Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-10-02
2001-03-27
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S810000, C435S091500, C536S023100, C536S024100, C536S024300, C536S024310, C536S024320, C536S024330, C536S025300
Reexamination Certificate
active
06207381
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nucleic acid analysis, and more particularly to the determination of the binding of an oligonucleotide probe to a test nucleic acid sequence, especially for the detection of sequence variations and quantification of products obtained in amplification reactions.
BACKGROUND OF THE INVENTION
Clinical analyses of DNA sequences are typically directed to determining how a gene in a patient sample differs from a prototypical normal sequence. DNA sequencing through the chain termination method developed by Sanger and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA 1977; 74: 5463-5467), and the chemical degradation method developed by Maxam and Gilbert (Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 1977; 74: 560-564), or using techniques such as Sequencing By Hybridization (SBH) or Sequencing By Synthesis (see e.g. WO 93/21340) all have the potential to identify mutations and in the same process also reveal the consequence of the mutation at the level of protein coding etc.
Another sequencing approach is disclosed in EP-A-223 618 which described the use of an immobilised DNA template, primer and polymerase exposed to a flow containing only one species of deoxynucleotide at a time. A downstream detection system then determines whether deoxynucleotide is incorporated into the copy or not by detecting the difference in deoxynucleotide concentrations entering and leaving the flow cell containing the complex of DNA template and polymerase.
For screening purposes, however, it is often sufficient, at least initially, to identify deviations from the normal sequence but without directly revealing how a sequence differs from the normal one or only roughly locating the mutation. There are a number of such techniques which speed up analysis as compared to those that involve DNA sequence determination.
One such method uses “label-free” detection based on surface plasmon resonance (SPR) for determining the binding of a short oligonucleotide probe to a single-stranded target sequence immobilised to a sensor chip. Since a mismatch significantly affects the binding affinity, the presence of a sequence deviation may be determined. This method has, however, several disadvantages, such as that it requires immobilizing long target sequences, usually PCR products, to the sensor chip and that the sensor chip can not be regenerated.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a method for determining the binding of an oligonucleotide probe to a test sequence, which method is devoid of the above disadvantages and which may be used for mismatch (mutation) detection as well as amplification (such as PCR) product quantification.
In accordance with the present invention, the above and other objects and advantages are achieved by a method of determining the binding of an oligonucleotide probe to a test nucleic acid sequence, comprising the steps of:
a) providing the test nucleic acid sequence in single-stranded form,
b) contacting the test nucleic acid sequence under hybridising conditions with a solution containing an oligonucleotide probe which is complementary to a defined portion of a standard nucleic acid sequence,
c) immobilizing to a first solid support a nucleic acid fragment at least part of which is complementary to said oligonucleotide probe,
d) contacting the solution from step b) with said first solid support, and
e) determining the amount of binding of oligonucleotide probe present in said solution to its complementary nucleic acid fragment on the first solid support, said amount being inversely related to the amount of binding of the oligonucleotide probe to the test nucleic acid sequence.
In a preferred embodiment, the test nucleic acid sequence is immobilized to a second solid support. In this case, the probe-containing solution is preferably separated from this second solid support before being contacted with the immobilized complementary nucleic acid fragment.
This and other preferred embodiments of the invention are described in more detail below.
DETAILED DESCRIPTION OF THE INVENTION
The method of the invention is based on the fact that a mismatch base considerably affects the affinity of a short oligonucleotide to a complementary sequence of a single stranded target sequence. A basic feature of the invention is that the detection is performed on oligonucleotide probe that has not bound to the target or test nucleic acid sequence rather than on probe that has bound thereto. This inhibition type approach offers several advantages. Firstly, the problem of having to bind long PCR products to a sensing surface is eliminated. Secondly, the sensing surface will be regeneratable, since the bound probe can easily be removed from the surface, in contrast to a biotin/avidin-bound PCR product, for example. Thirdly, since the PCR product is not bound to the sensing surface, and in a preferred embodiment bound to another solid phase, high temperature treatment to melt apart secondary structures of single stranded DNA can conveniently be carried out, as will be described further below. Finally, as will also be described in more detail below, the method permits analyses to be performed in a multispot or multichannel format.
The term nucleic acid as used herein is to be interpreted broadly and comprises DNA and RNA, including modified DNA and RNA, as well as other hybridising nucleic acid-like molecules, such as e.g. PNA (peptide nucleic acid). This also applies to the term oligonucleotide probe. The size of the oligonucleotide probe is suitably within the range of, say, from 7 to 24 bases, e.g. from 13 to 17 bases, but longer or shorter probes are also possible.
In one embodiment of the invention, the method is adapted for detecting one or more sequence deviations, such as mutations, and the approximate position thereof in a nucleic acid fragment, usually a DNA fragment. Although the method may be performed with a single oligonucleotide probe for detecting a specific mutation, it is preferred to use at least two, and preferably a number of oligonucleotide probes so that the probes together cover a whole DNA sequence to be tested, such as a PCR-amplified DNA sequence. The probes should then overlap by at least one base, and preferably by two or three bases. Such an analysis may be performed as follows.
Target DNA, usually PCR-amplified DNA from a patient, is bound to a solid phase. The binding to the solid phase may, as is well known in the art, for example, be effected by including one member of a terminal specific binding pair in the PCR product and providing the other member attached to the solid phase. The PCR product may, for example, be biotinylated and the solid phase coated by avidin or streptavidin, such as streptavidin-coated magnetic beads which are commercially available.
The bound DNA is then made single-stranded, for instance by treatment with sodium hydroxide, and incubated with a mixture of short oligonucleotides, e.g. 13-17-mers, which together cover the whole PCR product, or a desired part thereof to be analysed, as mentioned above.
The immobilised single-stranded DNA may form a secondary structure, and to avoid that a part or parts of the DNA sequence will thereby be unavailable for hybridisation with the oligonucleotides, the incubating mixture is preferably heated, for example to 94° C., for a suitable time, such that the secondary structures are melted apart. On cooling, the oligonucleotides present will then compete with, and in most cases dominate the reformation of the secondary structure.
After completing the incubation, i.e. at equilibrium between oligonucleotide probe in solution and oligonucleotide probe bound to the immobilised PCR product, the solution is preferably separated from the solid phase. The concentrations of the respective oligonucleotide probes in the solution are then determined by contacting the solution with immobilised single-stranded DNA sequences, usually oligonucleotides, which are complementary to the oligonucleotide probes, to hybridise the probes to the immobilised DNA
Larsson Anita
Persson Bjorn
Biacore AB
Horlick Kenneth R.
Seed Intellectual Property Law Group PLLC
Taylor Janell E.
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