WO1996000796A1

WO1996000796A1 - Method for detecting mutations

Info

Publication number: WO1996000796A1
Application number: PCT/NL1995/000227
Authority: WO
Inventors: Erik Mullaart; Jan Vijg; Gerrit Johannis De Vos
Original assignee: Ingeny B.V.
Priority date: 1994-06-28
Filing date: 1995-06-28
Publication date: 1996-01-11
Also published as: NL9401082A; AU2684295A

Abstract

The present invention provides a method for detecting mutations in the genome of an individual, comprising amplifying at least one fragment from the genome for testing; linking a label unique to the individual to the DNA fragment(s); providing a reference fragment(s) provided with another unique label and corresponding with the fragment(s) for testing; mixing the fragment(s) and the reference fragment(s); subsequently separating the mixture by means of electrophoresis; making visible the unique label; and comparing the label pattern of the fragment(s) for testing with the label pattern of the reference fragment(s). It becomes possible with the method according to the invention to simultaneously test DNA samples of a number of individuals for mutations.

Description

METHOD FOR DETECTING MUTATIONS

The present invention relates to a method for detecting mutations in the genome of an individual.

The genetic material differs from individual to individual because each genome is unique. Genetic variation can be determined by means of hybridization analysis after electrophoretic separation of DNA fragments which are generated by fragmenting for instance the genomic DNA. The electrophoretic pattern can be transferred from the gel to a nitrocellulose filter. By means of for instance a radioactively labelled probe the parts of the genome which hybridize with the probe can be made visible using an X-ray photo of the filter. The position of the visualized fragment is a measure of the size of the fragment. Variation between individuals in the position of a band when one probe is used indicates polymorphism. This is caused by mutations in particular regions in the genome. Such mutations may be base substitutions but also insertions and deletions. The running behaviour of a fragment in a gel is influenced by such mutations.

Polymorphism can for instance be used to identify an individual such as in determining paternity and in forensic medicine.

A high degree of polymorphism is generally encountered in non-coding parts of the genome. Logically, less polymorphism occurs in the genes. Genes can however be slightly mutated. Such mutations can result in mutations in the coded proteins or even in total deactivation of the gene. Such phenomena can cause particular clinical pictures or other disorders.

Determined polymorphic regions located very closely to the mutated genes can be inherited together with these genes which for instance cause hereditary diseases. Polymorphism can be used in such cases to trace the defective genes.

Because not all defective genes are linked to polymorphic regions and because information relating to mutations in coding regions may itself be important for the analysis of the genetic basis of particular diseases, it is important to also be able to trace these mutations in the coding regions themselves.

As stated above, use is generally made in tracing variations in the genome of the electrophoretic separation of DNA fragments on a gel. The separation can take place in one or two dimensions, wherein a two- dimensional separation often gives a better resolution of the fragments. The method for determining DNA sequence variations by means of a two-dimensional polyacrylamide gel is known from European patent 349.024. In this method double-strand DNA is digested with one or more restriction enzymes. The different fragments are separated in two directions by means of electrophoresis on the basis of two independent criteria, for example length and base pair sequence. By transferring the separation pattern to a nitrocellulose filter, causing a radioactive probe to hybridize with the separated fragments and subsequent autoradiography of the filter, the separation pattern can be made visible.

In the known method the fragments appear in the form of a dark spot on the film. The position of a fragment is specific to its length and base pair composition. However, no distinction can be made between fragments which appear at the same position. With the known methods only one set of fragments can thus be analysed at any one time.

In addition, the known method is very labour- intensive due to the different steps. Moreover, a separate gel and a separate filter as well as an X-ray film are required for each set of DNA fragments for testing. These materials, and also the chemicals required to develop the film, of course also entail additional costs. Waste chemicals also form an impact on the environment and radioactive probes can damage the health of those operating them. Work with radioactive probes moreover results in radioactive waste. In addition, the result of the experiment can only be obtained after some time.

It is the object of the invention to provide a method for detecting mutations in DNA of one or more individuals, wherein in simple and rapid manner DNA of different individuals can be tested simultaneously. This is achieved with the invention by detecting mutations in the genome of an individual, comprising the steps of: a) amplifying at least one fragment from the genome for testing; b) linking a label unique to the individual to the amplified DNA fragment(s) ; c) providing (a) reference fragment(s) provided with another unique label and corresponding with the fragment(s) for testing; d) mixing the fragment(s) and the reference fragment(s) ; e) subsequently separating the mixture by means of electrophoresis; f) making visible the unique labels; and g) comparing the label pattern of the fragment(s) for testing with the label pattern of the reference fragment(s) . The advantage of the method according to the invention is that a number of persons or a number of genes can be scanned on a single polyacrylamide gel. This saves much time as well as material and less waste is generated. The result is moreover already available immediately after the separation.

