A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections.
Herein, a method is described to increase the information density of sequencing experiments used to deconvolute nucleic acid selections. The method is facile and should be applicable to any selection experiment. A critical feature of this method is the use of biotinylated primers to amplify and encode a BamHI restriction site on both ends of a PCR product. After amplification, the PCR reaction is captured onto streptavidin resin, washed, and digested directly on the resin. Resin-based digestion affords clean product that is devoid of partially digested products and unincorporated PCR primers. The product's complementary ends are annealed and ligated together with T4 DNA ligase. Analysis of ligation products shows formation of concatemers of different length and little detectable monomer. Sequencing results produced data that routinely contained three to four copies of the library. This method allows for more efficient formulation of structure-activity relationships since multiple active sequences are identified from a single clone.
Department of Chemistry and The New York State Center for Excellence in Bioinformatics and Life Sciences, University at Buffalo, Buffalo, New York 14260, USA.
Jessica L
JL
Childs-Disney
Matthew D
MD
Disney
D000818:Animals; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D015245:Deoxyribonuclease BamHI / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D017931:DNA Primers; D012313:RNA; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D015245:Deoxyribonuclease BamHI; D011088:DNA Ligases