Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1992-11-09
1996-09-03
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
436 94, 436501, 422 55, 422 56, 422 57, 422 58, 422 681, C12Q 168, G01N 3348
Patent
active
055522700
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to molecular biology, and particularly to a method for determining DNA nucleotide sequences, and a device for carrying this out.
BACKGROUND OF THE INVENTION
By now, several methods have been described for assaying a nucleotide sequence and identifying individual substitutions of bases by hybridization techniques (Cotton, R. G. H. // Biochem. J, 1989, V. 263, pp. 1-10).
The most widespread techniques are those in which a test DNA fragment is attached to a membrane and hybridized thereon with labelled oligonucleotides (Wallace, P. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., Itakura, K. //Nucleic Acid Res., 1979, V. 6 pp. 3543-3557).
Known in the art are a method and a device for determining a DNA nucleotide sequence (E. Southern et al., PCT/GB 89/00460, 1989), which method comprises synthesizing oligonucleotides on a glass support, effecting hybridization with radioactively- or fluorescently-labelled test DNA, washing in the duplex dissociation conditions, detecting the presence of individual substitutions in the test sequence by analyzing the autoradiographic patterns or the intensity of fluorescence at individual dots, and reconstructing the DNA nucleotide sequence on the basis of data analysis. A device for carrying out said method comprises a supporting film or glass plate and a matrix covalently attached to the surface thereof, the matrix comprising the whole set or a selected part of oligonucleotides of desired length, the latter oligonucleotides being capable of taking part in the hybridization reactions. The surface of the support to which the oligonucleotides are attached is made of glass.
The above method and device, however, have low sensitivity. The value of the signal from the labelled DNA (laid out in the near-surface layer of the support) obtained from each individual matrix element is limited by the substrate surface capacity (as regards the covalently attached oligonucleotides) and it cannot be raised without increasing either the area of the matrix element or the sensitivity of the labelling marker. These limitations reduce the resolving power of the method and device, make it difficult to miniaturize the matrix, increase the requirements imposed on the sensitivity and resolving power of the detector, and raise the consumption of the reagents. The method is rather complicated because, even in assaying a test DNA fragment, it requires a series of successive hybridizations to be performed, with additional rounds of oligonucleotide matrix synthesis to be performed at one-letter step, for each dot where hybridization has not yielded unambiguous information on the sequence and, therefore each time new optimal hybridization conditions (temperature, reagent concentrations, etc.) have to be chosen, which involves further experimentation and considerable expenditure of time and reagents.
DISCLOSURE OF THE INVENTION
It is an objective of the invention to alter the method and apparatus in such a way that to improve their efficiency, to increase sensitivity, accuracy and reproducibility, to simplify recognition of point mutations in the nucleotide sequence, and to reduce the expenditure of reagents.
In the present method for determining the DNA nucleotide sequence which comprises formation of an array of oligonucleotides, their hybridization with the labelled test DNA, washing under duplex dissociation conditions, identification of every single substitution of bases in the test DNA by analyzing distribution of the labelling marker, and eventual computerized reconstruction of the DNA nucleotide sequence, the above objective is achieved by that the invented method involves formation of an array of oligonucleotides at such concentrations thereof which ensure the desired temperature of duplex dissociation at the washing step.
To achieve reliable discrimination of fully complementary duplexes from duplexes having point mutations (mismatches), it is preferred to carry out the washing step at a fixed temperature gradient.
In order to impr
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Ershov Gennady M.
Florentiev Vladimir L.
Ivanov Igor B.
Khorlin Alexandr A.
Khrapko Konstantin R.
Horlick Kenneth R.
Institut Molekulyarnoi Biologii Imeni V.A.
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