A Quantitative Tool for Producing DNA-Based Diagnostic Arrays

Whitaker, Tom J.

Atom Sciences, Inc.

关键词: Guanine;    Quality Control;    Dna;    Decay;    Atomization;   

DOI  :  10.2172/934664
RP-ID  :  DOE/ER/82536-2 Final Report
RP-ID  :  FG02-98ER82536
RP-ID  :  934664

美国|英语

来源: UNT Digital Library

【 摘 要 】

The purpose of this project was to develop a precise, quantitative method to analyze oligodeoxynucleotides (ODNs) on an array to enable a systematic approach to quality control issues affecting DNA microarrays. Two types of ODN's were tested; ODN's formed by photolithography and ODN's printed onto microarrays. Initial work in Phase I, performed in conjunction with Affymetrix, Inc. who has a patent on a photolithographic in situ technique for creating DNA arrays, was very promising but did seem to indicate that the atomization process was not complete. Soon after Phase II work was under way, Affymetrix had further developed fluorescent methods and indicated they were no longer interested in our resonance ionization technique. This was communicated to the program manager and it was decided that the project would continue and be focused on printed ODNs. The method being tested is called SIRIS, Sputter-Initiated Resonance Ionization Spectroscopy. SIRIS has been shown to be a highly sensitive, selective, and quantitative tool for atomic species. This project was aimed at determining if an ODN could be labeled in such a way that SIRIS could be used to measure the label and thus provide quantitative measurements of the ODN on an array. One of the largest problems in this study has been developing a method that allows us to know the amount of an ODN on a surface independent of the SIRIS measurement. Even though we could accurately determine the amount of ODN deposited on a surface, the amount that actually attached to the surface is very difficult to measure (hence the need for a quantitative tool). A double-labeling procedure was developed in which 33P and Pt were both used to label ODNs. The radioactive 33P could be measured by a proportional counter that maps the counts in one dimension. This gave a good measurement of the amount of ODN remaining on a surface after immobilization and washing. A second label, Pt, was attached to guanine nucleotides in the ODN. Studies were conducted using this technique and comparing the results of the radioactive label vs SIRIS measurements of Pt as a function of ODN length and distance of the Pt label from the attachment end. The SIRIS signal was not proportional to the amount of oligo attached to the surface as determined by the decay of the 33P label. We intentionally tested conditions under which one might expect the atomization efficiency to change and we believe this is the problem. Different lengths of oligos, and different placement of the label in the oligo affected the final signal. This obviously makes use of SIRIS as a quantitative tool for oligonucleotides problematic except under highly controlled situations.