Temperature Gradient Gel Electrophoresis (TGGE) for the Detection of Polymorphic DNA and RNA

Henco, Karsten; Harders, Jutta; Wiese, Ulrich; Riesner, Detlev

doi:10.1385/0-89603-258-2:211

Cited by 9 publications

(5 citation statements)

References 5 publications

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“…Answer determination has also been accomplished by DNA sequencing (Lee et al 2004), or by the denaturation temperature gradient-polymerase chain reaction (DTG-PCR) associated with denaturing gradient gel electrophoresis (DGGE) and/or temperature gradient gel electrophoresis (TGGE) (Yamamoto et al 2002; Lee et al 2003). These answer determination methods are time-consuming, and can not be used to solve several types of DNA computing problems including a TSP (Lee et al 2003; Tanaka et al 2005) because the discriminatory powers of DTG-PCR, DGGE, and TGGE are dependent on the diversity of oligonucleotide components, especially the G and C content (Riesner et al 1992; Henco et al 1994). In addition, the feasible answers of a TSP exist as a heterogeneous composite of DNA sequences that represent optimal and sub-optimal answers in a single aqueous solution.…”

Section: Introductionmentioning

confidence: 99%

Padlock probe-mediated qRT-PCR for DNA computing answer determination

Xiong

Frasch

2010

Nat Comput

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Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg-20 ng linear detection range between thermal cycle threshold (C t value) and target content. The C t values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology.

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Section: Introductionmentioning

confidence: 99%

Padlock probe-mediated qRT-PCR for DNA computing answer determination

Xiong

Frasch

2010

Nat Comput

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“…Genome profiling consists of two basic technologies: random PCR and TGGE, which have been well established [ 22 , 23 , 24 ]. However, if it is to be used for the purpose of general and universal applications, well-defined standardization is absolutely required to obtain significant results.…”

Section: Methodsmentioning

confidence: 99%

A database for the provisional identification of species using only genotypes: web-based genome profiling

et al. 2002

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Correspondence: Koichi Nishigaki. E-mail: koichi@fms.saitama-u.ac.jp Abstract Background: For a long time one could not imagine being able to identify species on the basis of genotype only as there were no technological means to do so. But conventional phenotype-based identification requires much effort and a high level of skill, making it almost impossible to analyze a huge number of organisms, as, for example, in microbe-related biological disciplines. Comparative analysis of 16S rRNA has been changing the situation, however. We report here an approach that will allow rapid and accurate phylogenetic comparison of any unknown strain to all known type strains, enabling tentative assignments of strains to species. The approach is based on two main technologies: genome profiling and Internet-based databases.

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“…Um diese Faktoren zu reduzieren sind in der Analysestrategie zur Detektion von BRCA 1-Mutationen verschiedene Präscreeningverfahren, wie beispielsweise Single-strand-conformation-Polymorphismus (SSCP)-Analyse [17], Proteintruncation-Test (PTT) [11], enzymaticmutation-detection (EMD) [30], Denaturierungs-Hochdruck-Flüssigkeitschromatographie (DHPLC) [8], sowie allelspezifische Oligonukleotidhybridisierung [24], Denaturierungs-Gradienten-Gelelektrophorese (DGGE) [17], Temperatur-Gradienten-Gelelektro- phorese (TGGE) [9], Conformations-Sensitive-Gelelektrophorese (CSGE) [18] mit anschlieûender Sequenzierung des Bereichs der vermuteten Mutation, in der Testung. B. BRCA 1 und BRCA 2, bei denen keine präferentiellen Mutationsbereiche bekannt sind, stellt besondere Herausforderung an ein molekularbiologisches Labor.…”

Section: Techniken Zur Mutationsanalyseunclassified