Lawrence J. Wangh

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.

Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell

BackgroundCurrent methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost.ResultsThis paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres.ConclusionThis method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules.

Glucocorticoids act together with estrogens and thyroid hormones in regulating the synthesis and secretion of Xenopus vitellogenin, serum albumin, and fibrinogen

In Xenopus serum albumin and fibrinogen are proteins whose synthesis and secretion by the liver are regulated by dexamethasone, while the synthesis and secretion of vitellogenin are regulated by estradiol-17β. We have previously demonstrated (Wangh and Schneider, 1982, Develop. Biol. 89, 287–293) that triiodothyronine acts together with estradiol-17β in the induction of vitellogenin in liver cultures. We report here on the interactions of dexamethasone with estradiol-17β and triiodothyronine. When pieces of liver from an in vivo estrogen-induced animal were maintained in culture for more than 2 weeks in a defined hormone-free medium, synthesis of secreted proteins gradually decreased. Addition of estradiol-17β, even at 10−6M, reinduced very little secreted vitellogenin. In agreement with our earlier finding, addition of triiodothyronine together with estradiol-17β reinduced very high levels of secreted vitellogenin. Dexamethasone at a minimum effective dose of 10−8M reinduced secreted albumin and fibrinogen. If triiodothyronine was also added, the dose of dexamethasone required was 10−9M. Thus, triiodothyronine enhances the activity of either single steroid. Addition of dexamethasone together with estradiol-17β resulted in reinduction of vitellogenin as well as serum albumin and fibrinogen. Unexpectedly, addition of dexamethasone together with estradiol-17β and triiodothyronine resulted in reinduction of vitellogenin, but the failure to induce serum albumin and fibrinogen. It is known from in vivo studies that extensive treatment with estradiol-17β induces high levels of vitellogenin synthesis, but suppresses liver synthesis of most other secreted proteins, including serum albumin and fibrinogen. Thus, we have demonstrated that estrogen suppression of glucocorticoid regulated secreted proteins also occurs in isolated pieces of liver tissue and that suppression requires all three of the tested hormones.

Thyroid hormones are corequisites for estradiol-17β in vitro induction of Xenopus vitellogenin synthesis and secretion

Abstract Estradiol-17β administered to male frogs induces liver synthesis and secretion of vitellogenin, the precursor protein of the major egg yolk proteins. Estradiol-17β alone failed to induce this protein in cultures of liver tissue maintained for 1–2 weeks prior to addition of the hormone. If a “complex” defined culture medium, such as Coons modified Hams F12 medium, is used, efficient primary and secondary induction of vitellogenin synthesis and secretion occurs in the presence of estradiol-17β, triiodothyronine, and dexamethasone. Using Coons medium we investigated the role of both triiodothyronine and dexamethasone as corequisites of estradiol-17β induction of secreted vitellogenin. Control cultures given no hormones showed a gradual decrease in the level of secreted albumin and fibrinogen. Addition of dexamethasone, alone, induced increased synthesis of secreted albumin and fibrinogen as well as other proteins. Cultures given thyroid hormones, alone, showed an increased level of secreted albumin and fibrinogen at early time points in the culture period. Thus, at early times thyroid hormones appear to enhance the activity of endogenous glucocorticoids. Independent of their interaction with glucocorticoids, thyroid hormones also enhance the activity of estrogens. Long-term cultures given estradiol-17β, alone, failed to synthesize and secrete vitellogenin. In contrast, cultures given the estrogen together with thyroid hormones showed vitellogenin synthesis. These results imply that similar interactions of several hormones occur in vivo in adult animals treated with estrogens. In the accompanying paper the interaction of dexamethasone with estradiol-17β and triiodothyronine is described (L. J. Wangh, 1982, Develop. Biol. 89, 294–298) .

