Surveys of Gene Families Using Polymerase Chain Reaction: PCR Selection and PCR Drift

Wagner, Andreas; Blackstone, Neil W.; Cartwright, Paulyn; Dick, Matthew H.; Misof, Bernhard; Snow, Peter M.; Wagner, Günter P.; Bartels, Janet L.; Murtha, Mike; Pendleton, John W.

doi:10.1093/sysbio/43.2.250

Cited by 230 publications

(104 citation statements)

References 10 publications

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“…The number of PCR cycles was limited to 20 to avoid a reduction in the microbial diversity represented in the libraries ͑Bonnet et al Polz and Cavanaugh 1998͒ and to minimize alterations in community composition caused by "bias 1:1" ͑Suzuki and Giovannoni 1996͒ which can result in higher equitability in the final amplified community. In addition, the DNA template concentration was increased to 100 ng per 25 L reaction volume in order to overcome stochastic variations ͑Polz and Cavanaugh, 1998͒, and the products of five PCR amplification replicates were pooled prior to cloning in order to minimize PCR drift ͑Polz and Cavanaugh 1998; Wagner et al 1994͒.…”

Section: Pcr Amplification Of 16s Rrna Genesmentioning

confidence: 99%

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Piza

Prado

Manfio

2005

Antonie Van Leeuwenhoek

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Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments ͑Santos-São Vicente estuary, Brazil͒. The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.

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Section: Pcr Amplification Of 16s Rrna Genesmentioning

confidence: 99%

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Piza

Prado

Manfio

2005

Antonie Van Leeuwenhoek

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“…The PCR method has become an invaluable tool in that specific DNA segments can be amplified from a background of complex genomes (Arnheim and Erlich 1992;Erlich et al 1991). In studies of molecular evolution (Wagner et al 1994) and microbial ecology (Steffan and Atlas 1991) this property has facilitated the characterization of both single genes and gene families in single or multiple species. This is generally achieved by designing specific primers that target conserved regions of homologous genes, thereby accelerating the detection, amplification, and ultimately sequence analysis of the genes under study.…”

Section: Introductionmentioning

confidence: 99%

Optimization of PCR amplification for sensitive capture of Methanopyrus isoleucyl-tRNA synthetase gene in environmental samples

2010

Ann Microbiol

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Microbes are abundant in marine and terrestrial ecosystems. However, the available evidence indicates that most microbial species in the environment cannot be cultivated. Accordingly, methodologies for characterizing genomes in environmental samples directly without cultivation have become foremost in importance. In this study, experimental conditions using PCR amplification to characterize such genomes were investigated with respect to the detection of target sequences present at low frequencies.Amplification of the isoleucyl-tRNA synthetase (IleRS) gene in Methanopyrus isolates was optimized regarding the choice of DNA polymerase, target sequence size, PCR primer size and the use of degenerate primers. Detection of IleRS was most sensitive when the target sequence size was about 1 kb. Among KOD, Taq and Pfu DNA polymerases, Pfu displayed the lowest efficiency, while KOD and Taq showed similar efficiency. Primers no shorter than 18mers gave satisfactory results. In addition, PCR bias caused by primer degeneracy could be alleviated by varying the balance between different added primers. Employing a combination of Taq DNA polymerase, a target DNA sequence approximately 1 kb in size, and primers of 20mer in length, PCR amplification of the IleRS gene could be achieved with as little as 2 pg pure isolated DNA template.

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“…PCR based allele dosage effects must be done with caution in order to exclude potential PCR artifacts. Two major classes of processes may skew template/product ratios based on theoretical modeling of PCR: PCR selection and PCR drift (Polz and Cavanaugh 1998;Wagner et al 1994). PCR selection comprises all mechanisms that inherently favour the amplification of certain templates due to properties of the genes, of their flanking sequences, or of the overall genome.…”

Section: Introductionmentioning

confidence: 99%

Assessment of the origin of new citrus tetraploid hybrids (2n = 4x) by means of SSR markers and PCR based dosage effects

Ferrante¹,

Lucretti

Reale

et al. 2009

Euphytica

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We report the accurate determination of the allelic configurations of a total of eight new citrus tetraploid hybrids by means of SSR analysis, coupled with capillary electrophoresis, and PCR based dosage effects. Tetraploid hybrids were spontaneously obtained from different interploid crosses (2x 9 4x) between diploid 'Femminello' lemon and the allotetraploid somatic hybrid (2n = 4x = 36) 'Key' lime ? 'Valencia' orange, and between diploid 'Wilking' and 'Fortune' mandarins and an autotetraploid 'Dancy' mandarin (2n = 4x = 36). To understand the opportunity to employ them in further backcross programs, the cytological mechanisms underlying their ploidy level were unambiguously determined using six SSR primers. PCR conditions were optimized and skewness in template/product ratios were verified. Tetraploid allelic configurations were determined from PCR based dosage effects using electropherogram peak heights to estimate the copy number per allele. In all the tetraploid hybrids we found out that diploginy (2n eggs) has occurred, contributing the extra haploid genome in the tetraploids. According to the marker genotypes, it was further inferred that the 2n eggs in 'Femminello' lemon resulted from first division restitution (FDR), while in 'Wilking' and 'Fortune' mandarins 2n eggs occurred in second division restitution (SDR). These new genotypes, with their improved genetic female background, can be therefore considered very valuable in our citrus genetic improvement program as pollen donors in backcrosses suitable to eliminate negative traits.

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Surveys of Gene Families Using Polymerase Chain Reaction: PCR Selection and PCR Drift

Cited by 230 publications

References 10 publications

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Optimization of PCR amplification for sensitive capture of Methanopyrus isoleucyl-tRNA synthetase gene in environmental samples

Assessment of the origin of new citrus tetraploid hybrids (2n = 4x) by means of SSR markers and PCR based dosage effects

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