WO2000014280A1

WO2000014280A1 - Method for detecting nucleotide sequences and/or nucleotide-binding proteins

Info

Publication number: WO2000014280A1
Application number: PCT/NL1999/000540
Authority: WO
Inventors: Alexander Adrianus Moen
Original assignee: Alexander Adrianus Moen
Priority date: 1998-09-04
Filing date: 1999-09-01
Publication date: 2000-03-16
Also published as: AU5535399A; NL1010012C2

Abstract

The invention relates to a method for detecting nucleotide sequences or nucleotides-binding proteins in a sample, comprising of bringing the sample into contact with a standard solution which contains as components one or more known probes in an incubation mixture, in order to enable complexing between the probe and the nucleotide sequence and/or the nucleotide-binding protein to be detected in the sample, and determining whether complexing has taken place by comparing the molecular weight of the probe(s) from the incubation mixture with the molecular weight of the probe(s) in the standard solution, wherein determining whether complexing has taken place is brought about by column-chromatographic separation of the components of the incubation mixture, separation in the same manner of the components of the standard solution and comparing the elution patterns of both separations, wherein a change in the elution speed of one or more probe(s) in the incubation mixture relative to the elution speed of one or more probe(s) in the standard solution indicates the presence of complexing.

Description

METHOD FOR DETECTING NUCLEOTIDE SEQUENCES AND/OR NUCLEOTIDE-BINDING PROTEINS

The invention relates to a method for detecting nucleotide sequences and/or nucleotide-binding proteins in a sample, comprising of bringing the sample into contact with a standard solution which contains as components one or more known probes in an incubation mixture, in order to enable complexing between the probe and the nucleotide sequence to be detected and/or the nucleotide-binding protein to be detected, and determining whether complexing has taken place by comparing the molecular weight of the probe (s) from the incubation mixture with the molecular weight of the probe (s) in the standard solution. The invention likewise relates to an embodiment wherein a nucleotide sequence to be detected is demonstrated in a sample using a known nucleotide-binding protein as a probe. The method according to the invention is important for instance in determining in a test subject, such as a patient or an animal to be examined, the presence of a certain DNA sequence (a gene or part of a gene) , RNA sequence (for instance messenger RNA, antisense RNA) or a nucleotide- binding protein (for instance a transcription factor) .

The DNA sequence to be detected may originate from a 'healthy' gene or may be an abnormal nucleotide sequence. The term 'abnormal' is understood to mean that the nucleotide sequence displays at least one or more differences from the corresponding nucleotide sequence in a healthy individual. The presence of an abnormal gene in a test subject then demonstrates for instance that the test subject is the carrier of an abnormal gene which may be responsible for a certain genetic disease, or forms the definitive diagnosis that the test subject is suffering from this hereditary disease. Diseases wherein it is possible to search for an abnormal gene with the method according to the invention are for instance cystic fibrosis (mucoviscidosis) , particular forms of leukaemia and muscular dystrophy.

Demonstrating certain DNA or RNA nucleotide sequences or nucleotide-binding proteins can also be important in the detection of micro-organisms in a test subject, particularly in those cases which relate to micro-organisms which are difficult or impossible to culture. Using specific probes it is then possible to demonstrate the presence of genetic or protein material of a micro-organism, and therefore the presence of this micro-organism, in a sample originating from the test subject.

The detection of nucleotide-binding proteins can be important in determining the individual sensitivity of patients to steroids. This sensitivity is related to changes in the binding of certain proteins to the DNA and is a function of both the quantity of protein and the binding force of the protein relative to the DNA. Both parameters can be determined with the method according to the invention.

In the above described molecular diagnostic methods particular use is currently being made of electrophoretic techniques to separate the nucleotide sequences. In order to identify a certain gene isolated DNA is for instance cleaved into specific DNA fragments using restriction enzymes, which fragments are then separated to size by means of electrophoresis, whereafter the fragments can be identified with for instance Southern blotting. Electrophoresis is a separation technique which is based on the movement of charged molecules in an electrical field. The electrophoretic mobility of a charged molecule depends among other things on the net charge, size and shape of the molecule. Different molecules hereby move at different speeds and in this manner the components of a mixture are separated from each other. Electrophoresis forms the most commonly used method for the separation of nucleic acids. At an alkaline pH linear DNA and RNA molecules have a uniform net charge per unit of length. By means of electro- phoresis in a medium which acts as a molecular sieve, for instance an agarose or polyacrylamide gel, the DNA fragments or RNA molecules can be mutually separated on the basis of their relative size. Smaller molecules migrate more rapidly than larger molecules and will hereby come to lie at a different position in the gel. After electrophoretic separation of the nucleotide sequences the different components must be made visible. This takes place by immersing the gel in ethidium bromide which binds to the nucleotides by intercalation between the base pairs. The ethidium bromide can also be included in the gel at an earlier stage. During irradiation with UV light the nucleotide fragments are then visible as separate bands on the gel due to the orange-red fluorescence of the ethidium bromide.

