THE GENERATION OF RADIOLABELED DNA AND RNA PROBES WITH POLYMERASE CHAIN-REACTION
THE GENERATION OF RADIOLABELED DNA AND RNA PROBES WITH POLYMERASE CHAIN-REACTION
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DOI：
10.1016/0003-2697(89)90019-5
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发表时间：
1989-02-15
影响因子：
2.9
通讯作者：
SOMMER, SS
中科院分区：
文献类型：
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作者：
SCHOWALTER, DB;SOMMER, SS
通过在聚合酶链反应（PCR）期间包括放射性三磷酸，可以产生具有非常高的比活性的探针。PCR标记的优点包括：（1）具有5 × 10 - 6的比活性的均匀标记; 109 cpm/μ g或更高（检测灵敏度：每24小时0.028 pg靶DNA）;（2）易于调节产生的标记探针的比活性和量;（3）有效标记小于500 bp的片段;（4）在宽范围的输入DNA模板上有效掺入;（5）用亚纳克量的输入DNA标记;（6）基因组DNA的直接标记。最小量的输入DNA允许在前述非标记条件下对通过一次扩增产生的DNA进行几乎无限数量的PCR标记反应。这避免了对CsCl梯度或其他大规模DNA制备方法的需要。除了非常高的比活性之外，上述优点也可以通过扩增后的转录物标记来实现，其中一个或两个PCR引物含有噬菌体启动子序列。
By including a radioactive triphosphate during polymerase chain reaction (PCR), probes of very high specific activity can be generated. The advantages of PCR labeling include (1) uniform labeling with a specific activity of 5 .times. 109 cpm/.mu.g or higher (sensitivity of detection: 0.028 pg of target DNA per 24 h); (2) ease of regulation of both the specific activity and the amount of labeled probe produced; (3) efficient labeling of fragments less than 500 bp; (4) efficient incorporation over a wide range of input DNA template; (5) labeling with subnanogram amounts of input DNA; and (6) direct labeling of genomic DNA. The minimal amount of input DNA allows a virtually unlimited number of PCR labeling reactions to be performed on DNA generated by one amplification under the previously described nonlabeling conditions. This obviates the need for CsCl gradients or other large scale methods of DNA preparation. The above advantages except for the very high specific activity can also be achieved by transcript labeling after an amplification where one or both of PCR primers contain a phage promoter sequence.