Restriction generated oligonucleotides utilizing the two base recognition endonuclease CviJI

• Published in: Nucleic acids research Vol. 22; no. 8; pp. 1470 - 1475
• Main Authors: SWAMINATHAN, N, GEORGE, D, MCMASTER, K, SZABLEWSKI, J, VAN ETTEN, J. L, MEAD, D. A
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 25-04-1994
• Subjects: Base Sequence; Biological and medical sciences; Clostridium perfringens - genetics; Deoxyribonucleases, Type II Site-Specific - metabolism; Diverse techniques; DNA - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Micrococcus luteus - genetics; Molecular and cellular biology; Molecular Sequence Data; Oligodeoxyribonucleotides - metabolism; Oligonucleotide Probes; Polymerase Chain Reaction; Sequence specificity; Enzyme; Cleavage site; DNA; Restriction endonuclease; Enzymatic digestion; Hydrolases; Restriction fragment; Esterases; Molecular probe; Method; Oligodeoxyribonucleotide
• ISSN: 0305-1048; 1362-4962

The conversion of an anonymous DNA sample into numerous oligonucleotides is enzymatically feasible using an unusual restriction endonuclease, CviJI. Depending on reaction conditions, CviJI is capable of digesting DNA at a two or three base recognition sequence. CviJI normally cleaves RGCY sites between the G and C to leave blunt ends. Under 'relaxed' conditions CviJI* cleaves RGCY, and RGCR/YGCY, but not YGCR sites. In theory, CviJI* restriction of pUC19 (2686 bp) should produce 157 fragments, 75% of which are smaller than 20 bp. Instead, 96% of the CviJI* fragments were 18-56 bp long and none of the fragments were smaller than 18 bp. Thermal denaturation of these fragments generates sequence specific oligonucleotides homologous for the cognate template. The enzymatic conversion of anonymous DNA into sequence specific oligomers has implications for several conventional and novel molecular biology procedures.