Kenneth E. Pierce

Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFΔ508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

Two-temperature LATE-PCR endpoint genotyping

BackgroundIn conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.ResultsTwo-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles.ConclusionSNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.

Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus

Aims:  A novel molecular assay for the detection of foot‐and‐mouth disease virus (FMDV) was developed using linear‐after‐the‐exponential polymerase chain reaction (LATE‐PCR).

Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays.

mis. 2000. The continued evolution of two-hybrid screening approaches in yeast: how to outwit different preys with different baits. Gene 250:1-14.tions of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast 7:253-263. 1999. Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252.tification of kinase-phosphatase signaling modules composed of p70 S6 kinase-protein phosphatase 2A (PP2A) and p21-activated ki-nase-PP2A.lecular cloning of CoA synthase. The missing link in CoA biosynthesis. S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. PCR is sufficiently sensitive to detect single copy genes from single cells. This extreme sensitivity is also the basis of one of its potential problems; even a single product molecule from a previous amplification can lead to a false positive result. Substituting dUTP for dTTP during PCR and treating subsequent reactions with uracil-DNA glycosylase (UDG) prior to amplification is one strategy for limiting carryover contamination (1). However, total elimination of contaminants is not always accomplished using this technique, particularly where PCR product length is short (2), which is a common situation in real-time PCR assays. In addition, there is the possibility, particularly when the initial sample contains only one or a few target molecules, that inclusion of UDG may reduce amplification efficiency and thereby delay or prevent detection. As a first step toward implementing a general protocol for using UDG at the level of single copy genes in single cells, we investigated conditions for preventing contamination during amplification of a 133-bp segment within the multicopy testis-specific protein gene (TSPY) from single male lympho-cytes. Cell lysis and real-time PCR with molecular beacons were carried out as described previously (3), except that the extension step of thermal cycling was increased to 30 s and the dTTP was replaced with dUTP at a 3-fold higher concentration. The resulting PCR product contained 27 uracil residues in the sense strand and 28 uracil residues in the antisense strand. Initial experiments compared the efficiency of amplification in the presence of dUTP or dTTP in terms of the mean detection cycle (C T) value (i.e., the point at which the molecular beacon fluorescence intensity reaches a threshold of 200 U) and the final fluorescence after 45 cycles. Amplification plots are shown in Figure 1A. The mean C T values of 33.9 and 34.4 for dUTP and dTTP …

Linear-After-The-Exponential Polymerase Chain Reaction and Allied Technologies

Accurate detection of gene sequences in single cells is the ultimate challenge to polymerase chain reaction (PCR) sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of linear-after-the-exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at different concentrations, but with novel design criteria to ensure high efficiency and specificity. Compared with conventional PCR, LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single cell genetic diagnosis.

Rapid Detection of TEM-Type Extended-Spectrum β-Lactamase (ESBL) Mutations Using Lights-On/Lights-Off Probes with Single-Stranded DNA Amplification

Rapid identification of specific TEM-type β-lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the blaTEM gene, as well as detecting widely spaced extended-spectrum β-lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25°C to 75°C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.

LATE-PCR and allied technologies: real-time detection strategies for rapid, reliable diagnosis from single cells.

Accurate detection of gene sequences in single cells is the ultimate challenge of PCR sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of Linear-After-The-Exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at -different concentrations, but with novel design criteria to insure high efficiency and specificity. LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single-cell genetic diagnosis.

Rapid detection of sequence variation in Clostridium difficile genes using LATE-PCR with multiple mismatch-tolerant hybridization probes.

A novel molecular assay for Clostridium difficile was developed using Linear-After-The-Exponential polymerase chain reaction (LATE-PCR). Single-stranded DNA products generated by LATE-PCR were detected and distinguished by hybridization to fluorescent mismatch-tolerant probes, as the temperature was lowered after amplification in 5(°)C intervals between 65°C and 25°C. Single-tube multiplex reactions for tcdA, tcdB, tcdC, and cdtB (binary toxin) sequences were initially optimized using synthetic targets and were subsequently done using genomic DNA; each target was detected and characterized by hybridization to one or more probes of a different fluorescent color. In the case of tcdC, three probes, each labeled with a Quasar fluorophore, hybridize to different locations with known mutations, including the deletion at nucleotide 117 in ribotype 027 strains and the premature stop codon mutation at nucleotide 184 in ribotype 078 strains, each of which is associated with hypervirulent infections. These and other tcdC mutations were distinguished from the reference sequence, as well as from each other by changes in the fluorescent contour generated from the combined Quasar-labeled probes. Specific variations in tcdA and tcdB were also identified in the multiplex assay, including those that identified strains lacking toxin A production. This single closed-tube assay generates substantially more information about virulent C. difficile than currently available commercial assays and could be further expanded to provide strain typing.