Contaminating insert degradation by preincubation colony PCR: a method for avoiding false positives in transformant screening

Colony PCR is a convenient alternative to conventional plasmid isolation and restriction digestion for high-throughput screening of recombinant colonies. However, an insert carryover from the ligation mix, adhered to colony or agar plate, generates a substantial number of false positives. To avoid this, a simple single-tube technique involving pre-PCR nuclease incubation has been developed by optimizing a buffer system that provides nuclease action and PCR amplification sequentially. Results presented in this work provide a technique that is amenable for high-throughput screening of recombinant colonies.     

A simple and rapid method for screening transformant yeast colonies using PCR.

A simple and rapid procedure for screening transformant yeast colonies is described. In this method, a trace amount of plasmid DNA is isolated from a small amount of yeast cell mass; then, the presence of the exogenous DNA in each yeast colony is detected by PCR amplification.     

PCR microfluidic devices for DNA amplification

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055–1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92–98; Schneegaβ, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101–121; deMello, A.J. DNA amplification: does ‘small’ really mean ‘efficient’? Lab Chip 2001; 1: 24N–29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77–87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28–29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820–825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.     

Direct colony PCR for rapid identification of varied microalgae from freshwater environment

Molecular identification of microalgal species is vital and involves sequencing of specific markers present in the genome, which are unique to a genus. PCR is a vital tool for identification of microalgae; the preparation of template DNA for PCR by traditional methods requires large amount of algal cells and a time-consuming process. One simple way to reduce these complications is to use the microalgal colonies directly for amplification of required DNA fragments from the genome. In this study, a simple cell-disrupting method, using autoclaved glass powder, has been used for direct colony PCR of microalgae. Four morphologically different microalgal strains were chosen from freshwater samples, and the PCR amplification reaction was evaluated with three different molecular markers (rbcL, internal transcribed spacer 2, and 18S rDNA). PCR amplification was optimized with less number of cells (0.04?×?10⁵), minimal quantity of glass powder (0.5 mg), and in the presence of Milli-Q water for internal transcribed spacer marker. The isolated strains were identified as Desmodesmus sp. JQ782747, Coelastrum proboscideum JQ898144, Chlorella sorokiniana JQ898145, and Scenedesmus sp. JQ782746 based on sequence similarity. This direct microalgal colony PCR proves to be a simple and rapid method for detection of varied microalgal species.     

Novel method for high-throughput colony PCR screening in nanoliter-reactors

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.     

Pre-germination genotypic screening using PCR amplification of half-seeds

A simple and rapid PCR-based method has been developed for determining the genotype of seeds before germination. Single half-seeds of rice (Oryza sativa L.) and wheat (Triticum aestivum L. em. Thell.) were preincubated, without grinding, in an aqueous extraction buffer. The resulting supernatants were then used in polymerase chain reaction (PCR) with oligonucleotide primers corresponding to rice single-copy sequences or a wheat microsatellite repeat. PCR products of identical size were amplified using either the half-seed extract or DNA isolated from leaf tissue. The remnant half-seeds can be maintained in ordered arrays using microtiter plates allowing the recovery of selected genotypes. Pre-germination genotypic screening of seed populations as described in this report should be useful for a variety of applications in plant breeding and genetics studies.     

One-step isolation of plant DNA suitable for PCR amplification

We report a one-step extraction technique for the isolation of plant DNA, DNA suitable for amplification by PCR can be produced from leaf material smaller than 0.3 mm² in less than 20 min, with no tube changes. The method was tested on several plant specA00AK020ies. The described method was found to extract DNA that could be amplified without any further purification or treatment. The isolated DNA was amplified using a universal chloroplast primer set. The method was validated by comparing size of PCR products generated by the novel method to PCR products generated using standard DNA isolation techniques.     

Methods for microbial DNA extraction from soil for PCR amplification

Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.     

Single-tube classical PCR for Candida auris and Candida haemulonii identification

Background

Candida auris and Candida haemulonii are emerging and multiresistant pathogens. C. auris has produced hospital outbreaks and is misidentified by phenotypic-based methods. The only reliable identification methods are DNA sequencing and MALDI-TOF.

Fast preparation of fungal DNA for PCR screening

Rapid DNA preparation for the quick screening is highly demanded in diverse research fields. Here, we combined an extraction buffer and heat treatment to generate DNA templates from yeast and filamentous fungal materials for PCR. This method may be widely applicable to diverse fungal species in clinical and basic studies.     

An improved colony PCR procedure for genetic screening of <Emphasis Type="Italic">Chlorella</Emphasis> and related microalgae

A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.     

DNA amplification using PCR with abutting primers

Molecular Biology - DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is...