CA2283188A1 - Method for detecting mutated alleles - Google Patents

Abstract

The invention relates to a method for detecting mutated allels in an excess of wild type allels. According to said method, said wild type allels are isolated by means of a separation process using a carrier which is bonded to one or several oligonucleotides, said oligonucleotides being complementary to the wild type allels.

Description

Method for Detecting Mutated Allels The present invention relates to a method for detecting genetic modifications, particularly a method for detecting few mutated allels in an excess of wild type allels.
The evidence of point-mutated allels in an excess of wild type allels comprises a significant diagnostic potential.
The fields of application to be mentioned are e.g..
Detection of tumor cells in the stool of patients suspected of having colorectal carcinomas;
- Detection of tumor cells in the sputum and bronchial lavage of patients suspected of having bronchial carcinomas;
- Detection of tumor cells in the urine of patients suspected of having bladder carcinoma;
- Detection of tumor cells in tissue biopsy samples taken.
Only the well-calculated amplification of defined point mutations has been possible by now, it being necessary for this purpose to precisely know the point mutation as regards the location and appearance. In this connection, the allel-specific oligonucleotide hybridization of cloned PCR
products (cf. Sidransky, D. et al., Science 256, pp. 102-105 (1992)) or the "Mutant Enriched PCR" (cf. Nollau, P. et al., Int. J. Cancer 66, pp. 332-336 (1996)) can be used as methods. However, these methods are not suited to detect few point-mutated allels in an excess of wild type allels when the position of the mutation, e.g. point mutation, deletion, is not known in advance.

