Aatsinki J T, Lakkakorpi J T, Pietilä E M, Rajaniemi H J
University of Oulu, Finland.
Biotechniques. 1994 Feb;16(2):282-4, 286-8.
A one-step (all reactants added simultaneously) reverse transcription and polymerase chain reaction (RT-PCR) procedure for amplification of full-length open reading frames (ORFs) of relatively rare transcripts was developed. It was applied for cloning rat luteinizing hormone/chorionic gonadotropin receptor cDNA isoforms larger than two kb. In the procedure developed, manual work is minimized, thus large numbers of samples can be handled, since after denaturation of template RNA and the primers and addition of other reagents, no further manual steps are needed. No inhibitory effect of avian myeloblastosis virus (AMV) reverse transcriptase (RT) on Thermus aquaticus (Taq) DNA Polymerase was found. This was because, under the conditions described, Taq DNA Polymerase effectively amplified picogram amounts of plasmid DNA or template reverse transcribed from nanograms of total ovarian RNA in the presence of AMV-RT. Even a large excess of AMV-RT did not inhibit Taq DNA Polymerase. Thus, our coupled one-step RT-PCR procedure amplifies fast and reproducibly full-length ORFs from nanogram amounts of total RNA.
开发了一种一步法（所有反应物同时添加）逆转录和聚合酶链反应（RT-PCR）程序，用于扩增相对罕见转录本的全长开放阅读框（ORF）。该程序用于克隆大于2 kb的大鼠促黄体生成素/绒毛膜促性腺激素受体cDNA异构体。在所开发的程序中，人工操作被减至最少，因此可以处理大量样本，因为在模板RNA和引物变性并添加其他试剂后，无需进一步的人工步骤。未发现禽成髓细胞瘤病毒（AMV）逆转录酶（RT）对嗜热栖热菌（Taq）DNA聚合酶有抑制作用。这是因为，在所描述的条件下，Taq DNA聚合酶在AMV-RT存在的情况下能有效扩增皮克量的质粒DNA或从纳克总卵巢RNA逆转录的模板。即使大量过量的AMV-RT也不会抑制Taq DNA聚合酶。因此，我们的一步法RT-PCR联用程序能从纳克量的总RNA中快速且可重复地扩增全长ORF。
