TRANSCRIPTION-BASED AMPLIFICATION SYSTEM AND DETECTION OF AMPLIFIED HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 WITH A BEAD-BASED SANDWICH HYBRIDIZATION FORMAT
TRANSCRIPTION-BASED AMPLIFICATION SYSTEM AND DETECTION OF AMPLIFIED HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 WITH A BEAD-BASED SANDWICH HYBRIDIZATION FORMAT
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DOI：
10.1073/pnas.86.4.1173
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发表时间：
1989-02-01
影响因子：
11.1
通讯作者：
GINGERAS, TR
中科院分区：
文献类型：
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作者：
KWOH, DY;DAVIS, GR;GINGERAS, TR
具有重要生物学意义的核酸的体外扩增主要通过一种DNA复制策略进行。聚合酶链反应是首个实现这一目标的方案。在本报告中，描述了一种基于转录的扩增系统（TAS）。TAS的每个循环由两个步骤组成。第一步是cDNA合成步骤，对于每份RNA或DNA靶核酸，会产生一份双链DNA模板拷贝。在这个cDNA合成步骤过程中，一个被依赖DNA的RNA聚合酶识别的序列被插入到待扩增的靶序列的cDNA拷贝中。第二步是通过将cDNA模板转录为多份RNA来扩增靶序列。该程序已应用于检测1型人类免疫缺陷病毒（HIV - 1）感染的细胞。经过4个TAS循环后，测得HIV - 1 RNA基因组的vif区域平均每个循环扩增38到47倍，导致原始靶序列的拷贝数增加2 - 5×10⁶倍。通过TAS方案的这种扩增使得在10⁶个未感染的CEM细胞群体中能够检测到少于1个HIV - 1感染的CEM细胞。通过基于磁珠的夹心杂交方案可以很容易地检测到来自HIV - 1感染细胞的TAS产生的RNA，这为鉴定扩增的HIV - 1特异性序列提供了额外的特异性。
The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RAN polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immundeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 .times. 106-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 106 uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily by accomplished by means of a bead-based sandwhich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.