. 2 Therefore, it was the object of the present invention to provide a method serving for detecting, and separating, few mutated allels in an excess of wild type allels.
The object is achieved by a method according to claim 1.
Advantageous embodiments follow from the subclaims.
A "mutated allel" will result if as compared to the wild type point mutations, deletions, insertions, inversions and substitutions, respectively, of relatively small or relatively great gene regions occur.
Examination samples which are suitable for testing for the presence of mutated genes are e.g.. blood, urine, stool, saliva, sputum, bronchial lavage, smear material and biopsy material.
The invention is based on the principle of an allel-specific oligonucleotide hybridization. For this purpose, oligonucleotides of 12-25, preferably 16-20, base pairs (bp) are bonded to a suitable carrier material, the oligonucleotides as probes being complementary to sections of the wild type allel. The oligonucleotide probes are present in great excess as compared to the target sequences.
Suitable carrier materials to which the oligonucleotides are bonded, are e.g. glass, such as silicates, gel materials such as agarose and dextran, or polymer materials such as polypropylene or polyacrylamide. The oligonucleotides are preferably bonded in covalent manner, since an adsorptive bond is hardly ever strong enough. Methods of covalently bonding oligonucleotides to carrier surfaces are described in Khrapko, K.R. et al., DNA Seq. 1, pp. 375-388 (1991);
Fodor S.P.A et al., Science 251, pp. 767-773 (1991) and Maskos et al., Nucl. Acid Research 20, pp. 1639-1648 (1992), for example.
The examination sample which contains both wild type allels (in excess) and mutated allels (few), is now subjected to a separation process, e.g. chromatography or electrophoresis, via the carrier to which the oligonucleotides are preferably bonded covalently. A person skilled in the art is familiar with the conditions for carrying out the separation processes and conventional separation apparatus, e.g. in the form of chromatographic columns, electrophoresis cabinets, electrophoresis tubes or capillaries, are used. For the hybridization of the wild type allel with the bonded oligonucleotide, the amplified DNA must be present as a single strand. This can be achieved e.g. by buffers having a corresponding salt content, e.g. SSC or SSPE. The previous isolation of a DNA strand via a correspondingly labeled primer is better suited. When e.g. a biotin-labeled primer is used, the corresponding DNA strand can be isolated via the bond to the streptavidine beads.
Since the oligonucleotides are only complementary to the wild type sequences but not to those of the mutant, they either hybridize exclusively with the wild type allel or retard the mobility thereof in a selective manner. In the case of an exclusive bond of the wild type, the mutated fragments are found in the sample fraction which is not bonded to the oligonucleotides on the carrier. In the case of the preferred bond of the wild type the mutated fragment is eluted before the wild type fragment thus being isolated therefrom.
The sensitivity of the method can be further increased if in addition to the sense strand of the wild type allel the anti-sense strand is also considered in the analytics.
The methodically simplest case is the case in which an allel only has point mutations and the sites where they usually occur are known. This applies e.g. to the KRAS gene which in the case of colorectal carcinomas exclusively has mutations in codon 12 or 13. In this case, only one oligonucleotide of about 20 by must be synthesized which is complementary to the region of the wild type allel where codons 12 and 13 are located. The oligonucleotide is bonded to a carrier which is suited for separation purposes, e.g. chromatographic or electrophoretic purposes. The gene of interest, here: KRAS
gene, is isolated from the examination sample and labeled in the form of restriction fragments or PCR products which cover codons 12 and 13, e.g. by means of radionuclides, fluorescent dyes, biotin/avidine system, and subjected to the chosen separation process. Regarding the separation process it is advantageous for the DNA to be present as a single strand. This can be achieved e.g. by labeling one of the two DNA strands using biotin and isolating it by bonding it to avidine. If exclusively the wild type is bonded, the mutated fragments will be found in the non-bonded fraction and can be analyzed after the collection thereof. In a preferred bond of the wild type a buffer is used which elutes the mutated fragment before the wild type fragment from the carrier. Here, salt solutions and temperature must be chosen such that the wild type allel is retarded as compared to the mutated allel. A particularly suitable salt is tetramethylammonium chloride, since the stability of CG
and AT base pairings is comparable. The temperature should be within the range of the melting temperature of the wild type allel. When e.g. 20meric bonded oligonucleotides and 3.0 M tetramethylammonium chloride are used, the melting temperature of a fully complementary hybrid is 60°C.
Another method has to be applied in the case in which the detection for genes with multiple heterogeneous point mutations is concerned. The p53 gene can be mentioned as an example. The p53 gene contains multiple mutations which are distributed over differing exons. In order to solve the problem of mutation detection in the p53 gene and as regards the mutation type of comparable genes, the following pathways are in principle possible:
- parallel arrangement of separation means, e.g. columns or capillaries, to the separation material (carrier) of which various oligonucleotides are bonded each, - series arrangement of separation means, to the separation material of which various oligonucleotides are bonded each, - binding of oligonucleotides, which hybridize with various sections of a gene, to a carrier.
Parallel arrangement of separation means Carrier materials for separation means are used to which an oligonucleotide is bonded which is complementary to a certain region of the wild type allel. If sense and anti-sense strands are considered for the analytics, two separation means having the corresponding complementary oligonucleotide are required in each case for a certain gene section. Each separation means contains an oligonucleotide (and two oligonucleotides, respectively, if sense and anti-sense are used) whose base sequence differs from that of the oligonucleotides of other separation means. With an oligonucleotide length of 20 bp, e.g. at least 30 separation means would have to be provided to cover the entire region of a target wild type sequence of 600 bp. Following PCR
amplification or corresponding restriction digestion by obtaining such a target sequence having 600 bp, it is converted according to standard methods into single strand, labeled, e.g. using fluorescent dyes, radionuclides or avidine/biotin system, and the 30 separation means are charged in parallel. The mutated allel is separated from the wild type allel by the separation means which uses a carrier having a mismatch with respect to the mutated allel. By portioning the sample to be investigated, the sensitivity is somewhat reduced when the separation means are arranged in parallel as compared to the series arrangement.
Series arrangement of separation means In this case, the separation means each of which have a certain oligonucleotide differing from the oligonucleotides of the other chambers, are arranged in series. This procedure presupposes that the wild type fragments bond quantitatively and the mutated fragments do not bond at all.
One valve each is mounted between each separation means.

