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KMID : 1101320040360010033
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Korean Journal of Clinical Laboratory Science
2004 Volume.36 No. 1 p.33 ~ p.37
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The Effect of Temperature and Cycles on Amplification of DNA by PCR
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Kim Chong-Ho
Shin Sang-Hee
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Abstract
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In order to study the effect of temperature of denaturation, annealing and extension and cycles on amplification of DNA by PCR method, We isolated the hepatitis B virus DNA from hepatitis B patient blood and compared the density of DNA amplified by Reference PCR Program (denaturation at 94℃ for 30 sec., annealing at 60℃ for 1 min., extension at 72℃ for 1 min., holding at 72℃ for 5min., 30 cycles) that is usually used in laboratory to the density of DNA amplified by PCR program changed only the denaturation temperature or annealing temperature or extension temperature. We amplified about 341bp of hepatitis B virus DNA by Reference PCR Program from hepatitis patient blood, but the DNAs denatured at 72℃ or 60℃ were not detectable on photoradiography film. The DNA amplified at 37℃ of annealing temperature was not detectable, but the DNA annealed at 72℃ was detectable the lower density of DNA than the DNA amplified by Reference PCR Program. Each DNA amplified by PCR program changed only the extension temperature to 37℃ or 60℃ was almost same density as DNA amplified by Reference PCR Program. We compared the density of hepatitis B virus DNA amplified by Reference PCR Program for 30 cycles, 20 cycles, 10 cycles, and 5 cycles. The DNA cycled for 20 cycles was not amplified well as cycled for 30 cycles, but the DNA was detectable on the photoradiography film. The DNAs amplified for 10 cycles or 5 cycles were not detectable on photoradiorgaphy film. The concentration of hepatitis B virus DNA amplified in Reference PCR condition for 30 cycles, 20 cycles, 10 cycles, and 5 cycles were 72㎍/㎖, 83×10-3㎍/㎖, 27×10-6㎍/㎖, and nondetectable, respectively.
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KEYWORD
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PCR, Hepatitis B virus DNA
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