[A method for obtaining the normalized cDNA libraries based on the effect of suppression of polymerase chain reaction]. / Metod polucheniia normalizovannykh bibliotek kDNK, osnovannyi na éffekte supressii polimeraznoi tsepnoi reaktsii.

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 allow the selection of the equalized single-stranded fraction resulted 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 reaction (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 interval terminal repeats in PCR with a primer corresponding 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.

[Analysis of effectiveness of cDNA synthesis, induced using complementary primers and primers containing a noncomplementary base matrix]. / Analiz éffektivnost' sinteza kDNK, initsiirovannogo s komplementarnykh praimerov i praimerov, soderzhashchikh nekomplementarnye matritse osnovaniia.

We have studied the efficiency of DNA synthesis catalyzed by M-MLV reverse transcriptase or Thermus aquaticus DNA polymerase for primers (4-17 nucleotides long) either completely matched or possessing a single mismatched base pair at all possible positions in the primer. It has been shown that DNA synthesis efficiency depends not only on the position of mismatched base pair but on the length and primary structure of the primer. The enzyme, template, and primer concentrations determine the relative level of mismatched DNA synthesis.

[Derivatives of ddUTP, modified at the 5-position of uridine, as substrate terminators of reverse transcriptase. Hydrolysis of oligonucleotides, terminated by these analogs, by phosphodiesterase I]. / Proizvodnye ddUTP, modifitsirovannye v 5-polozhenii uridina, kak substratnye terminatory obratnykh transkriptaz. Gidroliz oligonukleotidov, terminirovannykh étimi analogami, fosfodiésterazoi I.

Synthesis of 2',3'-dideoxyuridine 5'-triphosphate analogues with fluorescent and biotin residues at C5 of uracil base was carried out. The substrate properties of these analogues were studied with AMV, M-MLV, and HIV reverse transcriptase. All 5-derivatives studied were shown to be incorporated into the 3'-terminus of oligonucleotide. The stability of oligodeoxyribonucleotides terminated with ddUTP analogues modified at the 5-position of the pyrimidine residue to the exonuclease action of phosphodiesterase I and Klenow enzyme was more than 1000 times higher than that of nonterminated oligonucleotides.