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常见的反转录酶主要有： M-MLV 、 AMV 、 Tth 。通常 MLV 用于普通的 RT 反应， AMV 用于基因比较复杂、有二级结构或 GC 含量较高的 RT 反应， T th 用于单酶体系、基因结构较复杂的 RT 反应；如果 cDNA 要用于克隆表达， 建议使用 MLV 或 AMV ；如果用于基因表达水平的检测或杂交探针的制备，建议使用 Tth 。
RT 反应，引物的选择
随机引物 | 适用于长的或具有发卡结构的 RNA ；适用于 rRNA 、 mRNA 、 tRNA 等所有 RNA 的反转录反应；主要用于单一模板的 RT-PCR 反应。 |
Oligo dT | 适用于具有 PolyA 尾巴的 RNA 。（原核生物的 RNA 、真核生物的 Oligo dT rRNA 和 tRNA 不具有 PolyA 尾巴。）由于 Oligo dT 要结合到 PolyA 尾巴上，所以对 RNA 样品的质量要求较高，即使有少量降解也 会使全长 cDNA 合成量大大减少。 |
特异性引物 | 与模板序列互补的引物，适用于目的序列已知的情况。 |
影响 RT 反应的因素
影响 RT 反应的因素比较多，主要是 RNA 的质量，要求没有 RNA 酶的污染、没有基因组的污染；另外还有引物的选择是否合适；酶的浓度是否合适等。
FicoScript®M-MLV Transcriptase(反转录酶)
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FicoScript®Reverse Transcriptase（反转录酶） is an engineered version of M-MLV RT with reduced RNase H activity and increased thermal stability. The enzyme is purified to near homogeneity from E. coli containing the modified pol gene of Moloney Murine Leukemia Virus . The enzyme can be used to synthesize first-strand cDNA at temperatures up to 48°C, providing increased specificity, higher yields of cDNA, and more fulllength product than other reverse transcriptases. It can generate cDNA from 100 bp to >12 kb.It is suitable for qPCR.
1 、 Enzyme Storage Buffer: M-MLV Reverse Transcriptase is supplied in 20mM Tris-HCl (pH 7.5), 200mM NaCl, 0.1mM
EDTA, 1mM DTT, 0.01% Nonidet P-40 and 50% glycerol.
2 、 M-MLV Reverse Transcriptase 5X Reaction Buffer: When the M-MLV Reverse Transcriptase 5X Reaction Buffer supplied with this enzyme is diluted 1:5, it has a composition of 50mM Tris-HCl (pH 8.3 25°C ), 75mM KCl, 3mM MgCl 2 and 10mM DTT.
3 、 Source: Purified from an E. coli strain expressing a recombinant clone.
4 、 Storage Conditions: Store at – 20°C . Avoid exposure to frequent temperature changes.
5 、 Unit Definition: One unit is defined as the amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37°C . The reaction conditions are: 50mM Tris-HCl (pH 8.3), 7mM MgCl 2 ,
40mM KCl, 10mM DTT, 0.1mg/ml BSA, 0.5mM [ 3 H]dTTP, 0.025mM oligo(dT) 50 , 0.25mM poly(A) 400 and 0.01% NP-40.
6 、 Usage Note: M-MLV Reverse Transcriptase is less processive than AMV Reverse Transcriptase, and therefore, more units of the M-MLV enzyme are required to generate the same amount of cDNA as in the AMV reaction. Thus, starting with 1μg of mRNA
in a first-strand cDNA synthesis, 200 units of the M-MLV enzyme are recommended as opposed to 25 units of the AMV enzyme.
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