Limitations of current techniques

Great strides have been made at developing sensitive assays for the detection of DNA damage. However, many laboratories do not have access to the sophisticated analytical instruments, such as mass spectrometry, that are necessary for these assays. Consequently the applications of these tools have not been fully realized. Furthermore the large amounts of DNA required by several of the assays makes sample size a concern. The 32P-postlabeling assay has a high sensitivity but requires high levels of radioactivity. An ideal biomarker of DNA damage should have the following attributes to allow widespread utility. First, the biomarker should be robust with the ability to

Table 11.1 Techniques for measuring DNA damage

Technique

Adduct

Sensitivity

DNA

Problems/Comments

Comet Assay

Wide range

1/1010

per cell

Must score at least 100 cells

Electrochemical Detection

8-oxodG

1/106—107

50 ^g

Notoriously high background

Fluorescence Spectroscopy

AFB, PAH

1/105-106

100 ^g

Relatively low sensitivity

Immunoassays (ELISA,

AFB, PAH, 8-oxodG, UV

1/107-109

50-100 ^g

Need large amounts of DNA

IH, IB)

Mass Spectrometry (MS)

Wide range oxidative

1/105-106

100 ^g

Very sensitive, but equipment is

GC-IDMS

nitrosamines

1/1012

expensive and technically challenging

Accelerator MS

32P-Post-labeling

PAH, oxidative lesions

1/109—1010

5-15 ^g

Must use high levels of 32P

QPCR

Wide range

1/105

15 ng

Gene-specific

detect a large variety of lesions. Second, the assay should only require small amounts of DNA, keeping tissue sample size to a minimum. Third, the method should have high sensitivity so to detect biological relevant levels of damage. Fourth, the assay should have the ability to examine adduct frequencies in defined regions of DNA, such as mitochondrial genome or specific nuclear genes. Fifth, the methd should use common routine procedures without the need for expensive equipment or radioactivity making it widely accessible to research laboratories. Finally, the assay should have high throughput so that hundreds of samples could be processed in a day. Over the last decade, my laboratory has been working on an assay that is based on the QPCR. We recently have succeeded in measuring the amounts of PCR products using a 96-well fluorescence plate reader, circumventing the need for analysis by agarose gel electrophoresis gel or the use of radionucleotides. Thus, the QPCR assay fulfills all six of the criteria outlined above.

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