Richard Lai

A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step

BackgroundSite-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.ResultsThe strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants.ConclusionThis strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.

A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus

BackgroundThe emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes.ResultsBioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y.ConclusionOur one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

A New, Multiplex, Quantitative Real-Time Polymerase Chain Reaction System for Nucleic Acid Detection and Quantification

Quantitative real-time polymerase chain reaction (qPCR) has emerged as a powerful investigative and diagnostic tool with potential to generate accurate and reproducible results. qPCR can be designed to fulfil the four key aspects required for the detection of nucleic acids: simplicity, speed, sensitivity, and specificity. This chapter reports the development of a novel real-time multiplex quantitative PCR technology, dubbed PrimRglo™, with a potential for high-degree multiplexing. It combines the capacity to simultaneously detect many viruses, bacteria, or nucleic acids, in a single reaction tube, with the ability to quantitate viral or bacterial load. The system utilizes oligonucleotide-tagged PCR primers, along with complementary fluorophore-labelled and quencher-labelled oligonucleotides. The analytic sensitivity of PrimRglo technology was compared with the widely used Taqman(®) and SYBR green detection systems.

Application of the primRglo assay chemistry to multiplexed bead assays

In this unit, we describe a multiplex microsphere quantitative PCR. The system is based on the use of two additional oligonucleotides within a single tube PCR reaction. The first oligonucleotide is modified with a single base pair mismatch and is otherwise equivalent to a universal sequence added to the forward PCR primer. Further, this first extra oligonucleotide is coupled to Luminex microspheres. The second additional oligonucleotide is designed to be complementary to the universal sequence, and is modified with the fluorescent dye Cy3. As the PCR reaction proceeds, the second oligonucleotide is able to bind to the microspheres. Thus, quantitative monitoring of PCR progress takes place. The microsphere‐mediated Cy3‐detection is measured using flow cytometry directly after the PCR reaction. This allows a flow cytometer analysis from up to 150 different spheres and, therefore, multiple genes in one reaction. The multiplex microsphere qPCR is demonstrated using three target genes from Influenza A and Neisseria meningitidis. The multiplex microsphere system will enable a higher degree of multiplexing than is possible with currently available qPCR systems. Curr. Protoc. Cytom. 69:13.13.1‐13.13.10.

Use of the Real Time Polymerase Chain Reaction Method to Establish a Tightly Regulated Ecdysone Inducible System in Mammalian Cells

Introduction: The ecdysone inducible system potentially allows the study of conditional expression of the ex- ogenous reporter genes that may be cell lethal or alter the phenotype during the selection of transfectants. The system re- lies on two independent transfections of plasmids named pVgRXR and pIND. Disruption of the regulatory element within the plasmid during stable integration can result in silenced or high background expression of the exogenous reporter gene. Previous studies (1) have reported a transient luciferase reporter assay to screen the cell lines stably transfected with pVgRXR plasmid. However, there is no suitable method to screen the subsequent pIND transfection. In this study, we demonstrate a real time polymerase chain reaction (PCR) strategy to screen for background expression problems associ- ated with the ecdysone expression system and to simultaneously allow discrimination between the products of endoge- nously expressed and transfected genes. Method: Two screening methods were applied sequentially in order to establish a functional ecdysone expression system. Firstly, the HCT116/VgRXR#8 (a human colon cancer cell line stably transfected with pVGRXR plasmid) was estab- lished by utilising the previously reported luciferase reporter assay (1). Finally, the functional ecdysone system (HCT116/VgRXR#8/mutant p53) was established by a real time PCR screening strategy. This PCR based screening method for the exogenous reporter gene was made possible by utilizing the unique BGH polyA tail from the exogenous reporter gene. Result: Even when the same parental cell line (HCT116/VgRXR#8) was used in the subsequent transfection, background expression was still a common phenomenon. This can be monitored by efficient and sensitive real time PCR. Further- more, the primers designed in this study have high specificity for the exogenous reporter gene. Conclusion: The combined use of luciferase and real time PCR methods was necessary to enable the establishment of a tightly regulated ecdysone inducible system in mammalian cells.