Fluorogenic Real Time PCR

3.7.1. Reaction Mixture

To prevent PCR artifacts it is important to use "hot-start" DNA polymerase that is activated at high temperature (see Note 3). Usually "hot-start" enzyme is a thermostable DNA polymerase mixed with the specific antibodies that inhibit enzyme activity until heated at 95°C. It is also useful to include uracil DNA glycosylase and dUTP in the reaction because it will decrease the chances of carryover contamination of subsequent reactions with the amplicon (see Note 4). PCR reagents could be bought separately or in premade mixtures that also contain the enzymes. All the reagents are kept at -20°C.

To set up a reaction from the pre-made mixtures: defrost and combine the following reagents at room temperature, setting the reaction on ice could facilitate primer-dimer formation (see Note 5). Aliquots of Platinum Quantitative PCR SuperMix-UDG could be stored at 4°C for 2 wk. The 50 |L contain: 1 mL Platinum Quantitative PCR SuperMix-UDG (2X) 25 |L, ROX reference dye (50X); 1 |L each of primers (10 |M); and 10 |L template (10-107 copies per reaction).

To set up a reaction from the individual components: defrost and combine the following reagents at room temperature, setting the reaction on ice could facilitate primer-dimer formation (see Note 5). The 50-|L reactions contain: 20 mM Tris-HCl, pH 8.4; 50 mM KCl, 2-3 mM MgCl2 (see Note 6); 200 |M each dATP, dGTP, dCTP, and 400 |M dUTP (see Note 7); 0.2 |M each primer; 1X ROX reference dye (optional, see Note 8); 1 U uracil DNA glycosylase;

0.5-1.0 U of Platinum Taq DNA polymerase; and template (10—107 copies per reaction).

The volume of the reaction could be decreased to 25 or 20 ^L without the adverse effect on the results. Companies other than Invitrogen also provide "hot-start" enzymes and other reagents for real-time PCR. For example, see Bio-Rad, Roche, and Applied Biosystems.

3.7.2. Quantitative Real-Time PCR

Amplification reactions were conducted in a 96-well spectrofluorometric thermal cycler (ABI PRISM 7700, Applied Biosystems). We used two- or three-step temperature mode with similar results. For three-step cycling, reactions were incubated at 25°C for 2 min, 95°C for 2 min, then cycled using 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s, then reactions were incubated at 25°C for 2 min. Exceptions were made for amplicons longer than 500 bp. Two-step cycling consisted of 95°C for 15 s and 60-65°C for 30 s. The results are usually similar and a two-step PCR is quicker. The three-step mode may be recommended when an annealing temperature lower than 60°C is required. Fluorescence was monitored during every PCR cycle at the annealing step.

Fluorogenic PCR may be routinely used to quantify 100 or less copies of target in a background of nonspecific templates over a broad dynamic range of 107 to 10 copies. In Fig. 1 10-fold, serial dilution of cloned, c-myc cDNA ranging from 10 to 107 copies are discriminated by two-step real-time PCR using primer set 3 (Table 1). A linear relationship (r2 = 0.999) exists between the threshold cycle (CT) and starting copy numbers between 10 and 107 (standard curve, Fig. 1B). Similar fluorogenic PCR with comparable results was performed using 10-fold serial dilutions of IL-4 cloned cDNA (primer set 2, Table 1). Samples containing three-fold, serial dilutions (three replicate reactions per dilution) of cloned IL-4 cDNA ranging from 22 to 106 copies are discriminated by three-step, fluorogenic PCRs (primer set 2, Table 1). The correlation coefficient of CT vs copy number was 0.999.

Amplicons of various sizes could be detected with fluorogenic PCR primers. FAM-labeled primers were designed for amplicons of c-myc with sizes 66, 562, 1107 bp and GAPDH with sizes 74, 570, and 956 bp (Table 1). The fluorogenic PCRs (three-step cycling) were performed using cDNA from firststrand synthesis reactions (20 ^L) using HeLa total RNA (2.5 ng) as a template. PCRs were performed with 2, 0.2, 0.02, or 0 ^L of these first strand reactions. For all primer sets, cDNA dilutions were determined with evenly spaced CTs (data not shown), which confirm that PCR efficiency is comparable for the amplicons of various sizes.

o 10 20 30 40 Cycle Number

40

V.

35 ■

>> o

30

c

25 ■

20

H

15 ■

10

10" 10' !0: JO1 JO"1 10- ]0<' 107 10" Target copy number

Fig. 1. Sensitivity, precision, and dynamic range of fluorogenic real-time polymerse chain reaction (PCR). Tenfold serial dilutions of c-myc cDNA were amplified and detected using a fluorescein-labeled fluorogenic primer in two-step PCR on an ABI 7700 as described in Heading 3. (primer set 5, Table 1). (A) Amplification plot. (B) Initial complementary DNA concentrations vs CT, standard deviations are shown as error bars (12 replicates per dilution).

3.7.3. Multiplex Quantitative PCR

The fluorogenic primer method was also applied for the simultaneous detection of two sequences using FAM- and JOE-labeled primers. FAM-labeled primer set was used to detect the amount of a gene that is variable, either c-myc or IL-4, and JOE-labeled for a gene that is relatively constant and used as a reference. The results in Fig. 2A demonstrate the discrimination between threefold, serial dilutions of cloned IL-4 cDNA (primer set 2, Table 1) ranging from 22 to 300,000 copies, with each dilution containing 1 million copies of cloned GAPDH cDNA (primer set 12, Table 1). Similar results are shown for c-myc (primer set 5, Table 1) as the variable gene and GAPDH as the constant gene (Fig. 2B). cDNAs other then other than GAPDH may be used as the reference.

