Electrophoretic Separations of Large DNA Molecules by Periodic Inversion of the Electric Field

Carle, Georges F.; Frank, Mark Barton; Olson, Maynard V.

doi:10.1126/science.3952500

Cited by 902 publications

(349 citation statements)

References 14 publications

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“…Field inversion gel electrophoresis (FIGE) was used to separate rare-cutter fragments (Carle et al, 1986). Digested DNA was separated on 1% GTG agarose (FMC BioProducts, Rockland, ME) gels run in an HE100 horizontal gel apparatus (Hoefer Scienti®c, San Francisco, CA) cooled with circulating water at 88C, using 0.56TBE bu er and an applied voltage of 200 V. Switching was performed using a PPI-200 v200.20 programmable electrophoresis controller and Program 7 (MJ Research, Inc., Watertown, MA).…”

Section: Field Inversion Gel Electrophoresismentioning

confidence: 99%

Altered expression of Erg and Ets-2 transcription factors is associated with genetic changes at 21q22.2-22.3 in immortal and cervical carcinoma cell lines

1997

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Human Papillomavirus (HPV) type 16 is the most frequently detected HPV in cervical cancer. Although epidemiologic and experimental evidence indicates a prominent role for HPV infection in the development of this disease, other factors are also involved. Altered expression of the ets family transcription factors erg and ets-2 was found associated with the development of cervical carcinoma. Overexpression also occurred in a HPV-16-immortalized cervical cell line, CX16-2, which has HPV integrated at a translocation breakpoint t(19;21) involving 21q22.2-22.3, where these genes have been mapped. Six of 10 cervical carcinoma cell lines overexpressed ets-2 RNA suggesting an association of overexpression with cervical cell neoplasia. A clonally related pair of cervical carcinoma cell lines, C-4I and C-4II, showed di erential expression of erg and ets-2. C-4I overexpressed ets-2 RNA compared to normal cervical cells and C-4II. C-4II expressed a 5.3 kb erg transcript not seen in C-4I, ectocervical cells or other cervical carcinoma cell lines examined. Pulsed ®eld gel electrophoresis was used to analyse changes in DNA fragments related to structural changes and to construct a physical map encompassing erg and ets-2. Alterations in erg and ets-2 RNA expression in each of three di erent cell lines examined were associated with translocations. Association between altered expression of erg and ets-2 and altered regional structural suggests that these genes are important targets in cervical carcinogenesis.

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Section: Field Inversion Gel Electrophoresismentioning

confidence: 99%

Altered expression of Erg and Ets-2 transcription factors is associated with genetic changes at 21q22.2-22.3 in immortal and cervical carcinoma cell lines

1997

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“…However, the accurate analysis of the profile of the continuous fragment distribution is not trivial Kraxenberger et al, 1994;Cedervall et al, 1995). In addition, PFGE could be complicated by paradoxical migration patterns (Carle et al, 1986;Chu, 1991;Löbrich et al, 1993). Therefore, PFGE and constant-field gel electrophoresis (CFGE) are preferably used to quantify only the fraction of DNA fragments released (FDR) from the bulk DNA (Blöcher et al, 1989;Stamato and Denko, 1990;Iliakis et al, 1991a, b).…”

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confidence: 99%

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

2003

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Nine human tumour cell lines (four mammary, one bladder, two prostate, one cervical, and one squamous cell carcinoma) were studied as to whether cellular radiosensitivity is related to the number of initial or residual double-strand breaks (dsb). Cellular sensitivity was measured by colony assay and dsb by means of constant-and graded-field gel electrophoresis (CFGE and GFGE, respectively). The nine tumour cell lines showed a broad variation in cellular sensitivity (SF2 0.17 -0.63). The number of initial dsb as measured by GFGE ranged between 14 and 27 dsb/Gy/diploid DNA content. In contrast, normal fibroblasts raised from skin biopsies of seven individuals showed only a marginal variation with 18 -20 dsb/Gy/diploid DNA content. For eight of the nine tumour cell lines, there was a significant correlation between the number of initial dsb and the cellular radiosensitivity. The tumour cells showed a broad variation in the amount of dsb measured 24 h after irradiation by CFGE, which, however, was not correlated with the cellular sensitivity. This residual damage was found to be influenced not only by the actual number of residual dsb, but also by apoptosis and cell cycle progression which had impact on CFGE measurements. Some cell line strains were able to proliferate even after exposure to 150 Gy while others were found to degrade their DNA. Our results suggest that for tumour cells, in contrast to normal cells, the variation in sensitivity is mainly determined by differences in the initial number of dsb induced.

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“…DNA molecules in an approximate size range of 20 to 1,500 kb are easily fractionated. Alternatively, samples of a defined size range were assayed by field-inversion gel electrophoresis (FIGE); (Carle et al, 1986). The switching regime used was a ramped forward switch time of 3 to 60 sec and a ramped backward switch time of 1 to 5 sec, with a total run time of 17 hr.…”

Section: Methodsmentioning

confidence: 99%

“…The switching regime used was a ramped forward switch time of 3 to 60 sec and a ramped backward switch time of 1 to 5 sec, with a total run time of 17 hr. The transfer of DNA from agarose gels to nitrocellulose, and details of hybridization have been described elsewhere (Carle et al, 1986;Sherman et al, 1983).…”

Section: Methodsmentioning

confidence: 99%

“…If coupled to the known genetic map, such an ordered library would provide information on genetic versus physical map distances and be a source of mapped fragments for nucleotide sequencing of DNA. In attempt to prepare an ordered human chromosome library, the pulsed-field gradient gel electrophoresis (PFGE), which can separate DNA molecules of hundreds to thousands of kilobase pairs in length (Schwartz and Cantor, 1984;Chu et al, 1986;Carle et al, 1986), and the yeast artificial chromosome vector (YAC) for the cloning of large fragments of human DNA have been developed (Burke et al, 1987). As a step towards the ultimate goal, a YAC was used to construct a complete human genome DNA library.…”

Section: Introductionmentioning

confidence: 99%

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Encapsulation of cells in agarose beads for use in the construction of human DNA libraries as yeast artificial chromosomes (YAC)

et al. 1990

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SummaryA simple and general method for the molecular cloning of fragments of over one hundred kilobase pairs of exogenous DNA, by the encapsulation of cells in agarose beads, was developed for the construction of a human genomic DNA library in a yeast artificial YAC chromosome vector (in situ YAC construction). The main advantages of this method for use in the construction of a human genome library are as follows. First, linear DNA molecules of up to several hundred kilobase pairs in size can easily be prepared by the partial restriction enzyme digestion of the DNA encapsulated in agarose beads in vitro. Second, less than 2 x l0 G cells scraped from tissue culture plates are sufficient for preparation of the linear DNA molecule for construction of the genome library. The technical manipulations involved in construction of clones of very large segments of DNA, including encapsulation of cells in agarose beads, restriction enzyme digestion, ligation with the YAC vector, transformation into host yeast cells, and stable propagation are discussed.

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Electrophoretic Separations of Large DNA Molecules by Periodic Inversion of the Electric Field

Cited by 902 publications

References 14 publications

Altered expression of Erg and Ets-2 transcription factors is associated with genetic changes at 21q22.2-22.3 in immortal and cervical carcinoma cell lines

Altered expression of Erg and Ets-2 transcription factors is associated with genetic changes at 21q22.2-22.3 in immortal and cervical carcinoma cell lines

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

Encapsulation of cells in agarose beads for use in the construction of human DNA libraries as yeast artificial chromosomes (YAC)

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