Russian Journal of Bioorganic Chemistry, Vol. 23, No. 11, 1997 p. 785

In vitro Cloning of DNA Fragments Using One Polymerase Chain Reaction

K. A. Lukyanov and S. A. Lukyanov1

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, GSP-7, 117879 Russia

Received April 16,1997; in final form, June 9, 1997

Abstract: An improved version of the in vitro DNA cloning method described earlier is proposed. The method allows amplification by polymerase chain reaction (PCR) of individual DNA molecules of an unknown primary structure followed by sequencing. The modifications described here provide for in vitro cloning by means of a 40–45-cycle PCR (the original protocol required two consecutive amplifications). In addition, the in vitro cloning is suggested to be carried out in special 96-well plates in the presence of ethidium bromide; upon UV irradiation, the wells containing amplified DNA fluoresce to make the analysis of all 96 wells unnecessary. The improved protocol makes the preparation of individual in vitro clones more straightforward and less expensive.

Key words: in vitro cloning, PCR suppression, subtractive cDNA hybridization, differential screening, ethidium bromide, 96-well plate