Use of an automated sequencer to determine the sequence specificity of DNA damage

Hardman, Leigh C.; Murray, Vincent

doi:10.1080/15216549700202751

Cited by 4 publications

(4 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, the use of capillary electrophoresis permits increased sensitivity and hence more accurate intensity determination of damage bands compared with slab gels. There have been previous attempts to utilize automated DNA sequencers to examine the sequence selectivity of DNA binding agents in slab gels (Hardman and Murray, ; Yindeeyoungyeon and Schell, ; Bando et al , ) and nonautomatic capillary gels (Fundador et al , ) with varying degrees of success.…”

Section: Resultsmentioning

confidence: 99%

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences

Paul-Heng

Murray

2011

Biomedical Chromatography

Self Cite

View full text Add to dashboard Cite

The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences.

show abstract

Section: Resultsmentioning

confidence: 99%

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences

Paul-Heng

Murray

2011

Biomedical Chromatography

Self Cite

View full text Add to dashboard Cite

show abstract

“…With regard to other techniques, Hardman and Murray (28) employed thermal cycling (linear amplification) and Taq DNA polymerase to investigate the sequence specificity of haedamycin and cisplatin on an automated DNA sequencer. This methodology permitted a longer DNA sequence to be analysed with higher precision and accuracy.…”

Section: The Investigation Of Dna-damaging Agents Using Automated Dnamentioning

confidence: 99%

The Use of Automated Sequencing Techniques to Investigate the Sequence Selectivity of DNA‐Damaging Agents

Murray¹,

Nguyen

Chen

2012

Chem Biol Drug Des

Self Cite

View full text Add to dashboard Cite

In this review, the use of automated DNA sequenc-ing techniques to determine the sequence specific-ity of compounds that interact with DNA is discussed. The sequence specificity of a DNA-damaging agent is an essential element in determining the cellular mechanism of action of a drug. A number of DNA-damaging compounds are mutagenic, carcinogenic, as well as being widely used as cancer chemotherapeutic agents. The distribution of lesions in a sequence of DNA can give vital clues in the determination of the precise mechanism of interaction of the agent with DNA. The DNA sequence specificity of a number of DNA-damaging agents has been delineated using automated DNA sequencing technology, and these studies are discussed in this review. The current state-of-the-art methodology involves capillary electropho-resis with laser-induced fluorescence detection usually on an Applied Biosystems ABI 3730 capillary sequencer. This current technique has higher resolution, greater sensitivity, higher precision, more rapid separation times, is safer and easier to perform than previous methods. The two main methods to determine the DNA sequence selectiv-ity of compounds that interact with DNA are described: end labelling and the polymerase stop assay. The interaction of the antitumour drug, bleomycin, with DNA is utilized to illustrate the recent technological advances. The determination of the DNA sequence specificity of a DNA-damaging agent is crucial in understanding the cellular mechanism of action of a drug. A large number of DNA-damaging agents have been shown to have mutagenic and carcinogenic properties, and several are in widespread clinical use as antitumour drugs. The sequence selectivity of a DNA-damaging agent can be an essential component in elucidating the exact mechanism of interaction of the agent with DNA. The DNA sequence specificity of a number of mutagenic and carcinogenic compounds has been determined including UV light, DMS and other alkylating agents (1). Similarly, the DNA sequence specificity of several DNA-damaging agents that are clinically used as antitumour drugs has been determined including cisplatin, bleomycin and chlorambucil (1). These studies have produced important information on the mode of action of these drugs as mutagenic, carcinogenic and cancer chemotherapeutic agents. There are two main methods to determine the DNA sequence selectivity of compounds that interact with DNA: end labelling (Fig-ure 1A) and the polymerase stop assay (linear amplification) (Fig-ure 1B). The end-labelling technique can detect DNA damage caused by agents that result in strand breakage (or that can easily lead to strand breakage). The polymerase stop assay can detect DNA damage that does not cause strand breakage but can hinder the passage of DNA polymerase (or other enzyme). With the end-labelling technique, a sequence of DNA is modified at one end to incorporate an easily detectable label, for example a radioactive atom or a fluorescent molecule (Figure 1A). The usual fluorescent label is 6-FAM (6-carboxyfluores...

show abstract

“…There are a range of methods that can be utilised to determine the sequence specificity of cisplatin and related analogues, such as the linear amplification reaction (LAR) (combined with automated DNA sequencing) [ 33 ] and single-strand ligation PCR (sslig-PCR) [ 34 ]. The LAR in particular, is advantageous for detecting bulky DNA adducts, which involves Taq DNA polymerase extending from a radioactively- or fluorescently-labelled oligonucleotide primer until its activity is terminated by a DNA adduct [ 35 – 37 ].…”

Section: Introductionmentioning

confidence: 99%

Characterisation of the DNA sequence specificity, cellular toxicity and cross-linking properties of novel bispyridine-based dinuclear platinum complexes

2016

Self Cite

View full text Add to dashboard Cite

BackgroundThe anti-tumour activity of cisplatin is thought to be a result of its capacity to form DNA adducts which prevent cellular processes such as DNA replication and transcription. These DNA adducts can effectively induce cancer cell death, however, there are a range of clinical side effects and drug resistance issues associated with its use. In this study, the biological properties of three novel dinuclear platinum-based compounds (that contain alkane bridging linkers of eight, ten and twelve carbon atoms in length) were characterised to assess their potential as anticancer agents.MethodsThe properties of these compounds were determined using a DNA template containing seven tandem telomeric repeat sequences. A linear amplification reaction was used in combination with capillary electrophoresis to quantify the sequence specificity of DNA adducts formed by these compounds at base pair resolution. The DNA cross-linking ability of these compounds was assessed using denaturing agarose gel electrophoresis and cytotoxicity was determined in HeLa cells using a colorimetric cell viability assay.ResultsThe dinuclear compounds were found to preferentially form DNA adducts at guanine bases and they exhibited different damage intensity profiles at the telomeric repeat sequences compared to that of cisplatin. The dinuclear compounds were found to exhibit a low level of cytotoxicity relative to cisplatin and their cytotoxicity increased as the linker length increased. Conversely, the interstrand cross-linking efficiency of the dinuclear compounds increased as the linker length decreased and the compound with the shortest alkane linker was six-fold more effective than cisplatin.ConclusionsSince the bifunctional compounds exhibit variation in sequence specificity of adduct formation and a greater ability to cross-link DNA relative to cisplatin they warrant further investigation towards the goal of developing new cancer chemotherapeutic agents.

show abstract

Use of an automated sequencer to determine the sequence specificity of DNA damage

Cited by 4 publications

References 2 publications

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences

The Use of Automated Sequencing Techniques to Investigate the Sequence Selectivity of DNA‐Damaging Agents

Characterisation of the DNA sequence specificity, cellular toxicity and cross-linking properties of novel bispyridine-based dinuclear platinum complexes

Contact Info

Product

Resources

About