Ethidium bromide is however a highly carcinogenic substance which must be handled very carefully. The wearing of gloves and special provisions for ethidium bromide waste are for instance necessary. UV light moreover represents a potential danger to the people working therewith, for which additional safety measures must be taken.

Due to possible unevenness which can occur when the gels are poured it is furthermore possible that the different nucleotide fragments which are visible through the ethidium bromide do not all lie on one line, whereby problems may occur during reading. These problems will occur particularly when reading takes place in automated manner, since computers are unable to distinguish between an actually displaced band on the gel and a band which as a result of an unevenness in the gel or due to for instance ' smiling' of the gel, has come to lie at another position in the gel. It is therefore the object of the present invention to obviate these drawbacks.

This object is achieved with the invention by providing a wholly new method for detecting nucleotide sequences and/or nucleotide-binding proteins in a sample, based on bringing the sample into contact with a standard solution, which contains as components one or more probes in an incubation mixture, in order to enable complexing between the probe and the nucleotide sequence and/or the nucleotide-binding protein te be detected in the sample, and determining whether complexing has taken place by comparing the molecular weight of the probe (s) from the incubation mixture with the molecular weight of the same probe (s) in the standard solution, wherein determining whether complexing has taken place is brought about by column-chromatographic separation of the components of the incubation mixture, column-chromatographic separation in the same manner of the components of the standard solution and comparing the elution patterns of both separations, wherein a change in the elution speed of one or more probe(s) in the incubation mixture relative to the elution speed of the same probe (s) in the standard solution indicates the presence of complexing. The components to be measured are preferably nucleotide- probes.

'Probe ' is therefore understood to mean a known nucleotide sequence which is present in the standard solution and incubation mixture and is used to demonstrate a specific nucleotide sequence and/or specific nucleotide-binding protein. A nucleotide sequence can be DNA, RNA or antisense-RNA. The probe can also be a known nucleotide-binding protein with which a specific nucleotide sequence (DNA/RNA) can be demonstrated. The standard solution is a solution of known composition which contains as components one or more probe (s) which are specific to the nucleotide sequence (s) and/or nucleotide-binding protein (s) te be detected. The sample is the fluid or substance to be tested which comes from the test subject. Added together, the sample and the standard solution form the incubation mixture.

The use of nucleotide probes is based on recognition of a characteristic piece of DNA or RNA, i.e. a specific nucleotide sequence in the DNA or RNA, or a specific nucleotide-binding protein. Complexing (hybridization) of the probe and the nucleotide sequence te be detected occurs in principle only if the nucleotide sequence of the probe is exactly complementary to the nucleotide sequence and/or nucleotide-binding protein te be detected. Using probes it is possible to demonstrate the presence of a specific nucleotide sequence among many other nucleotide sequences because probes are absolutely specific and very efficient in finding the sequence to which they are complementary.

In accordance with the method according to the invention the presence of complexes between probe and the nucleotide sequence and/or nucleotide-binding protein te be detected is determined by separating the components of the incubation mixture and standard solution by means of column chromatography and comparing the respective elution patterns. The column chromatographic separation preferably takes place by means of gel permeation. Selection herein takes place according to differences in size between the probe (s) in the standard solution and the same probe (s) in the incubation mixture. When a probe in the incubation mixture has formed a complex with a nucleotide sequence or nucleotide-binding protein originating from the sample, the molecular weight of the probe will be greater than the molecular weight of the same, non-complexed probe from the standard solution, resulting in a different elution pattern. The presence of the probe or the complex between probe and nucleotide sequence or nucleotide-binding protein te be detected is preferably determined spectrophotometrically.

In order to increase the sensitivity of the method according to the invention it is possible to detect formed complexes by labeling the probes via known per se labeling techniques. Because only the labeled molecules will be detected, the other molecules present in the mixture are precluded. Different diagnoses can moreover be made simultaneously by using different labels. This option is called "multiplex-testing" .

As labels can be used not only for instance radioactive or fluorescent labels, but also for instance colouring agents or other chemical molecules. Separating the different components of the standard solution and incubation mixture with the method according to the invention limits the use of ethidium bromide and UV light to visualize the nucleotide sequences and/or nucleotide-binding proteins te be detected.

The quality of the chromatography column used moreover has no influence on the final outcome of the detection method according to the invention, because each sample and each standard solution can be tested with the same column. Possible errors in determining the result are extremely minimal, since contamination and other factors adversely affecting the functioning of the system do not result in an incorrect diagnosis, but in a recognizably different elution pattern which immediately indicates that the system has not operated as it should. If such a difference is ascertained, the test can be performed again. The electrophoretic method used heretofore lacks such a recognizable reliability.

