A simple ligation-based method to increase the information density in sequencing reactions used to deconvolute nucleic acid selections

RNA. 2008 Feb;14(2):390-4. doi: 10.1261/rna.576908. Epub 2007 Dec 7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Biotin / chemistry
  • DNA / chemistry*
  • DNA / genetics
  • DNA Ligases / chemistry
  • DNA Primers / chemistry
  • Deoxyribonuclease BamHI / chemistry
  • Humans
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • RNA / chemistry*
  • RNA / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, RNA / methods*
  • Streptavidin / chemistry

Substances

  • DNA Primers
  • RNA
  • Biotin
  • DNA
  • Streptavidin
  • Deoxyribonuclease BamHI
  • DNA Ligases