Identification and Eradication of a Denatured DNA Isolated during Alkaline Lysis-Based Plasmid Purification Procedures

Sayers, Jon R.; Evans, Debbie; Thomson, John F.

doi:10.1006/abio.1996.0397

Cited by 28 publications

(17 citation statements)

References 2 publications

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“…Earlier models of the T5FEN-DNA interaction threaded single-stranded DNA through the arch. 5,14 However the recent observation that hFEN can catalyse structure-specific cleavage of bifurcated DNA structures that have an oligodeoxynucleotide hybridised to their single-stranded regions and therefore no free 5′ terminus, 20 and that T5FEN can cleave closed circular DNA substrates, 36,38,39 suggests that threading cannot be operational in these substrates. It would therefore appear more likely that 5′-singlestranded DNA passes in front of the helical arch, or is clamped by the helical arch folding over the DNA.…”

Section: Discussionmentioning

confidence: 99%

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Williams

Sengerová

Osborne

et al. 2007

Journal of Molecular Biology

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Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.

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Section: Discussionmentioning

confidence: 99%

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Williams

Sengerová

Osborne

et al. 2007

Journal of Molecular Biology

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“…The NID and alkali minipreps produced similar amounts and molecular forms of DNA, except for some “irreversibly denatured” (fast migrating, single stranded) DNA forms in the alkali method (Figure 2A, lanes 1,2 vs. 3,4, arrow) [1], [25], [29]. The plasmid used for these experiments contains 2 SacI sites separated by approximately 400 bp, and appearance of this band was analyzed by densitometry.…”

Section: Resultsmentioning

confidence: 98%

“…Restriction digests with the indicated amount of restriction enzyme for the indicated length of incubation at 37°C are shown in lanes 5, 7, 9, 11 for the alkali method and lanes 6, 8, 10, 12 for the NID protocol. Arrow indicates “irreversibly denatured” DNA [1], [25], [29]. Arrowhead indicates the 400 bp band, and densitometry analysis of this band is reported for lanes 5–12.…”

Section: Resultsmentioning

confidence: 99%

A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

et al. 2011

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Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E.coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

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“…Plasmid DNA libraries are usually prepared in Escherichia coli. Most plasmid isolation procedures use strongly alkaline conditions to facilitate lysis of the host bacteria (1) and such preparations tend to be contaminated to varying degrees with genomic bacterial DNA and with nicked and linearized plasmid (2). We have already demonstrated that T5 exonuclease can be used to selectively remove linear, nicked, and single-stranded DNA from a plasmid preparation (2).…”

Section: A Methods For Enhancing the Transfection Efficiency Of Miniprmentioning

confidence: 99%

“…A total amount of 1 g of DNA was used, consisting of 0.5 g of reporter construct (pCMV-d2EGFP or pTK-RL) and 0.5 g of either cDNA library plasmid preparation or pCDNA 3.1(ϩ). DNA was diluted in a total of 30 l of serum-free DMEM, 2 2.5 l of SuperFect was added, and the sample was vigorously mixed. The mixture was incubated for 7-10 min at room temperature, supplemented with 150 l DMEM containing 10% FBS, and added to the cells.…”

Section: A Methods For Enhancing the Transfection Efficiency Of Miniprmentioning

confidence: 99%

A Method for Enhancing the Transfection Efficiency of Minipreps Obtained from Plasmid cDNA Libraries

Kiss-Tóth

Dower

Sayers

2001

Analytical Biochemistry

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Identification and Eradication of a Denatured DNA Isolated during Alkaline Lysis-Based Plasmid Purification Procedures

Cited by 28 publications

References 2 publications

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

A Method for Enhancing the Transfection Efficiency of Minipreps Obtained from Plasmid cDNA Libraries

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