Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-08-18
2001-09-04
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S196000, C435S197000, C435S199000, C435S287200, C435S288300, C435S288700, C536S023100, C536S024100, C536S024330, C536S025320
Reexamination Certificate
active
06284463
ABSTRACT:
The present invention relates to a method and a kit for detecting a mutation from a non-mutated sequence of a target polynucleotide. More specifically the invention relates to a method for detecting mutations by using a mismatch binding protein.
The development of methods for the detection of mutations in DNA is important in diagnostic tests since a large number of human genetic diseases are caused by single base substitutions or by small additions or deletions in the genome. For example, the possibility to detect mutations is an important tool in early detection of cancer.
There are a lot of methods available for detection of mutations. Single strand conformation polymorphism (SSCP)for example, is based on the fact that single stranded DNA that differs by as little as a single base can take up different conformations in a non-denaturing environment, which allows for separation by electrophoresis. In Denaturing gradient gel electrophoresis (DGGE) the altered melting characteristics, exhibited by base pair changes, are used. The melted duplexes are separated when moving into a gel of increasing denaturation concentration. These methods as well as others require gel electrophoresis separation.
Many methods worked out to detect mutations, consisting of one or a few bases, are based on hybridization between a standard DNA (or RNA) and a test DNA. The test sample is mixed with wild-type sample, which can be labelled, and the mixture is denatured and allowed to renature. The mutation is then exposed as a mispaired or unpaired base in a heteroduplex molecule. Different ways are known for the detection of the mispaired or unpaired base. Cleaving of by chemical or enzymatic treatment is one commonly used method. The fragments are observed by gel electrophoresis. Several patent applications describe the use of mismatch binding proteins to detect the heteroduplex molecule (e.g. WO 93/02216, WO 95/12689). DNA mispairing occurs in vivo and is recognized and corrected by a family of repair proteins. Included are proteins which recognize and bind to mismatch containing DNA, so called mismatch binding proteins. It has been found that the mismatch binding proteins forms specific complexes with any of the eight possible mismatched base pairs. The mismatch repair system has been studied in bacteria such as
E.coli
and Salmonella. MutS is one such protein identified in the
E.coli
mismatch repair system. The detection of binding of a mismatch binding protein to the DNA is usually made by some type of a label. Recently a rapid method for detection of point mutations using a label free technique was reported (by Ivan Babic et al on the 5
th
BIA symposium in San Diego, September 1995). The interaction of MutS with short oligonucleotides containing various base-pair mismatches was examined using optical biosensor technology.
The detection methods according to the state of the art based on gel electrophoresis or labelling procedures are time-consuming methods. Therefore, in developing a time-effective assay, these methods are difficult to apply for rapid screening of samples for the detection of mutations. These methods are also difficult to automate.
According to WO 95/12689 and also to Babic et al as mentioned above, MutS binds to DNA containing mispaired bases, but does not bind to DNA without mismatches or single stranded DNA. However, it was found in the present invention that MutS binds non-specifically. Thus, it was found that MutS binds both to single stranded DNA and to double stranded DNA without mismatches. This means that the accuracy of the MutS detection method is not satisfactory for detection of mutations, because non-specific binding of MutS can cause false positive.
The object of the present invention is to obtain an improved method for detecting a mutation by using a mismatch binding protein, without the draw-back mentioned above.
A further object of the invention is to present a kit useful for detection of mutations.
The objects of the invention are achieved by the method and kit as claimed in the claims. According to the invention a method for detecting a mutation from a non-mutated sequence of a target polynucleotide in a sample is obtained by using a mismatch binding protein. The method comprises:
a) providing non-mutated and mutated target polynucleotide,
b) forming duplex of the non-mutated and mutated single strands of the target polynucleotide in a),
c) adding a single strand binding protein to the polynucleotide from b),
d) incubating the mismatch binding protein with an activating agent,
e) adding the incubated mismatch binding protein from d) to the polynucleotide from c), whereby the mismatch binding protein binds to the duplex formed by one non-mutated and one mutated single strand of the target polynucleotide,
f) detecting the presence of any mismatch binding protein bound to the target polynucleotide.
According to a further aspect of the invention a kit for detecting a mutation from a non-mutated sequence of a target polynucleotide in a sample is achieved. The kit comprises:
a) single strand binding protein,
b) activator for mismatch binding protein,
c) mismatch binding protein,
d) amplification primer(s),
e) solid support for binding of target polynucleotide or mismatch binding protein.
With the present invention it was surprisingly found that the non-specific binding of mismatch binding protein could be avoided. Binding of the mismatch binding protein to single strands is inhibited by the single strand binding protein. By activating the mismatch binding protein with an activator, before addition to the sample, binding to double strands lacking mismatches does not take place.
According to a preferred embodiment of the invention, the mutated or the non-mutated target polynucleotide is immobilized on a solid support before the duplex in step b) is formed. Most preferably the non-mutated target nucleotide is immobilized. Alternatively, the target polynucleotide is immobilized on the solid support after duplex formation in step b) or mismatch binding protein is immobilized on a solid support and binding of the target polynucleotide is detected. According to a further preferred embodiment the solid support is a sensor chip surface. The presence of the mismatch binding protein can then be detected by a label free technique. One such method is detection by surface plasmon resonance (SPR). An optical biosensor system based on SPR is described in WO 90/05295 filed by Pharmacia AB. By this method of detection the time-consuming detection with gel electrophoresis or labelling could be avoided.
In the first step of the invention a sample of the target polynucleotide, i.e. a sequence of a DNA or RNA having a mutation, is mixed with a non-mutated (wild-type) sample of the polynucleotide. The sample mixture is denatured to achieve single strands of the polynucleotide, if the samples are not already single stranded and then the single strands are allowed to renature to double strands. There will then be formed homoduplexes as well as heteroduplexes. A homoduplex is a duplex of two non-mutated strands or two mutated strands. A heteroduplex is a duplex of one non-mutated and one mutated strand. According to one embodiment of the invention the target polynucleotide in the sample can be amplified before the first step of the invention. Conventional amplification methods can be used, especially amplification by PCR (see U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159).
In one preferred embodiment a single stranded non-mutated polynucleotide is immobilized to a sensor chip surface and the sample suspecting of containing a mutation of the polynucleotide is added in single stranded form. The added sample is allowed to anneal to the polynucleotide on the sensor chip.
In the next step a single strand binding protein is added which binds to the single strands being left in the mixture. Before the mismatch binding protein is added to the thus produced sample mixture, the mismatch binding protein is incubated with an activator. It was found that by activating the mi
Goto Masanori
Hasebe Masahisa
Tosu Mariko
Amersham Pharmacia Biotech AB
Ronning, Jr. Royal N.
Wilder Cynthia B.
Zitomer Stephanie W.
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