Fig. 2. Multiplex fluorogenic polymerse chain reaction (PCR) on ABI PRISM 7700. Amplification plots for time PCR comprising a threefold serial dilution of cloned complementary (cDNA): (A) Interleukin (IL)-4 cDNA (gray) from 303,030 to 22 copies, (B) c-myc cDNA (gray) from 1 million to 22 copies; each dilution had 1 million copies of cloned GAPDH cDNA (black). Fluorescein-labeled fluorogenic primers were used to detect IL-4 (primer set 2, Table 1) and c-myc (primer set 5, Table 1) and a JOElabeled primer was used to detect glyceraldehyde-6-phosphate dehydrogenase (primer set 12, Table 1). Corresponding plots of initial cDNA concentrations (two duplicate reactions per concentration) vs CT are shown below the amplification plots.

Fig. 2. Multiplex fluorogenic polymerse chain reaction (PCR) on ABI PRISM 7700. Amplification plots for time PCR comprising a threefold serial dilution of cloned complementary (cDNA): (A) Interleukin (IL)-4 cDNA (gray) from 303,030 to 22 copies, (B) c-myc cDNA (gray) from 1 million to 22 copies; each dilution had 1 million copies of cloned GAPDH cDNA (black). Fluorescein-labeled fluorogenic primers were used to detect IL-4 (primer set 2, Table 1) and c-myc (primer set 5, Table 1) and a JOElabeled primer was used to detect glyceraldehyde-6-phosphate dehydrogenase (primer set 12, Table 1). Corresponding plots of initial cDNA concentrations (two duplicate reactions per concentration) vs CT are shown below the amplification plots.

For example, threefold serial dilutions of target concentration (IL-4) were discriminated by fluorogenic PCR when using either 1 million copies of cloned cDNA ß-actin (primer set 13, Table 1) or 18 S ribosomal RNA (primer set 14, Table 1) as the reference gene (R).

Furthermore, first-strand cDNA from HeLa cell total RNA was used as a source of the R in place of specific cloned cDNA. This was done to determine whether the PCRs would amplify their specific targets within a mixture of nonspecific cDNAs. For these experiments, the variable template was cloned IL-4

cDNA (threefold dilutions) and the constant template was a fixed amount of first-strand cDNA from the reverse transcription of HeLa total RNA. Standard curves yield r2 values of 0.997 for P-actin (primer set 13, Table 1), 0.996 for GAPDH (primer set 12, Table 1), and 0.999 for 18S (primer set 14, Table 1). The fluorogenic PCR primers amplified only their appropriate target. All the primer pairs used in the previous examples are highly specific as demonstrated by the lack of signal when no template is added to the PCR. Analysis of the PCR products by agarose gel electrophoresis revealed either insignificant or no nonspecific PCR products or primer-dimers.

All the previously mentioned results were obtained using the ABI PRISM 7700 system. Detection with similar sensitivity and dynamic range was observed on the iCycler (Bio-Rad) and the SmartCycler (Cepheid, Sunnyvale, CA).

It is not always necessary to obtain a standard curve in order to quantitative the sequence if interest. It was demonstrated that the results of quantitative PCR using fluorogenic primers might be analyzed by the comparative CT method. The comparative CT method is another commonly used method, besides the standard curve method, for quantifying an unknown amount of target cDNA in a sample (User Bulletin no. 2, ABI PRISM 7700 Sequence Detection System, P/N 4303859). This method of analysis does not require plotting a standard curve of CT vs starting copy number. Instead, the amount of target is calculated based on the difference between the CT of the target and an endogenous R gene (ACT).

1. Design primers for the target of interest (T) and an R. If fluorogenic primers for T and R are labeled with the same fluorophore the amplification will be performed in separate tubes, if FAM and JOE are used for two primers the reaction could be done in one tube.

2. Check the efficiency of two primer sets. Run 6-10 serial threefold dilutions of sample detecting target and reference. If DCT between T and R are approximately the same with different dilutions, the efficiency of amplification are also similar.

3. Run the amplifications of samples one and two, both should include T and R. The difference between (ACT) of two samples is called AACT. Then the difference in the target concentration of two samples will be calculated as 2-AACT . For example:

Target Ref ACT AACT Rel target

Tar CT-Ref CT S1 ACT-S2 ACT concentration

When the experiment is performed with multiple replicates, average CT will be used and standard deviations need to be included in the calculations.

Sample

Ct

Ct

S1

29.4

24.8

S2

25.6

23.4

3.7.4. End-Point Detection of Allele-Specific PCR With Fluorogenic Primers

Because the PCR product demonstrates enhanced fluorescence compared to nonincorporated primers, the reaction products could be detected not only in real-time but also at end point (see Note 9). To demonstrate end-point detection capability, allele-specific PCRs were performed using human genomic DNA as a template. Discrimination of the alleles is based on the ability of DNA polymerase to extend 3' mismatches much less efficiently than correct matches (31). The 3'-ends of allele specific primers are complementary to one of two alleles. As a result, only one primer will extend with homozygote template and both primers will give a signal with a heterozygote.

Here we show a detection of a cytosine to thymine (C/T) polymorphism at position 558 of the RDS gene (32). Two unlabeled, allele-specific forward primers with either a dC or a T at the 3'-end, and a fluorogenic, reverse primer were designed to detect either the dC or T polymorphism (primer set 15, Table 1). Two allele-specific PCRs were performed on each of two genomic DNA samples bearing different single-nucleotide polymorphisms (Fig. 3). Following PCR, the fluorescence was determined directly in the PCR tubes using either fluorescence plate reader (Polarion, TECAN, Durham, NC) or an UV-transilluminator. The results show that both alleles can be identified correctly with the appropriate primer and there is no signal increase in the absence of target.

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