentedtodate.Use of hoststrainswithvariousdeficienciesintherecombinationsystem could improve thestabilityofmammalian genomic libraries (2-5). Inadditiontothe majorhostrecombinationfunctions, DNA repairactivitiesand normallyminorpathwaysthatmediate recombinationinthethepresenceofDNA fragmentscontaining highlyreiteratedmammalianDNA sequencesin multiplecopies may be another factor causing frequent rearrangement and deletioninE.coli.Thesehostsystemscannotallbeeliminated sincethe viabilityandgrowthrate must be considered. Thereforewe have developeda new cosmid cloningsystem (referredtoasFosmid system)thatemploystheuse ofa low copynumber plasmidbasedonthe F factor replicationorigin. We have demonstrated the ability of this vector to stably propagatehumangenomicDNA insertsincomparisonwiththat ofotherconventionalmulticopycosmids....
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Kim_et_al_1992_-_Fosmids - NucleicAcids Research, Vol....

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