Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10 degrees C and (ii) utilization of annealing t...

A two-step strategy, named exclusive PCR or E-PCR, has been developed to overcome the main limitation of PCR, which is the detection of already-known sequences only. This strategy allows the ability to detect and further clone and sequence genes for which no specific primers are available and in which a variable region exists between two conserved regions. This approach has been applied to Baci...

Scarcely any invention has altered biological science so radically in such a short period as the polyme-rase chain reaction, or PCR. With this technique, minute amounts of DNA can be replicated very rapidly and thereby amplified to such an extent that the DNA becomes easy to detect, study and use for any given purpose. The potential of this technique in medicine has long been known, and ever mo...

We determined and compared the method detection limits (MDLalpha) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDLalpha and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer ...

Typically DNA used in PCR assays is usually extracted according to die phenol-chloroform method (1) or by an alternative 'saltingout' rapid purification (2). Moreover partially purified DNA obtained with rapid procedures have been reported to be suitable for PCR amplification (3). We describe here a more simple and efficient method to amplify DNA directly from whole blood samples without any pu...

We have developed a simple two-dimensional YAC pooling strategy to facilitate YAC library screening via STS and Alu-PCR approaches. The method has been implemented using the human total genomic YAC library of Olson and coworkers, and its validity tested by isolation of many chromosomes 19- and 21-specific YACs. The Alu-PCR approach is notable in that it is hybridization-based, such that PCR pri...

inverse PCR primers, P1 and P2, can be used to delete the same internal region from a series of DNA constructs containing the target region. Similarly, an internal deletion can be made by a PCR-ligation-PCR approach as described by Ali and Steinkasserer (1). According to this method, for example, the first-round PCR can be performed in two seperate reactions by primers V1/P2 and P1/V2, followed...

Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse tr...