32P-postlabeling assay for carcinogen-DNA adducts: description of beta shielding apparatus and semi-automatic spotting and washing devices that facilitate the handling of multiple samples.
The utilization of the 32P-postlabeling assay in combination with TLC for the sensitive detection and estimation of aromatic DNA adducts has been increasing in the past few years. The procedure consists of 32P-labeling of carcinogen-adducted 3'-nucleotides in the DNA digests using [gamma-32P]ATP and polynucleotide kinase, separation of 32P-labeled adducts by TLC, and their detection by autoradiography. During both 32P-labeling and initial phases of TLC, a relatively high amount of [gamma-32P]ATP (3.0-4.5 mCi) is handled when 30 samples are processed simultaneously. We describe the design of acrylic shielding apparatus, semi-automatic TLC spotting devices, and devices for development and washing of multiple TLC plates, which not only provide substantial protection from exposure to 32P beta radiation, but also allow quick and easy handling of a large number of samples, thus expediting the assay workup and making it less labor-intensive. Specifically, the equipment includes: (i) a multi-tube carousel rack (7.5 cm diameter and 7.7 cm height) having 15 wells to hold capless Eppendorf tubes (0.5 ml) and a rotatable lid with an aperture to access individual tubes; (ii) a pipet shielder; (iii) two semi-automatic spotting devices to apply radioactive solutions to TLC plates; (iv) a multi-plate holder for TLC plates; and (v) a mechanical device for washing multiple TLC plates. Item (i) is small enough to be held in one-hand, vortexed, and centrifuged to mix the solutions in each tube while beta radiation is shielded. Items (iii) to (iv) aid in the automation of the assay.
Reddy
M V
MV
Environmental Health and Safety Department, Mobil Oil Corporation, Princeton, NJ 08540.
Blackburn
G R
GR
D015197:Carcinogenicity Tests / Q000295:instrumentation*; D002273:Carcinogens / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D010761:Phosphorus Radioisotopes; D011835:Radiation Protection; D013048:Specimen Handling / Q000295:instrumentation*
D002273:Carcinogens; D010761:Phosphorus Radioisotopes; D004247:DNA