Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific single-stranded DNA and improved real-time detection

Results

Effect of Primer T_m Differences on Amplification Efficiency and Specificity. Real-time PCR permits a clear comparison of amplification efficiencies among reactions that contain the same number of starting target molecules but different pairs of primers. Under these conditions, reactions with the higher amplification efficiency generate signals that become detectable in fewer cycles, i.e., have lower C_T values. In the first sets of experiments, we focus on the conditions for achieving efficient amplification during the exponential phase of asymmetric PCR. Exponential amplification can be monitored by using the fluorescent dye SYBR green I, which intercalates double-stranded DNA but does not detect the accumulation of single-stranded DNA during the linear phase of LATE-PCR.

We previously demonstrated that amplification is inefficient when T _m ^L is below T _m ^X (4). Fig. 1A confirms that result and shows the relative amplification efficiencies of primer pairs with T _m differences over a wider range. Each curve depicts the mean fluorescence increase for replicate samples at each tested value of T _m ^L - T _m ^X, using one of six different limiting primers (always at 50 nM) with the same excess primer (always at 1,000 nM) (Table 1). For this set of samples (Series A), the annealing temperature was always 52°C, chosen based on preliminary tests with the excess primer (see Materials and Methods). The fluorescence increase occurred latest for samples with T _m ^L - T _m ^X equal to -3 (i.e., limiting primer with lower T _m). Detectable fluorescence increased earlier with T _m ^L - T _m ^X equal to 0 (primers of equal T _m)or +3 (limiting primer with higher T _m) and earliest with that value equal to +5 or +7.

The Series A differences in real-time kinetics were quantified by calculating their mean C_T values (Table 2). Each increase in T _m ^L - T _m ^X yielded a statistically significant decrease in mean C_T value (P < 0.05), with the exception of the 0 and +3 primer combinations. The difference between the samples with negative values and those with the highest positive values of T _m ^L - T _m ^X was almost four cycles, a difference in amplification efficiency of ≈15%. Melting analysis confirmed that the CFTR-specific amplicon was the major product in each sample, including those in which T _m ^L - T _m ^X was equal to +5 or +7. Substantial levels of nonspecific products were present in only 4 of the 49 total samples, including 1 of 15 samples in the +5 and +7 groups.

Series B (Fig. 1B) used the same primer pairs as Series A but annealing temperatures that were always 2°C below the T _m ^L of the particular limiting primer in the reaction. As in Series A, the mean C_T values when T _m ^L - T _m ^X was +5 or +7 were significantly lower than when T _m ^L - T _m ^X equaled +3, 0, or -3 (P < 0.01). Moreover, melting analysis revealed that samples with T _m ^L - T _m ^X equal to -3 included varying amounts of nonspecific products that contributed to the fluorescence increase and masked a relatively low amplification efficiency.

The variance in C_T values among replicate reactions decreased as T _m ^L - T _m ^X increased. In Series B, the variance for samples in which T _m ^L - T _m ^X = +5 was significantly smaller than for groups having lower T _m ^L - T _m ^X values (P < 0.01, P < 0.001, and P < 0.05 for comparisons with sample groups having T _m ^L - T _m ^X values of +3, 0, and -3, respectively). These differences in the reliability of replicate reactions suggest that quantification of the amount of starting DNA will be more accurate when T _m ^L values are relatively high.

Gel electrophoresis of the reaction products of Series B confirmed that the higher annealing temperature for samples with a T _m ^L - T _m ^X value of +5 enhanced amplification specificity (Fig. 2). In contrast, nonspecific products were common in samples with T _m ^L - T _m ^X values of -3 and annealing temperature of 49°C. Even in samples with a value of 0, one sample contained high levels of nonspecific product, and others showed low levels.

Effect of Amplicon and Excess Primer T_m Difference on the Rate and Extent of Linear Amplification. A separate set of experiments (Series C) was carried out to test how the difference between T _m ^A and T _m ^X affects the rate and extent of single-strand synthesis during the linear amplification phase of LATE-PCR. Reactions included a molecular beacon to monitor CFTR-specific single-stranded DNA synthesis. Primer pairs were designed so that T _m ^L - T _m ^X = +5° to +6°C and are listed in Table 1. Fig. 3 shows the molecular beacon fluorescence for samples with T _m ^A - T _m ^X ranging from +13 to +23. Mean C_T values and rates of signal increase (line slopes) are presented in Table 3. The greatest fluorescence increase was generated by samples with T _m ^A - T _m ^X = 13. The rate and extent of fluorescence increase were progressively lower as the value of T _m ^A - T _m ^X increased. Final fluorescence levels were significantly different for each of the primer pairs tested (P < 0.01). Three pairs of primers yielded similar C_T values, suggesting that exponential amplification efficiency was similar in each case, and that the observed differences in subsequent fluorescence increase were almost exclusively due to differences in the rate of linear amplification. Thus, the rate of linear amplification increases as the difference between T _m ^A and T _m ^X decreases, so long as the difference between T _m ^L and T _m ^X is maintained.

Quantifying Starting Template Concentration Using Optimized LATE-PCR. Real-time symmetric PCR is commonly used to measure the initial template concentration in a sample through the comparison of C_T values. Fig. 4A shows that real-time LATE-PCR conveys the same quantitative information with low variation among replicate samples. Samples containing 10–100,000 copies of human genomic DNA plus the best primer pair from Series C generated fluorescent signals that reached threshold in relation to that starting concentration. Each 10-fold decrease in starting template concentration delayed detection ≈3.3 cycles, as expected for efficient exponential amplification. In contrast to symmetric PCR, plots from LATE-PCR samples neither diverged nor plateaued at different fluorescent values. Instead, fluorescence increased linearly and uniformly over several cycles. Thus, fluorescent values at later cycles continued to convey quantitative information. Fig. 4B shows a standard curve for starting template concentration based on the fluorescent values at cycle 40. It should be noted that the analysis did not require setting a baseline over several cycles, as is typical for standard real-time analysis. Instead, background fluorescence in the different samples was normalized by using values from the first detection cycle. Thus, where real-time detection is unavailable, it may still be possible to quantify the starting template by measuring the final amplicon concentration in LATE-PCR reactions using fluorescent probes or other detection methods.

