A new technique for determining the distribution of <i>N</i>7-methylguanine using an automated DNA sequencer

Shoukry, Safwat; Anderson, Marshall W.; Glickman, Barry W.

doi:10.1093/carcin/12.11.2089

Cited by 6 publications

(4 citation statements)

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“…Moreover, minor binding sites can also be revealed. In addition, the rapid analysis of a large number of samples permits the examination of diverse sequences in an efficient manner’ (22). However, the research community did not immediately embrace this technique and continued to use radioactive labelling with conventional polyacrylamide gels to determine the DNA sequence specificity of DNA‐damaging agents (1).…”

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

confidence: 99%

“…The first example of the use of an automated DNA sequencer to determine the DNA sequence specificity of a DNA-damaging agent was in 1991 (22). They used an Applied Biosystems 370A automated DNA sequencer with polyacrylamide slab gels to investigate the DNA sequence selectivity of dimethyl sulphate damage in a fluorescently end-labelled PCR product.…”

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

confidence: 99%

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

Murray¹,

Nguyen

Chen

2012

Chem Biol Drug Des

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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...

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

confidence: 99%

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

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“…The introduction of automated sequencers has enabled a further advance to be made as detailed in this paper. Previous work with automated sequencers (6,11,12) has involved the purification of a fluorescently-labelled PCR product. Our procedure does not suffer from this drawback and can be used directly on damaged DNA -including damaged DNA from intact cells (8).…”

Section: Phonementioning

confidence: 99%

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

Hardman

Murray

1997

IUBMB Life

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An automated sequencer was used to determine the sequence specificity of DNA damage caused by hedamycin in the plasmid pUC19 using a linear amplification / Taq DNA polymerase method. Previously, manual DNA sequencers have been in widespread use to investigate the sequence specificity of a DNA damaging agent. Manual DNA sequencers are restricted in the length of DNA sequence that can be read at base pair resolution for densitometry. An automated sequencer can greatly expand on the length of analysable DNA sequence. An additional important capability of the automated sequencer, is the ability to quantitate the intensity of damage at each base pair site. Thus we have used the automated sequencer to elucidate the sequence specificity of DNA damage for 300 bp. We have carried out an extended analysis of the sequence specificity of hedamycin DNA damage and found that the sequences 5'-cG_ t-Y, t G_t and cG_g are preferentially damaged. The sequence specificity of cisplatin was also investigated.

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“…The design of the automated DNA sequencer determines the number and type of controls required. Oui laboratory's earlier efforts relied on the PerkinlElmer Applied Biosystems Division (PE/ABD) 370/373A system (Foster City, CA) (Sage et aI., 1992;Koehler et al, 1991;Shoukry et al, 1991). More recently we acquired Pharmacia Biotech A.L.E ™ DNA sequencers (Piscataway, NJ) and applied them to the study of sequence-specific DNA damage.…”

Section: Internal Standards and Sequencing Reactionsmentioning

confidence: 99%

Technologies for Detection of DNA Damage and Mutations

Pfeifer¹

1996

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A new technique for determining the distribution of N7-methylguanine using an automated DNA sequencer

Cited by 6 publications

References 0 publications

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

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

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

Technologies for Detection of DNA Damage and Mutations

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