J. C. Andle, D. J. McAllister, J. T. Weaver, C. P. H. Vary* and J. F. Vetelino**
BIODE, Inc., 4 Rockland Ct., Brewer, ME 04412-1254
While many DNA detection applications ideally require "single copy" sensitivity, the ability to reliably amplify DNA sequences via polymerase chain reaction (PCR) techniques allows low copy numbers of target DNA to be amplified and detected using available instrumentation. Typically, the target is defined by two flanking sequences (primers) and any sequence flanked by these two primers is amplified. In practice, gel electrophoresis is employed to verify the molecular weight of the amplificant, providing further verification of sequence. However, gel electrophoresis does not allow the researcher to detect polymorphisms of a gene, in which point mutations, deletions or additions do not substantially alter the molecular weight of the DNA sequence.
One may employ a third DNA sequence - internal to the target sequence and containing the region of the suspected mutation - to perform an affinity-based recognition of the target. Typically, point mutations have dramatic effects on the affinity of DNA probes for the corresponding target. By employing an array of probe sequences - each corresponding to an anticipated mutation of the gene and immobilizing each probe to a separate detector- it is possible to differentially detect the various mutations.
One class of detectors which hold promise for such arrays are the piezoelectric biosensors [1]. Piezoelectric biosensors offer the ability to selectively detect small quantities of specific DNA - currently on the order of a few nanograms per milliliter of solution- in the presence of overwhelming concentrations of nonspecific DNA- as much as 1000-fold excess. Typically, the detection process is complete within a few minutes. These sensors directly detect the added mass of the analyte as it hybridizes with complementary DNA probes or triplex probes which are covalently bound to the surface. Mass sensitivities of a few hundred picograms per mm[2] are reported.
Past work has been directed at detecting chemically denatured DNA using complementary probes. Current effort is directed at the use of peptide nucleic acid (PNA) probes for triplex capture. Phase I of this project will investigate a four element (three probes plus reference) detector array. A DNA sample will be evaluated for the level of cross-reactivity to the "wrong" detectors relative to the "proper" and "reference" detectors.
Work supported in part by DOE under grants DE-FG02-92ER-81350 and DE-FG02-95ER81933.
[1] "Acoustic Wave-Based Biosensors", Invited paper, J. C. Andle and J. F. Vetelino, Sensors and Actuators A 44, pp.167-176 (1994).
* Maine Medical Center Research Institute, 125 John Roberts Rd., Suite 8, So. Portland, ME 04106
** Dept. of Electrical and Computer Eng. And Laboratory for Surface Science and Tech., 5764 Sawyer Research Center, University of Maine, Orono, ME 04469-5764