RAPID PRODUCTION OF SINGLE-STRANDED SEQUENCING TEMPLATE FROM AMPLIFIED DNA USING MAGNETIC BEADS

This chapter presents a general method for producing a pure preparation of single-stranded DNA (ssDNA) sequencing template from double-stranded DNA (dsDNA) amplified using the polymerase chain reaction (PCR). The amplified DNA is synthesized using one biotinylated and one nonbiotinylated primer and is bound to streptavidin beads via the biotin molecule. The two DNA strands are then dissociated and the nonbiotinylated strand is released and recovered from the supernatant. It produces ssDNA from templates of any amplifiable length, without strand bias and with sufficient yield for several sequencing reactions. This method utilizes the sensitivity and yield of double-strand PCR reactions and provides pure templates that give optimal sequencing results. The double-stranded amplified DNA to be sequenced is generated in two separate, reciprocal reactions. The streptavidin capture method is applicable to any PCR product, in that the efficiency of recovery of ssDNA does not appear to be affected by the length, base composition, or secondary structure of the amplified region. The method allows direct sequencing of amplified DNA, which for many applications is preferable to sequencing individual, cloned PCR products because direct sequencing obscures any random errors induced by Taq polymerase. © 1993