The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension...

The objective of this study was to identify and construct a human natural phage single-chain antibody (scFv) library of human anaplastic thyroid carcinoma (ATC) using phage display technology. Total RNA was extracted from lymphatic tissue near an ATC and used to amplify variable heavy chain (VH) and variable light chain (VL) fragments with added linker sequences using reverse transcription-poly...

The standard methodology for titrating dengue viruses, the plaque assay, is slow, time consuming and relatively expensive. Other methods require machinery that may not be routinely accessible to all researchers, particularly those in developing nations. We therefore sought to develop a rapid, simplified semiquantitative polymerase chain reaction (PCR) methodology based on the use of a template ...

Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affec...

Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of Escherichia coli has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (...

The aim of this study was to construct a human single-chain variable fragment (scFv) gene library for gastric cancer, from which human anti-cancer-associated gene (CAGE) scFvs are selected. Human lymphocytes were isolated from the peripheral blood of 10 gastric cancer patients and whole human heavy and light chain genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR). VH...

To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The N...

The objective was to investigate the intracellular localization and association of pig major histocompatibility complex (MHC) class I subunits with invariant chain (Ii). Pig MHC class I subunit cDNAs were cloned by RT-PCR and eukaryotic expression plasmids of α and β2m were constructed with fusions to red or enhanced green fluorescent protein (pDsRed2-N1-α, pEGFP-N1-α, pDsRed2-N1-β2m, and pEGFP...