Bauer P, Rolfs A, Regitz-Zagrosek V, Hildebrandt A, Fleck E
Humboldt Universität, Berlin, Germany.
Biotechniques. 1997 Jun;22(6):1128-32. doi: 10.2144/97226st05.
The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.
通过逆转录聚合酶链反应（RT-PCR）对无内含子基因的稀有mRNA拷贝进行精确定量时，需要完全去除基因组DNA，因为无法通过PCR产物的不同大小来区分cDNA和DNA扩增产物。在RT-PCR之前，通过用无RNase的DNase I处理RNA样品来实现DNA的消除。RT之前，经DNase处理的RNA样品缺乏PCR产物通常被视为有效DNA破坏的证据。然而，这可能因DNase I切割中使用的金属辅因子而异。用DNase I和镁作为辅因子处理受DNA污染的RNA样品，消化后会产生一个阴性PCR对照，无需进一步RT。矛盾的是，在额外的RT-PCR之后，可能会再次产生原始的含内含子DNA片段大小。在存在锰作为辅因子的情况下，RT产生的DNA片段不会出现。这是因为在存在锰的情况下，DNase I在大约相同的位点切割两条DNA链，产生平端或末端仅突出一两个核苷酸长度的DNA片段。然而，用镁作为辅因子进行DNase消化会产生有重组潜力的重叠片段。由于在DNase I消化和RT后，无法区分由RNA产生的PCR信号和由重组DNA产生的PCR信号，所有此类DNase I检测都应用锰而不是镁进行。
