Direct detection of nucleic acid hybridization on the surface of a charge coupled device.
A method is described for the detection of DNA hybrids formed on a solid support, based upon the pairing of oligonucleotide chemistry and the technologies of electronic microdevice design. Surface matrices have been created in which oligonucleotide probes are covalently linked to a thin SiO2 film. 32P labeled target nucleic acid is then hybridized to this probe matrix under conditions of high stringency. The salient feature of the method is that to achieve the highest possible collection efficiency, the hybridization matrix is placed directly on the surface of a charge coupled device (CCD), which is used to detect 32P decay from hybridized target molecules (1, Eggers, M.D., Hogan, M.E., Reich, R.K., Lamture, J.B., Beattie, K.L., Hollis, M.A., Ehrilich, D.J., Kosicki, B.B., Shumaker, J.M., Varma, R.S., Burke, B.E., Murphy, A., and Rathman, D.D., (1993), Advances in DNA Sequencing Technology, Proc. SPIE, 1891, 13-26). Two implementations of the technology have been employed. The first involves direct attachment of the matrix to the surface of a CCD. The second involves attachment of the matrix to a disposible SiO2 coated chip, which is then placed face to face upon the CCD surface. As can be predicted from this favorable collection geometry and the known characteristics of a CCD, it is found that as measured by the time required to obtain equivalent signal to noise ratios, 32P detection speed by the direct CCD approach is at least 10 fold greater than can be obtained with a commercial gas phase array detector, and at least 100 fold greater than when X-ray film is used for 32P detection. Thus, it is shown that excellent quality hybridization signals can be obtained from a standard hybridization reaction, after only 1 second of CCD data acquisition.
Center For Biotechnology, Baylor College of Medicine, TX.
J B
JB
Lamture
K L
KL
Beattie
B E
BE
Burke
M D
MD
Eggers
D J
DJ
Ehrlich
R
R
Fowler
M A
MA
Hollis
B B
BB
Kosicki
R K
RK
Reich
S R
SR
Smith
D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004563:Electrochemistry; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D013499:Surface Properties
D015345:Oligonucleotide Probes; D004247:DNA