Ahmad Ashraf, Ghasemi Jahan
Department of Chemical and Biological Engineering-Molecular Biotechnology, Chalmers University of Technology, Göteborg, Sweden.
Biosci Biotechnol Biochem. 2007 Aug;71(8):1970-8. doi: 10.1271/bbb.70164. Epub 2007 Aug 7.
Real-time PCR is a potent technique for nucleic acid quantification for research and diagnostic purposes, the wide dynamic range being one of the advantages over other techniques like the microarray. Several additives and enhancers have been studied to expand the PCR dynamic range in order to be more efficient in quantifying low quantities of nucleic acids, increase the yield and improve reaction efficiency. Shown here is that a combination of new buffers with the regularly used Tris buffer makes it possible to expand the real-time PCR dynamic range and to improve the efficiency and correlation coefficient. Mixing HEPES, TEA or MOPS with Tris was more efficient than Tris alone. It was also found that, if the pH value of the Tris buffer was calibrated with phosphoric acid instead of hydrochloric acid, then the dynamic range was significantly improved and low quantities could be detected and quantified more efficiently. Mixing more than one compound with the Tris buffer was also effective for expanding the dynamic range and increasing the efficiency and correlation coefficient in quantitative real-time PCR.
实时荧光定量聚合酶链反应（Real-time PCR）是一种用于核酸定量研究和诊断的强大技术，其广泛的动态范围是优于其他技术（如微阵列）的优势之一。为了更有效地定量低量核酸、提高产量并改善反应效率，人们研究了几种添加剂和增强剂以扩大PCR动态范围。结果表明，新缓冲液与常用的Tris缓冲液组合能够扩大实时荧光定量聚合酶链反应的动态范围，并提高效率和相关系数。将HEPES、TEA或MOPS与Tris混合比单独使用Tris更有效。还发现，如果用磷酸而不是盐酸校准Tris缓冲液的pH值，则动态范围会显著改善，并且可以更有效地检测和定量低量核酸。在定量实时荧光定量聚合酶链反应中，将不止一种化合物与Tris缓冲液混合对于扩大动态范围、提高效率和相关系数也很有效。
