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Use of Multiplex Polymerase Chain Reactions to Indicate the Accuracy of the Annealing Temperature of Thermal Cycling by Inchul Yang; Young-Ho Kim; Ji-Young Byun; Sang-Ryoul Park is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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A condition for multiplex polymerase chain reactions (PCRs) of which outcomes sensitively indicate the actual annealing temperature of thermal cycling is reported. The multiplex reaction was designed to produce four diVerent amplicons of 200, 300, 400, and 480 bp. However, the degree of ampliWcation of each amplicon sensitively responds to a small change in the annealing temperature, by which one can predict the actual annealing temperature of thermal cycling. Deviations between the actual and the designated annealing temperatures as small as 0.5 °C were manifested by the banding patterns of the multiplex PCRs in simple agarose gel electrophoresis. For prediction of temperatures in a more objective manner, capillary electrophoresis was also applied to obtain numerical expressions of the relative intensities of the amplicons. By optimizing the multiplex PCR conditions, where concentrations of buVer, dNTPs, and primer pairs were major factors, satisfactory sensitivity and reproducibility of the band patterning were achieved. Blind tests demonstrated the accuracy of the prediction of actual annealing temperatures within §0.5 °C. The multiplex PCR approach will be further reWned and te

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Author

Inchul Yang; Young-Ho Kim; Ji-Young Byun; Sang-Ryoul Park

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2005

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)