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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 CF Δ 508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

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...

Xist RNA localizes to the inactive X chromosome in cells of late cleavage stage female mouse embryos (Sheardown et al., 1997: Cell 91:99-107). Fluorescence in situ hybridization (FISH), however, does not quantify the number of Xist transcripts per nucleus. We have used real-time reverse transcription-polymerase chain reaction (RT-PCR) to measure Xist RNA levels in single preimplantation embryos and to establish developmental profiles in both female and male samples. The gender of each embryo was readily established based on Xist RNA levels, by counting Xist gene copies per cell, and by independent detection of the presence/absence of Sry, a Y chromosome-specific gene. Xist expression in males was found to be very low at all stages, as suggested by FISH. In contrast, female embryos contained measurable levels of Xist mRNA starting at the late 2-cell stage and rapidly accumulated Xist transcripts until morula stage. Xist RNA accumulation per embryo then reached a plateau, while cell division continued. We propose that during early cleavage high enough levels of Xist mRNA are transcribed to generate a pool of unbound molecules. This pool would serve to temporarily maintain X chromosome inactivation without additional transcription while the trophectoderm and inner cell mass (ICM) differentiate. The ICM would then loose the paternally imprinted pattern of X inactivation originally present in all embryonic cells.

Human neutrophil nuclei typically consist of three of four large heterochromatic lobes joined by thin, DNA-containing filaments. In addition, some lobes exhibit appendages of various sizes and shapes. Classical genetic and cytological studies suggest that some appendages contain specific chromosomes. The studies reported here provide the first detailed analysis of the spatial relationship between individual chromosomes and recognizable structures in neutrophil nuclei using fluorescent in situ hybridization. Analysis of DNA sequences in chromosomes 2, 18, X, and Y demonstrate that specific lobes in a population of neutrophil nuclei do not have a fixed chromosome content. This result implies that chromosomes partition randomly among lobes during neutrophil differentiation. However, neutrophil nuclear topography is not entirely fortuitous. For instance, none of the sequences probed in this study mapped to a filament and most centromeres lie in clusters near the nuclear periphery. In addition, one of the X chromosome centromeres in females and the Y chromosome centromere in males consistently associate with specific nuclear appendages found in a subset of neutrophil nuclei. Chromosomes 2 and 18 occupy discrete nd separate territories within individual lobes and neither territory ever extends into a filament. Surprisingly, the sizes of these territories are not proportional to chromosome length, suggesting that individual neutrophil chromosomes vary in their degree of compaction. These results are discussed in the light of models that attempt to explain nuclear morphology in terms of chromosome spatial organization.