Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus

Poddar, S. K.

doi:10.1006/mcpr.1999.0278

Cited by 84 publications

(65 citation statements)

References 15 publications

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“…Depletion of the limiting primer during the exponential amplification results in the linear synthesis of the strand extended from the excess primer. Although asymmetric PCR generates brighter signals than symmetric PCR does (6), it is seldom used because it exhibits overall efficiencies of 60-70%, in contrast to symmetric PCR, which is typically 90% or more efficient (7,8). Asymmetric PCR also requires extensive optimization to identify the proper primer ratios, the amounts of starting material, and the number of amplification cycles that can generate reasonable amounts of product for individual templatetarget combinations.…”

mentioning

confidence: 99%

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

Sanchez

Pierce

Rice

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

199

172

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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.

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mentioning

confidence: 99%

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

Sanchez

Pierce

Rice

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

199

172

View full text Add to dashboard Cite

show abstract

“…To improve the sensitivity and reproducibility of our assay, we used asymmetric PCR technique to generate an excess of single-stranded DNA targets (32)(33)(34). Because PCR amplicons are invariably longer than the 17-base probe sequence, PCR primers were designed so that the recognition element is placed either at the 3Ј end of the PCR product or 48 bases from the 3Ј end (termed int-PCR).…”

Section: Methodsmentioning

confidence: 99%

Rapid, sequence-specific detection of unpurified PCR amplicons via a reusable, electrochemical sensor

Lai

Lagally

Lee

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

177

148

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We report an electrochemical method for the sequence-specific detection of unpurified amplification products of the gyrB gene of Salmonella typhimurium. Using an asymmetric PCR and the electrochemical E-DNA detection scheme, single-stranded amplicons were produced from as few as 90 gene copies and, without subsequent purification, rapidly identified. The detection is specific; the sensor does not respond when challenged with control oligonucleotides based on the gyrB genes of either Escherichia coli or various Shigella species. In contrast to existing sequence-specific optical-and capillary electrophoresis-based detection methods, the E-DNA sensor is fully electronic and requires neither cumbersome, expensive optics nor high voltage power supplies. Given these advantages, E-DNA sensors appear well suited for implementation in portable PCR microdevices directed at, for example, the rapid detection of pathogens.E-DNA ͉ methylene blue ͉ Salmonella gyrB ͉ polymerase chain reaction T he species-specific identification of pathogenic bacteria poses a pressing problem with impacts ranging from food safety to the detection of biowarfare agents. For example, it has been shown that the early identification of bacterial pathogens can significantly reduce the breadth and severity of outbreaks of food-borne diseases (1), outbreaks that are responsible for Ϸ76 million illnesses, 325,000 hospitalizations, and 5,000 deaths per year in the United States alone (2). Current methods for the detection and identification of bacteria, however, are complex and slow; because the minimum infectious doses of food-borne pathogens such as Escherichia coli, Listeria monocytogenes, and Salmonella are very low (3, 4), the detection of clinically relevant levels of contamination generally requires amplification of the infectious organism via laboratory culturing over the course of 1 to several days (5, 6).The PCR-based amplification of pathogen-specific DNA, rather than the pathogens themselves, offers a potentially promising means of avoiding cumbersome, time-consuming culturing steps and achieving the rapid and reliable identification of microbes. Unfortunately, however, the methods traditionally used for the detection of PCR-amplified DNA, which include Southern blots and capillary electrophoresis (CE), are rather unwieldy, and, thus, PCR-based assays are typically limited to laboratory settings. In response to this problem, a number of more convenient, field-portable PCR detection schemes have been described in recent years (7,8). In particular, because microfluidic techniques allow the miniaturization of PCR reactions to single-chip dimensions, there has been much interest in the development of a PCR-amplification͞detection platform integrated onto a single integrated microdevice (9). The first such approach used the optical detection of PCR products via intercalating dyes that report on the presence of double-stranded DNA (10, 11). PCR, however, is often promiscuous, producing spurious amplification products (12, 13) that produce false pos...

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“…The data in the present report establish that the Centricon Plus-20 ®lter system concentrates viruses of average diameter greater than or equal to that of adenovirus (75±90 nm). Thus, it is expected that suspensions of other viruses (in¯uenza, 90±100 nm; parain¯uenza, 125±200 nm; herpes virus, 180±200 nm; poxviruses, 200±250 nm; cytomegaloviruses, 900±1000 nm) [8] could also be concentrated by following this method and subsequently their nucleic acid could be extracted rapidly for application in various procedures, such as nucleic acid ampli®cation and molecular diagnostics [9].…”

Section: Discussionmentioning

confidence: 99%

A modified rapid method of nucleic acid isolation from suspension of matured virus: applied in restriction analysis of DNA from an adenovirus prototype strain and a patient isolate

Gray

Poddar

2001

Journal of Medical Microbiology

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This report describes a method for the isolation of nucleic acid from a suspension of matured virus. Nucleic acid (DNA) was isolated from a prototype strain of adenovirus type 7 and a clinical isolate of adenovirus type 7. Instead of the usual method of ultracentifugation, a ®ltration method was applied to concentrate the virus rapidly and nucleic acid was then isolated by a standard phenol/chloroform/isoamyl-alcohol extraction procedure. The DNA was found to be suf®ciently puri®ed to generate a reproducible restriction endonuclease digestion pattern. The clinical isolate of adenovirus type 7 revealed loss of restriction site for the endonuclease HindIII when compared with the prototype strain.

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Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus

Cited by 84 publications

References 15 publications

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

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

Rapid, sequence-specific detection of unpurified PCR amplicons via a reusable, electrochemical sensor

A modified rapid method of nucleic acid isolation from suspension of matured virus: applied in restriction analysis of DNA from an adenovirus prototype strain and a patient isolate

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