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

Abstract

Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX 5 degrees C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis.

Fig. 1.

Detection of double-stranded DNA by SYBR green fluorescence during asymmetric PCR reveals differences in exponential amplification efficiency with different limiting primers. Curves show the mean fluorescence increase in replicate samples and are colored to indicate the value of T_m^L - T_m^X: -3, red; 0, green; +3, orange, +5, blue; +7, purple. The dashed line indicates thresholds for determining C_T values. The starting template was 600 pg of human genomic DNA in each sample. (A) Series A amplifications used an annealing temperature of 52°C, which is 2°C below T_m^X. (B) Series B amplifications used annealing temperature 2°C below T_m^L, shown in Table 1.

Fig. 2.

Amplification products following electrophoresis through a 3% agarose gel and ethidium bromide staining. Numbers above the bars indicate T_m^L - T_m^X values for each set of replicate samples. Annealing temperature in each case was 2°C below T_m^L, and starting template was 600 pg of human genomic DNA. Specific CFTR product is the lowest band in each lane, 77–81 nucleotides. Double- and single-stranded products were not resolved on this gel. S, 50-bp DNA ladder size standards. Lengths are shown at left.

Fig. 3.

Detection of CFTR-specific product during the linear amplification phase of LATE-PCR. Curves show increases in molecular beacon fluorescence in individual samples with T_m^A - T_m^X values of 13 (green), 17 (blue), 20 (red), or 23 (orange). Starting template was 600 pg of human genomic DNA in each sample.

Fig. 4.

Detection of CFTR-specific product in samples containing different initial concentrations of DNA. (A) Optimized LATE-PCR was carried out by using 100,000 (red), 10,000 (green), 1,000 (orange), 100 (blue), and 10 (purple) copies of human genomic DNA. Curves show molecular beacon fluorescence increase in eight replicate samples at each starting template concentration. (B) Plots of initial DNA concentration vs. cycle 40 fluorescence demonstrates the quantitative nature of these endpoint values. R² = 0.974