Competitive PCR Guide - gene-quantification.de

1 Takara Shuzo Co., PCR Guide1 BIOMEDICALSC ompetitive PCR GuideTable of 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 .. the Results of Competitive PCRA. Considerations .. 8B. Examples1. Visual Estimation Method .. 82. Graphical Method .. 10 Competitive DNA Construction Kit (For DNA Competitor preparation) ..TAK RR017 Competitive RNA Transcription Kit (For RNA Competitor preparation) ..TAK 6125 Human -Actin Competitive PCR Set (For correction of RNA amount) ..TAK 6607 Competitive PCR Related Products*RT-PCR Kits*Takara RNA PCR Kit (AMV) TAK R019 Takara RNA LA PCR Kit (AMV) ** .. TAK RR012 BcaBEST RNA PCR TAK RR023 Takara One Step RNA PCR Kit (AMV).

2 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, , an authorized thermal cycler.** 5,436,149 for LA Technology owned by TAKARA SHUZO CO., # L0126 Rev. 8/99 Takara Shuzo Co., PCR Guide2 BIOMEDICALSI. What is Competitive PCR ?A. Difficulties of Quantitative Analysis in Normal PCRThe polymerase chain reaction (PCR) is a highly sensitive method for the detection of small amounts of DNA bystandard PCR or RNA by reverse transcription PCR (RT-PCR).

3 However, accurate quantitation of the target isdifficult with normal PCR, as the amount of amplified product does not necessarily reflect the amount of templateinitially present in the reaction. This is due to the "plateau phase" of PCR, which is caused by: Deactivation of Taq DNA polymerase Shortage of nucleotide substrates Shortage of primer Inhibition by pyrophosphate Re-annealing of amplified DNAsDuring the plateau phase of PCR, nearly the same amount of amplified products will be obtained after a sufficientnumber of PCR cycles, regardless of the initial amount of template (Figure 1). It is almost impossible to determinethe PCR conditions that will yield sufficient amount of amplified products for detection by gel electrophoresisbefore reaching the plateau phase. Consequently, for a group of samples with different template concentrations,quantitation of the target template is difficult.

4 Competitive PCR was developed to overcome these difficulties 1020304050 Number of PCR cyclesAmount of amplified productsFigure 1. The plateau phase of 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 Shuzo Co., PCR Guide3 BIOMEDICALSB. Principle of Competitive PCRIn Competitive PCR, a known amount of a DNA fragment (competitor) is added to the sample. This competitormust contain sequences for the same primers used to amplify the target. When the target DNA and competitorare amplified together, both templates will compete for the same set of primers. Because of this competition, theratio of the amounts of the two amplified products reflects the ratio of the amounts of the target DNA and competi-tor.

5 Since the initial amount of the competitor is known, the amount of the target DNA can then be estimatedaccording to the T:C ratio (Figure 2).T: amount of amplified product from target DNA or RNAC: amount of amplified product from competitorWhen the T:C ratio = 1, the initial amount of target DNA or RNA will correspond to the amount of ideal competitor for quantitative PCR should be: Amplified by the same primers as the target DNA. Distinguishable from the target DNA (different size, different restriction fragment pattern, etc.) Purified and obtained at a known quantity or concentrationFigure 2. Principle of Competitive 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 :C = 10 1 log(T:C) = 1 0 of Competitor (copy number)Takara Shuzo Co.

6 , PCR Guide4 BIOMEDICALSC. Competitive RT-PCRC ompetitive RT-PCR can be performed similarly to Competitive PCR. There are two different Competitive between cDNA and DNA CompetitorThis method uses a DNA competitor, after cDNA synthesis from the target mRNA has been completed. Therelative amounts of cDNA among different samples can be determined. Since the yield and efficiency of the RTreaction is not reflected in this method, the absolute quantity of the target mRNA cannot be between mRNA and RNA CompetitorThis method is based on competition between mRNA and an RNA competitor during the RT reaction. The relativeamounts of target mRNA among different samples can be determined. As the yield of the RT reaction is reflectedin this method, the absolute quantity of the target mRNA can be determined (Figure 3).

7 RT reactionPCRGel electrophoresisGel electrophoresis patternAmount of RNA12 3456 781234 5678 Amplified cDNA of target mRNAA mplified cDNA of competitor RNAC ompetitor RNAT arget mRNAF igure 3. Competitive 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 Shuzo Co., PCR Guide5 BIOMEDICALSD. Different types of competitorsHomologous CompetitorThis is a competitor that has the same nucleotide sequences as the target DNA (RNA) but contains a deletion oran insertion, or has a different restriction site introduced by site-specific mutagenesis (Figure 4). When designingthis type of competitor, care should be taken to avoid the formation of a heteroduplex, which would interfere withsubsequent CompetitorThis is a competitor that has nucleotide sequences different from the target DNA (RNA) except for the sequencesof the primer annealing sites (Figure 4).

8 A heteroduplex will not be formed. Since the primer sequences are thesame, the difference in amplification efficiency between the target and heterologous competitor is usually CompetitorTarget mRNAC ompetitorHeterologous CompetitorCompetitorTarget mRNAF igure 4. Types of Shuzo Co., PCR Guide6 BIOMEDICALSE. Competitive RT-PCR Using an Internal Reference Template(Normalization of the amount of RNA in different samples using the Human -actin Competitive PCR Set)When dealing with highly purified, intact RNA samples, total RNA can be estimated by absorbance (A260) , crude RNA samples are often used for RT-PCR. In crude samples, the actual amount of intact RNA isless than the total RNA amount estimated by absorbance values due to DNA contaminants and/or degradedRNA. Therefore, A260 values cannot be relied upon to accurately compare the amount of RNA among crude precisely measure the expression level of a target gene in cells, the RNA amounts applied in the assay shouldbe normalized to a fixed amount.

9 This can be achieved by performing Competitive RT-PCR and amplifying aninternal reference template such as a housekeeping gene. Takara s Human -Actin Competitive PCR Set usesthe human -actin mRNA as the internal reference template. The -actin mRNA is widely used as a standard,since it is a housekeeping gene that is expressed at a constant level in most cells and tissues. The Human -ActinCompetitive PCR Set can therefore be used with any kind of sample derived from human cell lines, whileproviding more precise correction than conventional :1. Wang. , Doyle, and Mark, (1989) Quantitation of mRNA by the polymerase chain Natl. Acad. Sci USA 86, Diaco, R. (1995) Practical considerations for the design of quantitative PCR assays. PCR Strategies Chapter7, Gause, and Admovicz, J.

10 (1995) Use of PCR to quantitate relative differences in gene expression. PCRPRIMER, Ferre, F. (1992) Quantitative or semi-quantitative PCR: Reality versus myth. PCR Methods Applic. 2, Raeymaekers, L. (1995) A commentary on the practical applications of Competitive PCR. PCR MethodsApplic. 5, Siebert, and Kellogg, (1995) PCR MIMICs: Competitive DNA fragments for use in quantitative Chapter 8, Shuzo Co., PCR Guide7 BIOMEDICALSII. Analyzing the Results of Competitive PCRA. ConsiderationsFrom the results of Competitive PCR, the initial amount of target DNA (RNA) template can be estimated from theratio of T to C (T = amount of amplified target template; C= amount of amplified competitor). By determining theamount of competitor which gives a T:C ratio of 1 for each sample, the relative amounts of target DNA (RNA) canbe obtained.