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 (Tm L ) relative to that of the excess primer (Tm X ) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when Tm L ؊ Tm X > 5°C. We also investigated how altering Tm X relative to the higher Tm of the double-stranded amplicon (Tm A ) affects the rate and extent of linear amplification. Excess primers with Tm X closer to Tm A 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.asymmetric PCR ͉ primer melting temperature ͉ quantitative PCR A symmetric PCR, as first described by Gyllensten and Erlich (1), can produce single-stranded DNA for sequencing, for use as probes, or for improving detection signals in real-time PCR. Unfortunately, traditional asymmetric PCR is highly variable and often requires extensive optimization to maximize the production of specific single-stranded product and minimize nonspecific amplification (2, 3).We recently demonstrated (4) that these problems can be understood by considering that the melting temperature (T m ) of any primer decreases when its concentration is lowered (5-7). If the concentration of a primer designed for symmetric PCR is simply lowered for use as a limiting primer, the efficiency of the resulting reaction decreases. A conceivable solution to this problem is to lower the annealing temperature of the reaction, but this, in turn, increases the likelihood of nonspecific amplification due to increased mispriming by the excess primer.Our preferred strategy, Linear-After-The-Exponential (LATE)-PCR, uses primers that are deliberately designed for use at unequal concentrations, such that the concentrationadjusted T m of the limiting primer (T m L ) is at least as high as the concentration-adjusted melting temperature of the excess primer (T m X ) (i.e., T m L Ϫ T m X Ն 0) (4, 8). LATE-PCR begins with an exponential phase in which amplification efficiency is similar to that of symmetric PCR. Once the limiting primer is depleted, the reaction abruptly sw...

Exploring the Fitness Landscape of an RNA Virus by Using a Universal Barcode Microarray

“…4A). The 5Ј and 3Ј barcode identifiers were then amplified from this template by asymmetric PCR (47,52). In our protocol, the reverse primer is end labeled with either Cy3 or Cy5 and present in 20-fold molar excess over the forward primer.…”

Use of Sloppy Molecular Beacon Probes for Identification of Mycobacterial Species

“…This raises a number of concerns for the selection of an effective design for the PCR assays, all of which could cause a decrease in the intensity of the fluorescence from weaker hybrids, including the following: (i) competition be- tween probes that form strong hybrids and probes that form weak hybrids might diminish the number of weaker hybrids that form; (ii) competition between the probes and the complementary amplicon strands for binding to the target strands might diminish the number of weaker hybrids that form; and (iii) secondary and tertiary structures that are present in the target strands might diminish the formation of weaker hybrids. We therefore decided to utilize linear-after-the-exponential (LATE)-PCR, which is an efficient asymmetric PCR format (13,15), in which so many target strands are synthesized that their number exceeds the number of sloppy molecular beacon probes present in the assay and in which many more target strands are synthesized than complementary strands. By eliminating sources of competition for the binding of probes to target strands, weaker hybrids are more abundant, and their fluorescence is therefore more likely to be sufficiently intense for their T m to be measured.…”

Closed-Tube Barcoding

“…Linear-After-The-Exponential (LATE) PCR is an advanced form of non-symmetric PCR (Sanchez et al 2004;Pierce et al 2005). It is used to generate single-stranded amplicons from one or more target genes of interest.…”