Non-radioactive chemical sequencing of biotin labelled DNA

Richterich, Peter

doi:10.1093/nar/17.6.2181

Cited by 18 publications

(3 citation statements)

References 18 publications

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“…Another nonradioactive labeling strategy that was stable during the chemical reactions uses a biotin marker molecule chemically or enzymically attached to an oligonucleotide primer or enzymically attached to an end labeling reaction of restriction enzyme sites (Richterich, 1989). After fragment separation by direct blotting electrophoresis, the membrane-bound sequence pattern can be visualized by a streptavidin-bridged enzymic color reaction.…”

Section: Maxam and Gilbert Methodsmentioning

confidence: 99%

Gene Technology: Applications and Techniques

2012

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Section: Maxam and Gilbert Methodsmentioning

confidence: 99%

Gene Technology: Applications and Techniques

2012

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“…A methodological simplification, making use of magnetic bead capture for the purification steps (Ohara et al, 1997) relies, however, on the availability of 5' biotin/5' fluorescein doubly labelled primers for labelling of the target DNA by PCR and subsequent solid phase capture. Richterich (1989) added a biotin label to DNA by endfilling a restriction site. Chemical degradation of this DNA, followed by blotting and colourimetric detection supplied satisfactory sequence information.…”

Section: Chemical Cleavage Reactionmentioning

confidence: 99%

Strategies for introducing non-radioactive labels during the automated sequence analysis of nucleic acids

Igloi¹

1998

Electron. J. Biotechnol.

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Rapid, large-scale genome as well as diagnostic DNA sequencing projects are, at present, dependent on the use of sensitive automated sequencers that rely on the detection of fluorescent signals. This emission is not an intrinsic property of the biomolecules but is a property of an optical label that must be incorporated before, during or after the sequence specific reaction. The choice of strategy, that is to be reviewed here, is a function of both the chemistry and the enzymology of the system as well as the nature of the fractionation and the physical parameters of the detection. One may differentiate beween the labelling of the primer, incorporation of the label during elongation or base specific termination with labelled terminators. In reviewing the development of each of these strategies, the conclusion is reached that, whereas labelled primers have universal applicability, the current generation of fluorescent terminators have in, conjunction with appropriate DNA polymerases, attained a refinement that strongly favours their use. However, since they are not available for all commercial sequencing systems, the alternative procedures are not merely of historic interest but are an essential component in DNA sequencing protocols. The possibility of automated direct sequence analysis of RNA has not been ignored completely.

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“…To detect the fragment, the gel is exposed to Xray film for autoradiography, resulting in dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred [102]. Another non-radioactive labeling strategy which is stable during the chemical reactions uses a biotin marker molecule chemically or enzymatically attached to an oligonucleotide primer or an end-filling reaction of restriction enzymes sites [119]. After fragment separation by direct northern blot electrophoresis, the membrane-bound sequence pattern can be visualized by a streptavidin-bridged enzyme colour reaction [120].…”

Section: Chemical Methodsmentioning

confidence: 99%

Macroscopic, Microscopic and Molecular Evaluations for the Identification of Erythrina Species Distributed in Thailand

Khaonim

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Plants in the genus Erythrina belong to the family FABACEAE. There are six species distributed in Thailand (Erythrina fusca Lour., Erythrina stricta Roxb., Erythrina crista-galli L., Erythrina subumbrans (Hassk) Merr., Erythrina variegata L., and Erythrina indica Lam.). Due to the similarity of the morphological characters and synonym in a vernacular name, the identification of these species is ambiguous. Therefore, an accurate investigation of their identities is essential. This research aimed to distinguish six Erythrina spp. through the macroscopic, microscopic, and molecular genetic analyses. The anatomical characteristics of each species (cross section of midrib) and the constant values of leaves including stomatal number, epidermal cell number, stomatal index, epidermal cell area, vein islet number and palisade ratio were investigated. The macroscopic characters and anatomical characteristics of the midrib of six investigated Erythrina species were illustrated. The stomatal type of all six species was paracytic type which was consistent with unique characteristics of plants in this family. In terms of microscopic leaf constant numbers, the stomatal number and stomatal indices in lower epidermis among these six species were overlapping, whereas the stomata in the upper epidermis were found only in E. crista-galli, E. subumbrans and E. variegata (60-136, 12-44 and 4-28 stoma/mm2 respectively). E. crista-galli demonstrated the highest number of upper stomata which could be used as an indicator for the identification. In addition, E. subumbrans and E. variegata exhibited distinct upper epidermal cell number (1080-1820 and 404-532 cell/mm2 respectively). This study revealed the overlapping of the palisade ratio among six Erythrina species. Nevertheless, vein islet number could be used to identify E. subumbrans from other species (12.75-20.75 and 3.50-11.00 cell/mm2 respectively). Moreover, the cross sections of the midrib of six investigated Erythrina species revealed the distinguished arrangement of tissue especially the vascular bundle. None of the trichome was found in these species. Additionally, the nucleotide sequences of five regions; ITS, matK, psbA_trnH, rpoC and ycf1 gene, were evaluated and compared among these species and 2 outgroups. The sequence lengths of gene among six Erythrina species were 650, 790, 375, 416 and 634 base pairs in length, respectively. The genetic relationship was demonstrated as phylogenetic tree constructed from each gene region. All studied of Erythrina were classified into 6 groups. E. stricta and E. subumbrans were close related these other species. In conclusion, leaf macroscopic and microscopic characteristics of six Erythrina species distributed in Thailand, both qualitative and quantitative, could be used as a tool for these plants authentication. Molecular genetic characteristics using ITS, matK, psbA_trnH, rpoC and ycf1 gene sequences provided valuable information to evidently support the identification of six Erythrina species.

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Non-radioactive chemical sequencing of biotin labelled DNA

Cited by 18 publications

References 18 publications

Gene Technology: Applications and Techniques

Gene Technology: Applications and Techniques

Strategies for introducing non-radioactive labels during the automated sequence analysis of nucleic acids

Macroscopic, Microscopic and Molecular Evaluations for the Identification of Erythrina Species Distributed in Thailand

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