生物化学技术

Most attempts to identify and isolate a novel cDNA result in the acquisition of clones that represent only a part of the mRNA’s complete sequence (Fig. 1). The approach described here to clone the missing sequence (cDNA ends) employs polymerase chain reaction (PCR). Since the initial reports of rap ...

The synthesis of complementary DNA (cDNA) from an mRNA template by the action of reverse transcriptase is a fundamental technique in molecular cloning. The objective is to make a large number of long cDNA copies. The quality and length of the cDNA product largely depends on the quality of the mRNA used as ...

In studying the expression of a gene in mammalian tissues, it is important to determine the levels of the corresponding mRNA. Several methods have been described for measuring the level of expression of a gene in a tissue. These methods are in situ hybridization using cDNA probe or riboprobe (1), slot- ...

Technical improvements of methods used for the nonradioactive detection of nucleic acids have replaced the 32P-based techniques in many laboratories. The most commonly used labels are digoxigenin, fluorescein, and biotin, which are linked through a spacer to a nucleotide and are inco ...

In plant cells, mitochondrial RNA (mtRNA) constitutes about only 1% of the total RNA. From this, most RNAs are ribosomal RNAs. Thus, isolation of high purified mtRNA is necessary, not only for construction of a mitochondrial cDNA library, but also for the analysis of plant mitochondrial transcri ...

Methods allowing sensitive and accurate quantitative analysis of defined RNA species are required in a wide variety of gene expression studies. Unlike the traditional hybridization methods, RNase protection or SI nuclease assays, the methods based on reverse transcription (RT) and ...

There is a need for specific and sensitive methods of characterization of RNA adapted to clinical applications. For this purpose, we have designed a procedure consisting of a continuous reverse transcriptase-polymerase chain reaction (RT-PCR) step coupled with a nonradioactive de ...

Unlike gene expression in prokaryotic cells, which is primarily under transcriptional control, gene expression in eukaryotic cells is subject to both transcriptional and post-transcriptional controls. Since transcription and translation in eukaryotic cells are separat ...

The reverse transcription-polymerase chain reaction (RT-PCR; 1,2) applies the power of amplification to the study of gene expression: the PCR is carried out on cDNA obtained by reverse transcription of mRNA. The protocol described here has been developed to allow a simple and highly sensiti ...

The S1 nuclease is an endonuclease isolated from Aspergillus oryzae that digests single- but not double-stranded nucleic acid. In addition, it digests partially mismatched double-stranded molecules with such sensitivity that even a single base-pair mismatch can be cut and hence dete ...

Primer extension is a relatively quick and convenient means by which gene transcription can be monitored. The technique can be used to determine accurately the site of transcription initiation or to quantify the amount of cap site-specific message produced.

The isolation and characterization of RNA polymerases from the Salmonella phage SP6 and the E. coli phages T7 and T3 has revolutionized all aspects of the study of RNA metabolism (1–6). Indeed, it is now possible to generate unlimited quantities of virtually any RNA molecule in a chemically pure form. ...

RNA has the tendency to form both secondary and tertiary structures that can impede its separation by electrophoresis. As such, identical species of RNA exhibiting varying degrees of intramolecular base pairing migrate at different rates and result in the smearing of distinct RNA molecu ...

The isolation of uncontaminated, intact RNA is essential for analyzing gene expression and for cloning genes. The presence of a large quantity of naturally occurring carbohydrates makes plant tissues one of the most difficult materials from which to isolate high-quality RNA with good yi ...

This chapter describes the detection of specific DNA sequences by hybridization to a labeled probe of complementary sequence. This method is suitable for the detection of a wide range of DNA concentrations down to single-copy genes within mammalian genomic DNA (little more than 1 pg of hybrid ...

The use of enzyme-labeled probes, in conjunction with chemiluminescence, allows a flexible approach to hybridization and detection procedures. This is shown by the diversity of applications in which the system can be used, including Southern blots (1,2), Northern blots (3), colony and pla ...

DNA probes can be labeled using a fluorescein-labeled nucleotide and subsequently detected with a dioxetane-based chemiluminescence system using an antifluorescein antibody conjugated to alkaline phosphatase (1,2). The use of chemiluminescence avoids the fading of resul ...

The selection of an appropriate labeling reagent for a particular experiment, for the most part, depends on the sensitivity and resolution required. For maximum sensitivity in membrane hybridization, a radioactive label is often the label of choice.

The direct labeling of nucleic acid probes with horseradish peroxidase (HRP) was first described by Renz at European Molecular Biology Laboratory (EMBL) in 1984 (1). The methodology was combined with enhanced chemiluminescence (2) (a light-producing HRP catalyzed reaction based on l ...

Nonradioactive oligonucleotide probes are increasingly being used in various applications, for example, polymerase chain reaction (PCR) product detection, in situ hybridization and colony and plaque screening (1). Oligonucleotide probes are short, defined sequences, 15– ...