The separation is preferably a two-dimensional separation, wherein the mixture is separated in one direction on the basis of differences in size and in the other direction on the basis of differences in base pair composition of the fragments. The separation in the second dimension can for instance take place in a denaturing gradient. Fragments with a high GC content will melt and stop at a higher concentration of the denaturing agent than fragments with a high AT content. In this manner differences in base pair composition are made visible. A two-dimensional separation thereby has a higher resolution than a one-dimensional separation. In a practical embodiment of the method the DNA fragments for testing are amplified using a DNA- amplification method. Such an amplification method is for instance the polymerase chain reaction (PCR) . With the amplified fragments a heteroduplexing is performed. The fragments of an individual are then provided with a label, for example a fluorochrome, specific to that individual. The fragments are thereafter separated in two directions on a polyacrylamide gel.

During the separation identical fragments of different persons will come to rest at exactly the same place in the gel. When the label is subsequently made visible, fragments which are mutated relative to the corresponding non- (or differently) mutated fragment of a second individual will come to rest at another position in the gel. It can now be seen which fragment contains one or more mutations since the two labels do not coincide.

The label is preferably a fluorochrome which, after being excited with visible or invisible light of a determined wavelength, returns while emitting light with a wavelength specific to that fluorochrome. In addition, any other chemical compound can be used which can be linked to a DNA molecule, can be excited in one way or another and returns within a short time to the initial state while emitting light of a specific wavelength. Excitation can optionally also take place using electromagnetic radiation. The labels can also be made visible independently of each other or in any desired combination. The patterns of a large number of patients could thus be compared one by one with the control. Further understood by. "unique label" is any other molecule which is distinguished from other corresponding molecules by means of a detectable characteristic. In addition to fluorochromes can be envisaged elements which each have a unique NMR spectrum. Examples of elements which can be used are gold, silver, nickel, iron etc. The fragments of a person are then provided with an element unique to that person which can later be distinguished using for instance NMR, X-ray diffraction or other techniques. In a preferred embodiment of the invention the label is formed by fluorochromes which, after excitation, each emit light with a clearly discernible colour. When the set of DNA fragments of one patient is labelled with a yellow fluorochrome and the control with a blue fluorochrome, coinciding fragments will result in green spots. In. the case of a mutation a blue spot will be detectable on the one hand at the position of the non- mutated gene and a yellow spot will be detectable on the other hand at a completely different position. When the DNA fragments of a number of patients which are labelled per person with a label specific to that patient are placed on a gel together with a control, the DNA of a large number of patients can be tested simultaneously. Not only is the time-consuming step of transferring the fragments to a nitrocellulose filter, the hybridizing and subsequent autoradiography hereby avoided but there now also exists the option of screening a large number of patients simultaneously using one gel. This saves time as well as material. An additional advantage is that, because the fragments do not have to be transferred to a filter, fragments can no longer be lost. _n„„_n^ 96/00796 ₆

The invention further relates to a device for performing the method according to the invention. The device consists of a substrate for a gel, a light source placed on one side of the gel which emits light of a wavelength range such that all the excitabfe TabeTs occurring in the label pattern can be excited therewith, a filter placed on the other side of the gel which allows through light emitted by the excitable labels but not the light emitted by the light source, and means for detecting the pattern of the light emitted by the excitable labels.

The label pattern can of course also be detected by eye. In the case of a small number of patients with clearly distinguishable colour labels this is still feasible. However, as the number of patients becomes larger and the labels less easy to distinguish from each other, the device preferably contains a scanner which can detect the wavelength emitted by each spot. In preference the device is further provided with a processing unit for analysing the light pattern detected by.the detecting means. When it is possible to standardize the separation the results can be compared automatically with data stored in the processor unit. Diagnostics can hereby be largely automated. When recording of the emitted label pattern is desired the device can also contain a recording unit. A photo or video camera can be envisaged here.

Applications of the device are to be found for instance in prenatal or preconceptive diagnostics. The principle of the invention is illustrated in the accompanying figures 1 and 2. Shown schematically in figure 3 is an embodiment of the device according to the invention.

Figures 1 and 2 are discussed in the following example.

Figure 3 shows a schematic view of an embodiment according to the invention. The gel 1 is situated on a substrate (not shown) The substrate can be a separate substrate but may also be formed by one or both glass plates of the original gel arrangement. In addition, the gel may also be placed directly onto the filter. The filter serves herein as substrate. A filter 2 is situated on one side of the gel. On the other side is situated a light source 3 with a suitable wavelength range, for instance an UV lamp. On the side of the filter remote from the gel is situated a scanner 4. Filter 2 does not allow the light emitted by the lamp 3 and falling through the gel through to the scanner 4. The light emitted by the fluorochromes in the gel 1 is however allowed through. Using the scanner it can be determined which wavelength is being emitted at which position. On the basis of this data the spot pattern can be analysed.

