Method enabling pyrosequencing on double-stranded DNA

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing meth od that takes advantage of co-operativity between four enzymes to monitor D NA synthesis (M. Ronaghi, M. Uhlen, and P. Nyren, Science 281, 363-365). Py rosequencing has so far only been performed on single-stranded DNA, In this paper different enzymatic strategies for template preparation enabling pyr osequencing on double-stranded DNA were studied. High quality data were obt ained with several different enzyme combinations: (i) shrimp alkaline phosp hatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exon uclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuc lease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide , single-stranded DNA-binding protein, and Klenow DNA polymerase improved t he sequence quality. Apyrase was the fastest and most efficient of the thre e different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyr osequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content. (C) 2000 Ac ademic Press.