Rapid sequencing of unpurified PCR products by thermal asymmetric PCR cycle sequencing using unlabeled sequencing primers.

Numerous methods have been developed for direct sequencing of single or double-stranded DNA amplified by PCR, and most require template purification steps prior to sequencing to remove excess unincorporated primers and dNTPs. Purification methods can be cumbersome, laborious and time consuming, or yield low DNA recoveries. Strategies for direct sequencing of doublestranded DNA products without purification steps have been described (1, 2), but these require use of end-labeled primers for sequencing, since primers carried over from the amplification reaction will generate superimposed sequence ladders if an unlabeled sequencing primer is used. Primer labeling has the disadvantage that the radioactive pattern of sequencing ladders decreases in strength with increasing distance from the primer. Furthermore, primer end labeling requires additional manipulations, and must be carried out periodically even when using the same primer. Here we report a new sequencing strategy that omits PCR product purification altogether, yet does not require end-labeled primers for generating clear sequencing ladders. A thermal asymmetric PCR method has been described for generating single-stranded DNA (3). Our sequencing strategy relies on thermal asymmetric PCR and utilizes one or two relatively short primers in the initial PCR amplification of target DNA. Subsequent sequencing is carried out by temperature cycling (4, 5, 6) using a longer sequencing primer. Sequencing cycles employ a high annealing temperature to avoid annealing and elongation of carried over short primers. The amplification reaction contains primers 15 -17 nt in length with Tms near or below 50°C as calculated by the formula: 69.3 + 0.41 (%GQ — 650/L, L = primer length (3). If the amplified product is to be sequenced from one end only, a single long primer at this end may be used in both the amplification and sequencing reactions. The PCR annealing temperature during amplification is set according to the Tm of the short primer(s) used. Following PCR, adequate amplification is confirmed by brief gel electrophoresis of 5 y\ aliquots and ethidium bromide staining. If product concentration is low, additional amplification cycles are carried out. When satisfactory, 10 fd of reaction mix (100-200 ng of product) is utilized directly in the subsequent sequencing reactions. The sequencing primer (20-25 nt) should have a Tm at least 10°C higher than the carried over primers. We routinely set the annealing temperature to the calculated Tm of the sequencing primer, or 1 -2°C higher or lower if carrying out simultaneous reactions with separate sequencing primers of slightly different Tms. Details of the amplification and sequencing reactions are described in the legend to Figure 1. Recently we developed a novel PCR method to recover genomic sequences flanking T-DNA insertions from transgenic Arabidopsis plant lines. This method utilizes a long T-DNA specific primer (Tm = 61 °C) and a short arbitrary, degenerate 16-mer (Tm = 44-49°C) to amplify the flanking sequences (manuscript in preparation). The amplified products from several transgenic lines were sequenced directly using the same T-DNA specific primer (Figure 1A). We also used this strategy to sequence nested deletion clones prepared in the pUCl 19 vector. Insert sizes of deletion clones were checked by PCR (30-35 cycles) carried out with bacterial cells directly (7), and aliquots from reactions indicating suitable deletion sizes were utilized directly in cycle sequencing. M13 forward and reverse primers used in the amplification reaction had calculated Tms of 51°C and 48°C, respectively, and the primer annealing temperature was set at 52°C. Cycle sequencing employed a long M13 forward (Tm = 62°Q or reverse (Tm =61°Q primer (Figure IB and 1C, respectively), with annealing in each cycle set at 62°C.