Discussion

The efficiency and specificity of double-stranded DNA amplification during the exponential phase of asymmetric PCR depend on the relative T _m of the limiting and excess primers and the annealing temperature of the reaction. This report confirms that reactions in which T _m ^L is below T _m ^X and the annealing temperature is set in relation to T _m ^X, as is generally the case for traditional asymmetric PCR primers, have lower efficiencies than reactions in which T _m ^L equals T _m ^X (4). The present findings also demonstrate that increasing T _m ^L above T _m ^X can increase efficiency and specificity even further. The improved specificity is of particular importance for applications intended to generate useful quantities of single-stranded DNA.

A priori, it seemed possible that increasing T _m ^L well above the annealing temperature might reduce amplification specificity. Our results demonstrate, however, that setting T _m ^L at 7°C or 9°C above the annealing temperature did not increase the amount of nonspecific product, and amplification efficiency was improved by the use of those limiting primers, as evidenced by a decrease in mean C_T values. In contrast, a large amount of nonspecific product was generated when T _m ^X was only 5°C above the annealing temperature. Thus, one advantage of having T _m ^L > T _m ^X is that the true optimal annealing temperature for both primers can be used, one sufficiently low relative to T _m ^L to allow for efficient utilization of the limiting primer but sufficiently high relative to T _m ^X to limit amplification of nonspecific products by the excess primer.

The use of primer pairs with T _m ^L above T _m ^X is likely to improve amplification efficiency for many target sequences, although the optimal T _m difference between limiting and excess primers may vary with the target and primer ratio. Similar tests amplifying the β-hexosaminidase gene (mutations of which are responsible for Tay-Sachs disease) with primer ratios of 40:1 show that increasing T _m ^L - T _m ^X from 0 to +4 increases amplification efficiency while maintaining specificity (unpublished data). We have incorporated the use of positive T _m ^L - T _m ^X values as a general strategy for LATE-PCR primer design.

Improving the rate and extent of single-stranded DNA synthesis can be accomplished by reducing the difference between T _m ^A and T _m ^X. Presumably, this relates to the ability of the excess primer molecules to hybridize to their target strand in the presence of an increasing concentration of competing single-stranded product. CFTR excess primers with calculated T _m ^X within 20°C of T_m ^A generated reasonable rates of single-stranded synthesis, with the best rates obtained when that difference was 13°C. We have also obtained good results amplifying sequences within the β-hexosaminidase and β-globin genes when these limits are maintained (ref. 4 and unpublished results). Those amplicons have higher T _m ^A than the CFTR amplicon generated here and thus require primers with higher T _m ^X and T _m ^L, suggesting this rule can be generally applied to other asymmetric amplifications. It may be difficult, however, to design primers that meet this criterion for amplicons that are long or GC-rich. In such cases, achieving good yields may require altering the primers or amplicon in other ways, for example, by incorporating nucleotide analogs that lower T _m ^A. It is also important to recognize that other factors besides primer T _m may influence the extent of linear amplification. For instance, subtle changes in the 5′ end of the limiting or excess primer can have a profound effect on the rate of single-strand production, even when those changes have minor effects on primer T _m.

One of the significant advantages of LATE-PCR over symmetric PCR is the increased intensity and reproducibility of real-time signals. In LATE-PCR, the concentration of the limiting primer is chosen such that it becomes depleted just as the product becomes detectable with a labeled probe. In contrast to symmetric PCR, the probe signals in LATE-PCR continue to increase at similar rates in all samples, and the highly variable reaction plateau is avoided. Like symmetric PCR, LATE-PCR is able to quantify the initial targets in a sample using C_T values, something that would be difficult or impossible using traditional methods of asymmetric PCR. In fact, there are few publications in which hybridization probes have been used with unequal concentrations of primers; most used low primer ratios insufficient to sustain linear amplification (16–19).

The highly reproducible linear increase in single-stranded product means that LATE-PCR end-point analysis can be used to quantify starting template concentrations. Quantification is possible for at least 5 orders of magnitude in starting template but depends on primer concentrations, reaction optimization, and number of cycles. For some applications, end-point analysis may provide an alternative to expensive real-time detection equipment and licensing fees.

Another advantage of LATE-PCR is the flexibility that it provides for detection methods. It is worth noting that the same molecular beacon probe was used for all samples, despite the use of primers with T _m over a wide range. This is not possible with symmetric PCR, because probes must be designed with a relatively high T _m to ensure that probe hybridization during the annealing step occurs before primer hybridization and extension. In contrast, LATE-PCR provides single-stranded product that is freely available to hybridize with the probe at whatever temperature is appropriate for that probe. This permits the use of hybridization probes with T _m below the primer T _m. Advantages of using probes with relatively low T _m include simplified design, lowered background signal, and increased allele discrimination (4). These improvements are accrued by using any type of probe that emits a signal upon hybridization.

Conclusion

LATE-PCR establishes straightforward primer design criteria for generating high yields of single-stranded DNA products without extensive reaction optimization. LATE-PCR effectively eliminates the need for multiple rounds of amplification or laborious steps for purifying single-stranded DNA. When combined with real-time or end-point detection using low-T _m probes, this method offers unique potential in forensics, bioweapons detection, and other fields where assay sensitivity and consistency are essential.