DNA SEQUENCING WITH THERMUS-AQUATICUS DNA-POLYMERASE AND DIRECT SEQUENCING OF POLYMERASE CHAIN REACTION-AMPLIFIED DNA
DNA SEQUENCING WITH THERMUS-AQUATICUS DNA-POLYMERASE AND DIRECT SEQUENCING OF POLYMERASE CHAIN REACTION-AMPLIFIED DNA
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DOI：
10.1073/pnas.85.24.9436
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发表时间：
1988-12-01
影响因子：
11.1
通讯作者：
BROW, MAD
中科院分区：
文献类型：
--
作者：
INNIS, MA;MYAMBO, KB;BROW, MAD
来自Thermus aquaticus（Taq）的高度热稳定的DNA聚合酶对于手动和自动DNA测序都是理想的，因为它快速、高度进行性、具有很少或没有3“-外切核酸酶活性，并且在广泛的温度范围内具有活性。提供了产生具有均匀条带强度的> 1000个碱基的可读延伸产物的测序方案。高反应温度和碱基类似物7-脱氮-2“-脱氧鸟苷的组合用于通过富含G+ C的DNA进行测序并解析凝胶压缩。我们修改了聚合酶链反应（PCR）的条件，直接DNA测序的不对称PCR产物，而无需中间纯化，通过使用Taq DNA聚合酶。通过不对称PCR和直接测序的模板制备的耦合应该促进大规模测序项目的自动化。
The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3''-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products > 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2''-deoxyguanosine was used to sequence through G+C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.