New insights into the mechanisms of nuclear segmentation in human neutrophils

During human neutrophil differentiation, large portions of the genome condense and associate with the nuclear envelope to form filament‐like structures. As a result, the nucleus of the mature neutrophil typically consists of a linear array of three or four lobes joined by thin, DNA‐containing filaments. Despite the medical significance of neutrophil nuclear morphology, little is known about the events regulating neutrophil nuclear differentiation and its pathological states. This work presents a new model of the mechanisms governing nuclear filament formation in human neutrophils. This model is based on recent chromosome mapping studies in human neutrophils and on studies of genetic and pathological conditions affecting neutrophil nuclear shape. According to this model, filament assembly is initiated by factors that interact with specific regions of the genome in a hierarchical and dose‐dependent manner. In this regard, the strategies governing the molecular interactions responsible for filament formation appear to resemble those involved in transcriptional silencing, a phenomenon that also affects the properties of extended chromosomal regions. According to the silencing paradigm, bound filament control Factors must recruit additional Filament Foehn factors which spread along adjacent DNA to mediate filament formation. A better understanding of the factors that shape the neutrophil nucleus may lead to new clinical tools for the diagnosis and manipulation of abnormal neutrophil differentiation. J. Cell. Biochem. 73:1–10, 1999.

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.

Fluorescent signatures for variable DNA sequences

Life abounds with genetic variations writ in sequences that are often only a few hundred nucleotides long. Rapid detection of these variations for identification of genetic diseases, pathogens and organisms has become the mainstay of molecular science and medicine. This report describes a new, highly informative closed-tube polymerase chain reaction (PCR) strategy for analysis of both known and unknown sequence variations. It combines efficient quantitative amplification of single-stranded DNA targets through LATE-PCR with sets of Lights-On/Lights-Off probes that hybridize to their target sequences over a broad temperature range. Contiguous pairs of Lights-On/Lights-Off probes of the same fluorescent color are used to scan hundreds of nucleotides for the presence of mutations. Sets of probes in different colors can be combined in the same tube to analyze even longer single-stranded targets. Each set of hybridized Lights-On/Lights-Off probes generates a composite fluorescent contour, which is mathematically converted to a sequence-specific fluorescent signature. The versatility and broad utility of this new technology is illustrated in this report by characterization of variant sequences in three different DNA targets: the rpoB gene of Mycobacterium tuberculosis, a sequence in the mitochondrial cytochrome C oxidase subunit 1 gene of nematodes and the V3 hypervariable region of the bacterial 16 s ribosomal RNA gene. We anticipate widespread use of these technologies for diagnostics, species identification and basic research.

Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

BackgroundThe formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR.ResultsWe constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression.ConclusionThis paper describes the first example of multiplex RT-LATE-PCR and its utility, when combined with PurAmp sample preparation, for quantitative analysis of transcript levels in single cells. With this technique, copy numbers of different RNAs can be accurately measured independently from their relative abundance in a cell, a goal that cannot be achieved using symmetric PCR. The technique illustrated in this work is relevant to a wide array of applications, such as stem cell and cancer cell analysis and preimplantation genetic diagnostics.

Replication of Xenopus erythrocyte nuclei in a homologous egg extract requires prior proteolytic treatment.

Reactivation and reinitiation of DNA replication in quiescent frog erythrocyte nuclei has been analyzed following incubation in extracts prepared from activated Xenopus eggs. Nuclear decondensation and DNA synthesis only occurred if nuclei were pretreated with low doses of trypsin. This protease treatment did not digest histones, but did degrade several nonhistone proteins. Activated erythrocyte nuclei swell and begin DNA synthesis by 30 min after being mixed with the egg extract. In some extracts virtually complete genome replication was achieved in all nuclei after 2-3 hr. Addition of several protease inhibitors during sperm nuclear isolation significantly reduced the template efficiency of these preparations. We concluded that proteolytic alteration of nonhistone nuclear structural proteins may be a general mechanism which permits quiescent nuclei to reenter the replication cycle. Erythrocyte nuclei and egg extracts provide an excellent experimental system in which to investigate the processes of nuclear reactivation.

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).