PCR product with strands of unequal length.

Biochemical properties of ssDNA mnolecules compare interestingly with those of RNA: ssDNAs capable of binding target molecules ( 1-3) or cataltzing relactions (4-5) have been isolated from synthetic pools of randomii-sequenLce ssDNAs by repeated cycles of ini vitro selection and PCR amplification. The neC.ative (-) strands of the PCR product, while necessary foiaimplificaltionl. can interfere with bindine or catalytic activities of positive (+) strands. One method for redUcine this interterence has heeni to use asymmnetric PCR to generate imore (+) strands than strands (22). Ideally, all (-) strands would be removed fromn an amplified (+) ssDNA pool. Toward this goal, specific biotinylation of the (strand followed by streptavidin septaration has been' used ( 1,4,5), and an approach involv lug a detachable primller has recently been described (6). Biotin-based ssDNA preparation may be undesirable if .another biotin-based separation is planned durinu a different step of the in vitro selectioll protocol. Her-e we present a simple mnethod for purification of the (+) PCR produCt strands that involves incorporatinct a terminatorof (+)-strand synthesis within the (-)-strand PCR primer, causing thte tw^o product strands to differ significantly in length (Fig. 1) The (+) ssDNA cain then be readily purified by gel electrophoresis. The (-) ss DNA can also be saved and used to regenerate the comlplete pool. The (-)-strand PCR primierl has ai tripLartite comlpositioll: 5'-lengthener-terminator-complemiient (Fig. 1 A). The comiiplement segiment serves the priming functioni, the lengthener segment is responsible for the site difference of the two straLnlds. and the intervening termiiinatoiis noni-niucleotide material that blocks (+)-strand elongation. The ter-miiinatoris coimposed of two successive triethyleneglycol phosphate units, incorporated using1 the Glen Research spacer phosphoramidite 9. The sequence of the flankina DNA was designed to enhance terminator function. The lengtheniersequence is poly-dA, and a purinie is excluded ait the 5' position of the complemnent segment so as to prevent the terminal 3' nucleotide of the (+) strand leither the legitimate templated terminal 3' nuCleotide or the non-temiiplated 3' adenosine residue frequently added by Thq DNA polvmnerase (7 fromi bridging the terminatoito pair with the lengthener aand primlle continued extension. Efficacy of the iprimer-terminator' is illustrated by strandspecific labeling of PCR products, atnid -analysis hy deniatLuilngg.el electrophoresis (Fig. B). The (-) strands (laine I ) migrate mLuch more slowly than do the (+) stra.nds (lane 2) and termillnator readthrough is only 0.3%/(. A slice Sufficiently hroad to include most anomiialously migrating (+) strands (barred zone of' lane contains <2c%c of the radioactivitv of the fUill-lengDth (-) strand. Figure 1. lnccjiaL.l striand lengrth in E'( 'R Lsimn 'a primrcl-tCeroinilator. (SSchemiati ot E'CR prodILnet. thC (-Stl--sltlrd jriHer--tCe-nilMntoris inten-n-LtCd h\ spacir manteal ( ternntiato) that prevneilts + )-str-and1 clongation. (B Strand scpal-ation inI .1 deIntorin-g1 vel. The )-str-atnd pr-inelwals eitheiprillnletcilinn itol (I incs and 2 or normal lane,,s -3 and 4). Positions ot mariker-s illdi-in X1Iij divest of lpBR32 DNA) arec indicateL: S( 1()it niDNA \%MIld miki l-ite kitthin thle /one indicated h! thte h-a.