Comparison and critical evaluation of PCR-mediated methods to walk along the sequence of genomic DNA

Abstract

Although researchers can access information on the entire genomic DNA sequence of typical research organisms, convenient genome walking methods in the laboratory are still needed. For the analysis of microorganisms, these methods are especially useful because the available genetic information is often scarce or limited.Many genomic walking methods are based on the polymerase chain reaction (PCR), and useful methods have been developed. This report reviews the methodologies of PCR-mediated genomic walking methods and evaluates their efficiency and usefulness to help microbiologists to select the appropriate method for each target microorganism. The concept and specific features, such as advantages and disadvantages, of five major PCR-mediated genomic walking methods (random PCR, inverse PCR, panhandle PCR, cassette PCR, and rapid amplification of genomic ends) are briefly described. The improved methods and their characteristics are listed, and a report of experimental comparison of such methods is also introduced briefly. Each of these methods has both advantages and disadvantages, and there is a trade-off between the specificity of target amplification and the ease of the method. The cassette PCR seems to be a standard method, but suitable method should be selected in consideration of the characteristics of the material.

Molecular Characterization of Transgenic Rice Producing Resveratrol

“…The multiple or incomplete integration events pose technical challenges for characterization of transgenes using PCR-based chromosome-walking techniques (Tonooka et al 2009;Zhang et al 2012). In this study, we were confronted with a severe challenge for conventional PCR amplification due to the poly G region of the ubiquitin promoter.…”

Identification of the Genomic Insertion Site of the Thyroid Peroxidase Promoter–Cre Recombinase Transgene Using a Novel, Efficient, Next-Generation DNA Sequencing Method

“…A number of PCRbased methods are available for chromosome walking, such as inverse PCR (4), ligation-mediated PCR (5,6), and specific-primer PCR (7,8), which identify transgene flanking sequences. However, these methods are inefficient, can generate nonspecific amplification products, and have other deficiences that make them difficult to apply (9). Other researchers have used a combination of microarray hybrid capture and next-generation sequencing to identify transgene insertion sites (10).…”

Rapid Catch and Signal (RCS) Technology Platform: Multiplexed Three-Color, 30 s Microwave-Accelerated Metal-Enhanced Fluorescence DNA Assays

“…After amplification, DNA material can be easily detected by common analytical methods. Despite the obvious advantage of PCR in DNA detection, this approach has some disadvantages [2,8,9], e.g., sensitivity to DNA material contaminants; misreading; quite high cost of analysis, reagents, and time to fulfill experiments; and, most importantly, limited utility as a general fast and easy point-ofcare method of specific DNA sequence quantification [2,3].…”