Nucleic Acid Blotting Techniques

Theory and Practice

Terry Amiss and Sharon Collins Presnell

1. INTRODUCTION

This chapter deals with basic concepts and techniques in nucleic acid blotting. Many of the techniques involved with Southern blotting and Northern blotting are similar. Negatively charged, purified nucleic acids from prokaryotic or eukaryotic cells are separated according to size by electrophoresis through an agarose gel matrix. The RNA or denatured DNA is subsequently transferred and immobilized onto a membrane composed of nitrocellulose or nylon. The nucleic acids on the membrane are then hybridized to a specific labeled "probe," which consists of homologous single-stranded nucleic acids that carry molecules, allowing detection and visualization of the hybridized probe. Hybridization between the immobilized nucleic acids and labeled probe allows detection of specific DNA or RNA sequences within a complex mixture of DNA or RNA. The specific method of detection and visualization is dependent on the nature of the labeled probe; radioactive probes enable autoradiographic detection, and probes labeled with enzymes facilitate chemiluminescent or colorimetric detection. Nucleic acid blotting yields valuable information pertaining to gene integrity and copy number (Southern blot) and provides a means of analyzing gene expression and mRNA size (Northern blot). These methods can be used to characterize tissues and cultured cells in the laboratory and often provide valuable information for clinical evaluation of patient samples.

2. NUCLEIC ACID BLOTTING IN THE 21ST CENTURY

With the discovery of polymerase chain reaction (PCR) and the myriad of RNA and DNA analysis technologies that have emerged in the past decade, most scientists no longer rely solely on traditional nucleic acid analysis tools such as Northern and Southern blotting techniques. With the proper controls, the presence of a specific gene can be determined by PCR, with far less DNA required as starting material. Mutations in specific genes can also be detected through the careful design and utilization of specific primers and/or sequence analysis of amplified DNA. PCR-based methods are still challenged by the complexity of human genomic DNA, and technical limitations of PCR have hindered its use in the analysis of long stretches of DNA, such as those produced by digestion with restriction enzymes. Limitations such as these are responsible for the persistence of Southern blotting as a technical approach to DNA analysis. Ed Southern himself explains that these difficult challenges were the problem that led to the development of the first blotting method, over 25 years ago (1).

The use of RT-PCR for gene expression analysis has superseded Northern blot analysis because of the development of methods that allow semiquantitative assessment of gene expression and the ability to conduct meaningful experiments with only a small amount of RNA. Although the quantity of RNA required and the time it takes to complete a Northern blot analysis make it an undesirable method for many routine studies of gene expression, there are unique advantages that Northern blotting offers. RT-PCR relies on the conversion of each copy of RNA into a corresponding cDNA, and then amplification of that cDNA into enough copies that they can be visualized; thus, there are two opportunities for mRNAs to be selected for or against: reverse transcription and amplification. Reverse transcription often selects against very small or very large mRNAs, and the success of amplification of nucleic acid relies heavily on the quality and effectiveness of the primers utilized. Furthermore, true quantitation of signal using an RT-PCR approach is more difficult than quantitation of a Northern blot signal for the above-mentioned reasons. The Northern blot has the unique advantage of detecting the true size of any mRNA and can be used to isolate novel transcripts from heterogeneous mRNA pools (2). Quantitation of the signal from the Northern blot is also more straightforward, because the strength of signal is related directly to the number of copies present in the original sample and is not an extrapolated value influenced by the efficiency of the steps in the process. Thus, there are still specific situations in which traditional blotting techniques are the best choice when analyzing nucleic acids.

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  • mika-matti
    What are nucleic acid blotting techniques?
    6 months ago

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