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The Determination of Hybridization Temperature Several Primers in Polymerase Chain Reaction (Penentuan Suhu Hibridisasi Beberapa Primer dalam Reaksi Rantai Polimerase)

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Hybridization temperature of PCR technique plays an important role in obtaining the successful amplification of templates DNA. The hybridization temperature depends on the composition and the number of oligonucleotides existing in the primers. In this work we have optimized the hybridization temperature of seven primers used to amplify the templete DNA. The following methods were applied in this research : 4 (number of C and G) + 2 (number of A and T) - 5 degree C and 22 + 1.46 (In) degree C. By using these formulas we estimated the succesful temperature of hybridization to be between 63 degree C and 67 degree C. At 67 degree C all primers were able to amplify the templete DNAs well. At 63 degree C one of the primers didn't work, but if concentration of the templete DNA was added, all the primers worked well. Based on the data obtained in this research we recommend the hybridization temperature used for all primers containing more than 20 nucleotides to be between 63 degree C and 67 degree C. Based on the results of this study it can be concluded, that the higher the hybridization temperature within this range the better the amplication results.

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Majalah Kedokteran Bandung,28(1)1996:20-24

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: ., 1996

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hybridization

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