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A positive control for detecting heteroduplexes in DGGE for microbial community fingerprinting.

Article Excerpt
Denaturing gradient gel electrophoresis in combination with PCR has found a wide application for analysis of genetic variants in many disciplines of bioscience, especially evaluation of microbial diversity in environmental samples. However, amplification of an environmental sample containing multiple DNA species can lead to formation of heteroduplexes (HDs), a PCR artifact. Appearance of HDs on the denaturing gradient gel can lead to a skewed interpretation of results and inaccurate conclusions. The present study is designed to develop a simple methodology to verify presence or absence of heteroduplexes in PCR samples which in conjunction with 'reconditioned' PCR can be used as an effective control to indicate presence and positions of HDs during DGGE analysis. Identification of HDs can allow effective design of reconditioned PCR by varying template and primer amounts and PCR cycle number.

Denaturing gradient gel electrophoresis (DGGE) is a powerful technique for separation of double-stranded DNA molecules of nearly identical size according to their nucleotide composition. This technique has a wide range of applications, from detection of point mutations to evaluation of microbial community diversity. DGGE exploits the phenomenon of discrete melting behavior of double stranded DNA. PCR-amplified fragments are separated during electrophoresis according to their melting behavior on a polyacrylamide gel with a linearly increasing denaturant gradient. The resulting DGGE band patterns reflect sequence diversity in a given sample. However, during the final PCR cycles when the concentration of primers is low, heteroduplexes (HDs) can form. HDs result in "phantom bands" on the denaturing gradient gel and often are erroneously interpreted as additional DNA species or sequence variants (Acinas et al. 2005). This can lead to overestimation of sequence population diversity. Cloning of PCR products from a mixed DNA template sample can exacerbate the problem. The non-directed mismatch repair system of E. coli generates a number of novel sequences from a single HD clone (Ruano 1992; Qiu et al. 2001). Other screening methodologies which utilize PCR products such as RFLP and Thermal Gradient Gel Electrophoresis (TGGE) are prone to the same problem (Osborn & Moore 2000).

In the past there were a number of reports of PCR artifacts pertaining to DGGE. With the use of constant denaturing capillary electrophoresis, formation of HDs was demonstrated and a simple "reconditioning PCR" suggested (Jensen 1993; Thompson & Marcelino 2002) to reduce the number of HDs produced. In another study to eliminate HDs, samples were treated by T7 endonuclease (Qiu et al. 2001). Surprisingly, a recent literature search revealed neither evidence of research being conducted according to suggested improvements nor a general awareness of the problem within the DGGE community. This is probably due to a fact that in spite of recommended methods, researchers lack control measures to judge the absence or presence...

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