摘要
从嗜热细菌ＦＤ３００９中克隆并分离纯化得到的耐热逆转录酶（ＦＤ－ＴＲＴ），逆转录反应在６５℃高温下进行，可克服常温逆转录酶的许多缺点，以酵母核糖体ｒＲＮＡ为模板，以与２５５和１８Ｓ ｒＲＮＡ匹配的寡聚脱氧核苷酸为引物，探明了ＦＤ－ＴＲＴ的最适反应条件： ２５ｍｍｏｌ／Ｌ Ｔｒｉｓ－ＨＣｌ（ｐＨ ８．５，２５℃），２５ ｍｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４，２ ｍｍｏｌ／Ｌ ＭｎＣｌ２，１００ μｇ／ｍｌ明胶．５ ｕｎｉｔ ＲＮａｓｉｎ，２５０ ｍｍｏｌ／Ｌ ４种 ｄＮＴＰ， １μＣｉ３ Ｈ－ｄＣＴＰ， １２ｕｇ ＲＮＡ，２５ ｐｍｏｌ引物，并在此条件下测得 ＦＤ－ＴＲＴ和 Ｔａｑ ＤＮＡ 聚合酶逆转录活力与聚合酶活力的相对比值分别为０．０５６，０．００４５．
Thermostable reverase transcriptas (TRT) used in reverse transcription at high temperature possess more advantages than the usual enzymes from AMV or MMLV. TRT can reverse-transcribe RNA efficiently at high temperature because there in less advanced structure of RNA at high temperature. An optimal assay method of FD-TRT was developed using the yeast ribosomal RNA as template. The FD-TRT showed optimal activity on the reaction condition of 25 mmol/L Tris-HCI (pH 8. 5, 25℃), 25 mmol/L (NH4 )2 SO4, 2 mmol/L MnCl2, 100 ug/ml gelatin, 5 unit RNasin, 250 umol/L each of four dNTPs, 1 uCi 3 H-dCTP, 12 ug RNA, 25 pmol primers. The activity ratios of reverse transcriptase to DNA polymerase were 0. 056 and 0. 0045 for FD-TRT and Taq DNA polymerase respectively.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
1998年第2期225-228,共4页
Journal of Fudan University：Natural Science
基金
国家自然科学基金
关键词
耐热逆转录酶
FD-TRT
DNA
逆转录反应
thermostable reverse transcriptase
reverse transcription at high temperature
enzyme activity assay
