Kalachikov S M, Kel' A E, Dymshits G M, Ishov A M, Borkhsenius S N
Mol Biol (Mosk). 1994 Mar-Apr;28(2):444-52.
To obtain the universal polynucleotide hybridization probes for testing mycoplasmal contaminations in cell cultures, we have cloned several DNA fragments from the srRNA gene of Acholeplasma laidlawii. Before cloning, in order to exclude cross-hybridization of these probes with eukaryotic rRNA, the thermodynamic parameters of duplex formation between DNA complementary to mycoplasmal rRNA and eukaryotic rRNA had been studied. Using a set of computer methods, the region which forms weak heteroduplexes with eukaryotic srRNA was revealed. This region occupies positions 250 to 550 position of the mycoplasmal srRNA. Three different DNA fragments which include the region were generated in PCR, cloned in pUC18, and their hybridization characteristics were evaluated. In appropriate hybridization conditions the probes hybridize with all mycoplasmal RNAs studied without cross-hybridization with eukaryotic ribosomal RNA and DNA, and allow one to detect virtually any mycoplasmas (or any prokaryote) in cell cultures. Blot-hybridization of universal probes with mycoplasmal DNA digested by BsuRI allows one to identify the different species of mycoplasmas.
为了获得用于检测细胞培养物中支原体污染的通用多核苷酸杂交探针，我们从莱氏无胆甾原体的srRNA基因中克隆了几个DNA片段。在克隆之前，为了排除这些探针与真核生物rRNA的交叉杂交，我们研究了与支原体rRNA互补的DNA与真核生物rRNA之间双链体形成的热力学参数。使用一套计算机方法，揭示了与真核生物srRNA形成弱异源双链体的区域。该区域位于支原体srRNA的第250至550位。在PCR中产生了包含该区域的三个不同DNA片段，克隆到pUC18中，并评估了它们的杂交特性。在适当的杂交条件下，这些探针与所有研究的支原体RNA杂交，而不与真核生物核糖体RNA和DNA交叉杂交，并能检测细胞培养物中几乎任何支原体（或任何原核生物）。通用探针与经BsuRI消化的支原体DNA进行印迹杂交，可鉴定不同种类的支原体。
