A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification.
A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification.
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DOI：
10.1016/j.gene.2012.01.031
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发表时间：
2012-04
期刊：
影响因子：
3.5
通讯作者：
D. Pratyush;Shalbha Tiwari;Ashok Kumar;S. Singh
中科院分区：
文献类型：
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作者：
D. Pratyush;Shalbha Tiwari;Ashok Kumar;S. Singh
含有高GC片段的组织特异性基因很难通过标准PCR扩增。我们报告了一种改进的方法，可以成功扩增具有bb0 - 70% GC碱基对的基因片段。这种新的触点PCR方法的不同之处在于初始退火温度（Ta）比引物熔化温度低1.5°C，每循环下降0.2°C，持续20个循环，然后在固定的Ta下继续15个循环。用该方法对不同的助溶剂进行了测试，结果表明甜菜碱的效果优于其他助溶剂。该方法经济、快速，对其他基因的GC富区扩增也具有特异性。
Tissue specific genes that contain high GC segments are difficult to amplify by standard PCR. We report an improved method for successful amplification of gene segment that has >70% GC base pairs. This new method of touch down PCR differed by having an initial annealing temperature (Ta) 1.5°C below the primers melting temperature that descended 0.2°C per cycle for 20 cycles and continued thereafter at fixed Ta for next 15 cycles. Different co-solvents were tested with this method to improve the result and betaine proved better than the other co-solvents. This new method is economical, fast and specific in amplifying GC rich region of other genes also.