Ayoub Rashtchian

Archaebacterial DNA Polymerases Tightly Bind Uracil-containing DNA

We show that archaebacterial DNA polymerases are strongly inhibited by the presence of small amounts of uracil-containing DNA. Inhibition appears to be competitive, with the DNA polymerase exhibiting ∼6500-fold greater affinity for binding the inhibitor than a DNase I-activated DNA substrate. All six archaebacterial DNA polymerases tested were inhibited, while no eubacterial, eukaryotic, or bacteriophage enzymes showed this effect. Only a small inhibition resulted when uracil was present as the deoxynucleoside triphosphate, dUTP. The rate of DNA synthesis was reduced by ∼40% when dUTP was used in place of dTTP for archaebacterial DNA polymerases. Furthermore, an incorporated dUMP served as a productive 3′-primer terminus for subsequent elongation. In contrast, the presence of an oligonucleotide containing as little as a single dUrd residue was extremely inhibitory to DNA polymerase activity on other primer-template DNA.

Immunochemical approaches to gene probe assays.

Antibodies specific for helical nucleic acids can be applied to assays for hybridized DNA and/or RNA. The assays can use either radioactive or nonradioactive detection systems. Antibodies specific for RNA-DNA hybrids are applicable to assays for measuring hybrid helices that are immobilized on plastic or nitrocellulose, whether the helices are preformed in solution or are formed on the solid-phase support. Alternatively, anti-RNA-DNA hybrid antibodies can be immobilized and used to capture hybrids formed in solution, resulting in an assay with a high signal-to-noise ratio. It has been applied to a test for the presence of ribosomal RNA of Campylobacter jejuni in biological samples.

Novel methods for cloning and engineering genes using the polymerase chain reaction

Use of the polymerase chain reaction (PCR) has become increasingly widespread in virtually all aspects of molecular biology. Recently, novel ligation-independent methods have been developed for the cloning of DNA fragments amplified using PCR. Ligation-independent cloning utilizing the enzyme uracil DNA glycosylase (termed UDG cloning) provides an efficient method for gene cloning and recombinant PCR. This technology is now being applied to site-directed mutagenesis, the generation of nested deletions, and the engineering of novel gene constructs. The ease and flexibility of this methodology, combined with PCR amplification, simplify gene cloning and engineering techniques.

Effective amplification of 20-kb DNA by reverse transcription PCR.

Abstract Polymerase chain reaction has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs. However, polymerase chain reaction amplification from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the integrity of the RNA template and the prevention of false priming events during reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain reaction conditions enabled us to improve the specificity and yield of product but they were not definitive. Finally, we have shown that the same reverse transcription polymerase chain reaction technology can be used for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance.

Molecular Cloning of PCR Products

It is often desirable to clone PCR products to establish a permanent source of cloned DNA for hybridization studies, to obtain high-quality DNA sequencing results, or to separate products when PCR amplification yields a complex mixture. The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments.

The Biotin System

Nucleic acid hybridization has been one of the most powerful techniques in molecular biology during the past two decades. The applications of nucleic acid hybridization range from determination of overall similarity between organisms (Brenner, 1989) to determination of even a single base mutation in a given gene. Nucleic acid hybridizations are basically performed in three general ways: (1) solution hybridization, (2) hybridization on membrane filters, and (3) in situ hybridization to cytological preparations.

Labeling and Detection of Nucleic Acids

Nucleic acid hybridization has been one of the most powerful techniques in Principle and molecular biology during the past two decades. The applications of nucleic applications acid hybridization range from determination of overall similarity between organisms (Brenner, 1989) to determination ofeven a single base mutation in a given gene. Nucleic acid hybridizations are basically performed in three general ways: (1) solution hybridization, (2) hybridization on membrane filters, and (3) in situ hybridization to cytological preparations.

16 – Ligation-Activated Transcription Amplification: Amplification and Detection of Human Papillomaviruses

This chapter describes an isothermal method for amplification of nucleic acids based on ligation of a promoter sequence to a target nucleic acid, resulting in target amplification through a transcribed RNA intermediate. This target amplification methodology has been combined with solution hybridization based enzymatic detection of amplified target nucleic acid to produce a highly sensitive method for the detection of nucleic acids. Application of these technologies to detection of human papillomaviruses (HPV) is described. The widespread utility of amplification in molecular biology and molecular diagnostic procedures has stimulated the development of other nucleic acid amplification methods. Some of these methods are dependent on the presence of a DNA target and amplify a target sequence; other methods are based on replicatable probes that are amplified after hybridization to reveal the presence of the probe. The ligation-activated transcription reaction is performed under isothermal conditions without thermocycling. The reaction is capable of using RNA or DNA as the starting template and produces both single-stranded RNA and DNA amplification products.