The present invention will be further elucidated with reference to the accompanying examples, which are herein given only by way of illustration and are not intended to limit the invention in any way.

EXAMPLE 1

In this example the gene which, when mutated, results in the disease cystic fibrosis is compared with the corresponding non-mutated gene. An amplification of the exons of the gene is carried out by means of PCR. Use is made herein of primers which hybridize with the terminals of the different exons. The thus formed fragments of the mutated gene, which substantially enclose the whole exons, are labelled with a yellow, light-emitting fluorochrome. The fragments which are formed starting from the non-mutated gene are labelled with a blue, light-emitting fluorochrome.

Both sets of fragments are then mixed and separated on a two-dimensional polyacrylamide gel. After separation the gel is exposed, whereby the fluorochromes are excited. The yellow and blue light subsequently emitted by the fluorochromes is detected as a green spot at positions where the fragments are identical, as a blue spot at a position where a fragment of the non-mutated gene has appeared and as a yellow spot at positions where the mutated fragments of the mutated gene are present.

The result is shown in figures lA-lC. Figure 1A is a gel having only the fragments of one patient. The fragments of the non-mutated gene are made visible in the gel of figure IB. Figure 1C shows the combination of both gels.

The different colours are designated herein as shown in the table below.

COLOUR SYMBOL

EXAMPLE 2

In this experiment a non-mutated gene is combined in the same, manner as in example 1 with two different mutated genes. The non-mutated gene is again labelled with a blue colour, the mutated genes are labelled with respectively red and green fluorochromes. The mixed colour of these three colours of light is white. After exposure white light is emitted at all positions where fragments of the three individuals are situated. The greater part of the fragments is not detectable. A mutated fragment of the red-labelled person is visible as a red light spot. Only the combination of blue and green remains at the original position of that fragment because the red light has disappeared there. At the positions where the green light has disappeared there remains a purple spot, while the green spot is visible at another position.

The result of the above experiment is illustrated schematically in figures 2A-2D. The different colours are designated herein as shown in the table below.

COLOUR SYMBOL blue θ red 0 green θ purple (red + blue) 0 blue green 3 brown ( red + green) θ white ( red + green + blue) 9

The present invention provides a method and device with which a large number of individuals can be tested for mutations in their genome in rapid and simple manner. The invention is of particular significance in diagnostics, but is not limited thereto.

Claims

1. Method for detecting mutations in the genome of an individual, comprising the steps of: a) amplifying at least one fragment from the genome for testing; b) linking a label unique to the individual to the DNA fragment(s); c) providing (a) reference fragment(s) provided with another unique label and corresponding with the fragment(s) for testing; d) mixing the fragmen (s) and the reference fragment(s) ; e) subsequently separating the mixture by means of electrophoresis; f) making visible the unique labels; and g) comparing the label pattern of the fragment(s) for testing with the label pattern of the reference, fragment(s) .

2. Method as claimed in claim l, characterized in that the separation is performed by means of two- dimensional electrophoresis, wherein the mixture is separated in one direction on the basis of differences in size and in the other direction on the basis of differences in base pair composition of the fragments.

3. Method as claimed in claim 1 or 2, characterized in that the DNA is amplified by means of the PCR technique.

4. Method as claimed in claim l, 2 or 3, characterized in that the unique label is a fluorochrome.

5. Method as claimed in claim 4, characterized in that the fluorochrome is made visible using light.

6. Method as claimed in claim 1, 2 or 3, characterized in that the unique label is a chemical element.

7. Method as claimed in claim 6, characterized in that the chemical element is made visible using NMR or X-ray diffraction techniques.

8. Device for making visible a label pattern, generated for instance using the method as claimed in any of the claims 1-5, comprising a substrate for a gel, a light source placed on one side of the gel which emits light of a wavelength range such that all excitable labels occurring in the label pattern can be excited therewith, a filter placed on the other side of the gel which allows through light emitted by the excitable labels but not the light emitted by the light source, and means for detecting the pattern of light emitted by the excitable labels.

9. Device as claimed in claim 8, characterized in that the means for detecting the emitted light pattern are formed by a scanner.

10. Device as claimed in claim 8 or 9, further comprising a processing unit for analysing the light pattern detected by the detecting means.

.11. Device as claimed in any of the claims 8- 10, further comprising a recording unit for recording the light pattern.