When a series of samples is tested successively with the same column, a reference measurement with the standard solution will have to be performed at least once.

The separation of the probe and the formed complex can be optimized by choosing different gel- permeation materials subject to the molecule te be detected. This results in great flexibility of the system. Furthermore, the column chromatographic separation can easily be performed in automated manner, whereby the speed of testing is increased. Automated chromatographic detection systems with recording equipment coupled thereto are per se known.

A fraction collector can advantageously be added to the column chromatographic separation in order to collect the separated fractions. In this manner a certain nucleotide sequence and/or nucleotide-binding protein can if required be isolated from the material originating from a test subject, for instance for the purpose of further scientific research.

The sample to be tested can in principle consist of all the material from the test subject in which genetic material in the form of nucleotides (DNA or RNA) is present, such as blood, semen or saliva, and all tissue cells. For this purpose the sample must first be processed in order to obtain the nucleotide material. For this purpose the cells must be broken open, whereafter the constituents of the contents of the cell which do not originate from the nucleus must be removed such that DNA and/or RNA remains which can be further purified and concentrated. Extraction and purifying of the nucleotide content of the cells takes place in accordance with standard protocols known to the skilled person. The present invention will be further illustrated in the examples and figures below, which serve solely for the purpose of elucidation and not as limitation.

EXAMPLES

EXAMPLE 1:

Using the method according to the invention the expression of corticosteroid-receptor proteins (DNA- binding proteins) was tested in a cell culture system. For this purpose the cells were divided into two portions wherein one of the portions was incubated in the presence of corticosteroids, while as control the other portion was incubated without corticosteroids. In order to detect the produced corticosteroid- receptor proteins in the sample a probe was made consisting of a specific nucleotide sequence which binds to the receptor protein in question and labeled with biotin and streptavidin-FITC. Both samples were incubated in PBS with the labeled probes for a number of minutes, and samples of 10 μl were then analysed with HPLC (running time: 6 minutes, mobile phase: PBS) . The gel filtration material used in the column was Superdex-200 (Pharmacia) . As standard solution was taken a sample of the probe only.

The formed complexes were detected using fluorescence detection (FIG 1) .

Claims

1. Method for detecting nucleotide sequences and/or nucleotide-binding proteins in a sample, comprising of bringing the sample into contact with a standard solution which contains as components one or more known probes in an incubation mixture, in order to enable complexing between the probe and the nucleotide sequence and/or the nucleotide-binding protein te be detected in the sample, and determining whether complexing has taken place by comparing the molecular weight of the probe (s) from the incubation mixture with the molecular weight of the probe (s) in the standard solution, characterized in that determining whether complexing has taken place is brought about by column- chromatographic separation of the components of the incubation mixture, column-chromatographic separation in the same manner of the components of the standard solution and comparing the elution patterns of both separations, wherein a change in the elution speed of one or more probe (s) in the incubation mixture relative to the elution speed of the same probe (s) in the standard solution indicates the presence of complexing.

2. Method as claimed in claim 1, characterized in that the probe is a known DNA sequence for detecting a DNA sequence, RNA sequence or a DNA-binding protein in a sample.

3. Method as claimed in claim 1, characterized in that the probe is a known RNA sequence for detecting a DNA sequence, antisense RNA sequence or an RNA-binding protein in a sample.

4. Method as claimed in claim 1, characterized in that the probe is a known antisense RNA sequence for detecting an RNA sequence in a sample.

5. Method as claimed in claim 1, characterized in that the probe is a nucleotide-binding protein for detecting a DNA sequence or RNA sequence in a sample.

6. Method as claimed in claims 1-5, characterized in that the DNA sequence, antisense RNA sequence, RNA sequence or the nucleotide-binding protein te be detected in the sample originates from the test subject him/herself.

7. Method as claimed in claims 1-5, characterized in that the DNA sequence, antisense RNA sequence, RNA sequence or the nucleotide-binding protein te be detected in the sample originates from a pathogen, for instance a bacterium or virus, which has invaded the test subject.

8. Method as claimed in claims 1-4, characterized in that the nucleotide-binding protein te be detected is for instance a steroid-receptor.

9. Method as claimed in claim 5, characterized in that the nucleotide-binding protein with which a nucleotide sequence is demonstrated is for instance a steroid-receptor.

10. Method as claimed in any of the claims 1-9, characterized in that the column chromatographic separation is gel permeation.

11. Method as claimed in any of the claims 1-10, characterized in that a fraction collector is coupled to the chromatography column in order to obtain the nucleotide sequence and/or nucleotide-binding protein te be detected in isolated form.