Capillary electrophoresis methodology for identification of cancer related gene expression patterns of fluorescent differential display polymerase chain reaction

George, Kimberly S; Zhao, Xilin; Gallahan, Daniel; Shirkey, Ann; Zareh, Ali; Esmaeli-Azad, Babak

doi:10.1016/s0378-4347(97)00115-1

Cited by 14 publications

(7 citation statements)

References 14 publications

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“…The mixture was denatured at 95°C for two minutes, and analysed through a DNA Sequencing Polymer (Applied Biosystems) with an ABI PRISM Genetic Analyser (Applied Biosystems), according to a previous method. 25 Data were analysed using Genescan 310 Software (Applied Biosystems).…”

Section: Clonality Assessmentmentioning

confidence: 99%

Polyclonal nature of diffuse proliferation of interstitial cells of Cajal in patients with familial and multiple gastrointestinal stromal tumours

Chen

Hirota²,

Isozaki³

et al. 2002

Gut

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Background: Diffuse proliferation of interstitial cells of Cajal (ICCs) in the myenteric plexus layer of the intestine has been described in patients with familial and multiple gastrointestinal stromal tumours (GISTs). However, it is not fully understood whether proliferation is polyclonal or monoclonal. Aims: To evaluate the clonal nature of diffuse ICC proliferation in familial and multiple GIST cases, we carried out clonal analysis using inactivation at the human androgen receptor (HUMARA) locus. Materials and methods: Diffuse ICC proliferation tissues from three female patients were microdissected using a laser capture microdissection (LCM) system. Normal intestinal mucosal tissues were also microdissected for polyclonal controls and GIST tissues for monoclonal controls from the same patients, and genomic DNA was extracted. After digestion by restriction enzyme HhaI, the HUMARA locus was amplified by a fluorescent polymerase chain reaction (PCR) procedure and the PCR products were analysed. Results: One case was uninformative because it was homozygous at the HUMARA locus. In the two other cases, PCR products from the diffuse ICC proliferation showed two alleles as well as those from normal intestinal mucosal tissues, indicating that ICC proliferation was polyclonal. In contrast, PCR products from associated GIST tissues showed only one allele, indicating that GISTs were monoclonal. Conclusion:The results suggested that diffuse ICC proliferation in familial and multiple GIST cases was non-neoplastic hyperplasia.

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Section: Clonality Assessmentmentioning

confidence: 99%

Polyclonal nature of diffuse proliferation of interstitial cells of Cajal in patients with familial and multiple gastrointestinal stromal tumours

Chen

Hirota²,

Isozaki³

et al. 2002

Gut

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“…Sorting by molecular weight was originally performed using gel electrophoresis but is nowadays carried out by capillary electrophoresis 7, 25. Originally, radioactive or optical labels were applied in four different terminator reactions (each sorted and read out separately), but today four different fluorophores, one per nucleotide (A, C, G, and T) are used in a single reaction 6.…”

Section: Introductionmentioning

confidence: 99%

High‐throughput DNA sequencing – concepts and limitations

2010

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Recent advances in DNA sequencing have revolutionized the field of genomics, making it possible for even single research groups to generate large amounts of sequence data very rapidly and at a substantially lower cost. These high-throughput sequencing technologies make deep transcriptome sequencing and transcript quantification, whole genome sequencing and resequencing available to many more researchers and projects. However, while the cost and time have been greatly reduced, the error profiles and limitations of the new platforms differ significantly from those of previous sequencing technologies. The selection of an appropriate sequencing platform for particular types of experiments is an important consideration, and requires a detailed understanding of the technologies available; including sources of error, error rate, as well as the speed and cost of sequencing. We review the relevant concepts and compare the issues raised by the current high-throughput DNA sequencing technologies. We analyze how future developments may overcome these limitations and what challenges remain.

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“…In further development of fluorescent DDRT-PCR, labeling random primers [12] was replaced by labeling anchored primers and capillary DNA sequencers were used for cDNA fragment detection [9,10,23]. Although the use of Genotyper Genetic Analysis software (Perking-Elmer ABI) facilitated semi-automatic recognition of unique bands (e.g., bands present in the treated sample but missing in the control), quantitative evaluation of data has not been demonstrated.…”

Section: Discussionmentioning

confidence: 99%

Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles

et al. 2005

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BackgroundGene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR) and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far.ResultsWe developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i) cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii) intensity of bands is determined by densitometry, (iii) densitometric values are normalized, and (iv) intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment) for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans.ConclusionWe developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in reseach on organisms for which microarray analysis is not available or economical.

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Capillary electrophoresis methodology for identification of cancer related gene expression patterns of fluorescent differential display polymerase chain reaction

Cited by 14 publications

References 14 publications

Polyclonal nature of diffuse proliferation of interstitial cells of Cajal in patients with familial and multiple gastrointestinal stromal tumours

Polyclonal nature of diffuse proliferation of interstitial cells of Cajal in patients with familial and multiple gastrointestinal stromal tumours

High‐throughput DNA sequencing – concepts and limitations

Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles

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