Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer.
Two DNA duplexes of identical sequence and 35 nt in length were synthesized by an original and a highly improved version of phosphoramidite chemistry. By base composition analysis, DNA synthesized by improved chemistry (termed DMTS-imp) contained no detectable modified bases while DNA synthesized by the original chemistry (termed DMTS-std) had a large number of modifications. Under optimal reaction conditions, HhaI and RsaI cleaved the DMTS-std duplex to 76-77% completion and the DMTS-imp duplex to 96-99% completion. Restriction analysis and piperidine treatment yielded estimates of approximately 3.0% modified nucleotides in DMTS-std and approximately 1.0% in DMTS-imp. Overall, the improvements in chemistry increased the restriction efficiency of synthetic DNA up to 10-fold.
Farrance
I K
IK
Department of Genetics, University of Georgia, Athens 30602.
Eadie
J S
JS
Ivarie
R
R
D000225:Adenine; D001482:Base Composition; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000032:analysis / Q000378:metabolism; D015254:DNA Modification Methylases; D004262:DNA Restriction Enzymes / Q000378:metabolism; D006147:Guanine; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009943:Organophosphorus Compounds; D010880:Piperidines; D015183:Restriction Mapping / Q000379:methods; D013379:Substrate Specificity; D013941:Thymine
D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D010880:Piperidines; D006147:Guanine; C049676:diisopropyl phosphoramidite; C032727:piperidine; D004247:DNA; D015254:DNA Modification Methylases; D004262:DNA Restriction Enzymes; D000225:Adenine; D013941:Thymine