Takara Shuzo Co., Ltd. - PDF document

Takara Shuzo Co., Ltd. Competitive PCR Guide 1 I.What is Competitive PCR?A.Difficulties of Quantitative Analysis in Normal PCR...........................................2B.Principle of Competitive PCR.............................................................................3C.Competitive RT-PCR..........................................................................................4D.Different Types of Competitors...........................................................................5E.Competitive RT-PCR Using an Internal Reference Template............................6II.Analyzing the Results of Competitive PCRA.Considerations...................................................................................................8B.Examples1.Visual Estimation Method...............................................................................82.Graphical Method.........................................................................................10For DNA Competitor preparation).................................(For RNA Competitor preparation)...................................(For correction of RNA amount)................................... Takara RNA PCR Kit (AMV) Ver.2.1.........................................................................TAK R019Takara RNA LA PCR Kit (AMV) Ver.1.1**...................................................................TAK RR012BESTª RNA PCR Kit......................................................................TAK RR023Takara One Step RNA PCR Kit (AMV).........................................................................TAK R024 *TaKaRa's PCR related products are sold under licensing arrangements with Roche Molecular Systems and F. Hoffman-La Roche Ltd. and ThePerkin-Elmer Corporation. Purchase of this product is accompanied by a limited license to use them in the Polymerase Chain Reaction (PCR) processfor research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front fee, either bypayment to Perkin-Elmer or as purchase, i.e., an authorized thermal cycler.**U.S.Patent 5,436,149 for LA Technology owned by TAKARA SHUZO CO., LTD.Lit. # L0126 Rev. 8/99 Takara Shuzo Co., Ltd. Competitive PCR Guide2 I.What is Competitive PCR ?A.Difficulties of Quantitative Analysis in Normal PCR 01020304050 Figure 1. The plateau phase of PCR.After approximately 30 cycles of PCR, almost the same amount of product will be obtained for samplesA and B, even though they initially contain different amounts of template. Takara Shuzo Co., Ltd. Competitive PCR Guide 3 B.Principle of Competitive PCR Figure 2. Principle of competitive PCR.Samples are analyzed by agarose gel electrophoresis and the amount of competitor required to give a T:Cratio = 1 is determined. In this example, the amount of target DNA corresponds to 107 copies of competitor. T:C = 10 3.2 1 0.32 log(T:C) = 1 0.5 0 -0.5TargetCompetitor105106107108Amount of Competitor (copy number) Takara Shuzo Co., Ltd. Competitive PCR Guide4 C.Competitive RT-PCR RT reactionPCRGel electrophoresisGel electrophoresis patternAmount of RNA 1234567812345678 Amplified cDNA of competitor RNACompetitor RNATarget mRNA Fixed amounts of RNA template are mixed with increasing amounts of competitor RNA. Reversetranscription is performed, followed by PCR. The samples are then analyzed as for competitive PCR. Takara Shuzo Co., Ltd. Competitive PCR Guide 5 D.Different types of competitors Homologous CompetitorTarget mRNACompetitorHeterologous CompetitorCompetitor Figure 4. Types of competitors. Takara Shuzo Co., Ltd. Competitive PCR Guide6 E. Competitive RT-PCR Using an Internal Reference Template1.Wang. A.M., Doyle, M.V. and Mark, D.F. (1989) Quantitation of mRNA by the polymerase chain reaction.2.Diaco, R. (1995) Practical considerations for the design of quantitative PCR assays.3.Gause, W.C. and Admovicz, J. (1995) Use of PCR to quantitate relative differences in gene expression. 4.Ferre, F. (1992) Quantitative or semi-quantitative PCR: Reality versus myth. 5.Raeymaekers, L. (1995) A commentary on the practical applications of competitive PCR. 6.Siebert, P.D. and Kellogg, D.E. (1995) PCR MIMICs: Competitive DNA fragments for use in quantitative PCR. Takara Shuzo Co., Ltd. Competitive PCR Guide 7 II.Analyzing the Results of Competitive PCRB.Examples1.Visual Estimation Method Takara Shuzo Co., Ltd. Competitive PCR Guide8 SampleAmount of Competitor (copy number)1Sample A102Sample B103Sample C10 For each sample (A, B, and C) the amount of competitor required to give a T:C ratio is determined afteragarose gel electrophoresis. Takara Shuzo Co., Ltd. Competitive PCR Guide 9 SampleAmount of CompetitorT:Clog(T:C)1Sample A102Sample A100.1-13Sample A100.01-24Sample B101015Sample B106Sample B100.1-17Sample C1010028Sample C101019Sample C10 2.Graphical method 10510610702-2log(T:C)Amount of Competitor (copy number) Sample CSample BSample A