Russian Journal of Bioorganic Chemistry, Vol. 22, No. 9, 1996, p. 587
A Method for Obtaining Equalized cDNA Libraries Based on Polymerase Chain Reaction Suppression
K. A. Luk’yanov*, N. G. Gurskaya*, M. V. Matts*, G. L. Khaspekov**, L. B. D’yachenko***, A. A. Chenchik***, S. G. Il’evich-Stuchkov, and S. A. Luk’yanov* 1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,ul. Miklukho-Maklaya 16/10, Moscow GSP-7, 117871 Russia
** Cardiological Center, Russian Academy of Medical Sciences, Moscow , Russia , *** Clontech, Palo Alto , California , USA
Abstract: A new efficient method for obtaining cDNA libraries with equal representation of all cDNA types (equalized libraries) in a single round of equalization was developed. The method is based on differences in the renaturation kinetics of double-stranded cDNAs of different genes and allows the selection of the equalized single-stranded fraction resulting from the incomplete reassociation of the total cDNA without laborious and inefficient physical separation. The equalized single-stranded fractions are selectively amplified by polymerase chain reac-tion (PCR). The amplification of other DNA molecules is inhibited due to PCR suppression, i.e., the suppression of amplification of the DNA molecules flanked with long inverted terminal repeats in PCR with a primer corre-sponding to the external moiety of the repeat. The efficiency of the developed method was estimated in obtaining an equalized cDNA library based on mRNA from the activated human T lymphocyte Jurkat cell line.
Key words: cDNA, representation of mRNA, equalization