Depending on the position of the valve, a measuring cell (valve position I) or the next separation means (valve position II) is charged. For example, a fluorescent photometer or a scintillation meter are in consideration as measuring cell. The bonded fragments are eluted according to standard methods, e.g. by means of heat or change of the salt concentration. The separation process takes place e.g.
as follows:
Valve downstream of separation means 1: position I
Charge of separation means 1 Charge of the measuring cell by non-bonded fraction Valve downstream of separation means 1: position II
Valve downstream of separation means 2: position I
Charge of separation means 2 Charge of the measuring cell by non-bonded fraction etc.
As compared to the parallel arrangement, the potential advantage of the series arrangement is a higher sensitivity.
Bonding of oligonucleotides which hybridize with various sections of the wild type allel to a carrier In principle, a separation will also function if oligonucleotides which are complementary to various sections of the target wild type sequence are linked to a carrier.
Here, the buffer conditions must be chosen such that the wild type allel is retarded as compared to the mutated allel. This can be achieved when the separation is carried out at a temperature which is within the range of the melting temperature and oscillates around the melting temperature, respectively. A reversible interaction between bonded oligonucleotides and wild type sequences occurs under these conditions. The region of the gene having a point mutation is not bonded. All in all, the interaction between mutated allels and bonded oligonucleotides is weaker than the interaction between wild type allel and bonded oligonucleotides. The principle of separation implies that the separation output decreases with increasing number of differently bonded oligonucleotides.
The present invention distinguishes itself in that few mutated allels can be detected in an excess of wild type allels. Furthermore, it is suited to detect heterozygous and homozygous mutations and polymorphisms, respectively. Thus, the present invention provides a product serving for analyzing genetic modifications of multifarious origin.
Therefore, the present invention is widely used for diagnosis. In addition, it is a basis for the development of new therapeutic approaches.
The invention is further illustrated by means of the figures, which show:
Figure l: separation of mutated p53 allels of p53 wild type allels by separation chambers arranged in parallel and having immobilized oligonucleotides, Figure 2a: valve positions in the series arrangement of separation chambers, Figure 2b: separation of mutated p53 allels of p53 wild type allels by separation chambers arranged in series and having immobilized oligonucleotides.
The invention is now explained in-more detail by means of the examples.
Example 1: Analysis of a mutation in the K-R.AS gene DNA can be extracted from tissues, body fluids, secretions or excretions. For this purpose, samples, e.g. stool, blood, pancreatic juice, urine or sputum, are suspended in an aqueous solution of 6 M guanidinum isothiocyanate. Following centrifugation, NP-40 is added (final concentration 1 0).
After an incubation period of at least 10 minutes at room temperature, 500 ~l of the suspension are fed into a commercially available cartridge having a glass filter to isolate the DNA. After centrifugation and two wash steps using cold ethanol (4°C), the DNA is eluted with hot water (70°C). In order to prevent degradation, prolonged storage in 10 mM Tris-HC1 (pH 7.4) is carried out.
For carrying out a PCR amplification, 500 ng DNA are transferred into 100 ~1 of a 10 mM Tris-HC1 buffer, pH 8.3.
The buffer contains the following additions: 1.5 mM MgCl2, 50 mM KC1 0.01 0 (w/v) gelatin, in each case 200 ~M dNTP, 2.5 U Taq polymerase and 0.3 ~mol of the respective primers.
The sequence of the primers is as follows: sense: 5'-GTATTAACCTTATGTGTGACATGTTC-3'; anti-sense: 5'-TCAAAGAATGGTCCTGCACC-3'. For concluding the oligonucleotide synthesis in an automatic DNA synthesizer, a biotinylated nucleotide is introduced into the anti-sense primer at the 5'-end and a nucleotide labeled with a fluorescent dye (e. g.
fluorescein) is introduced into the sense primer at the 5' end thereof. The labeled nucleotides are commercially available.
For the detection of mutations in codons 12 and 13 of the K-RAS gene, a 20meric oligonucleotide is synthesized to solid carriers and linked to solid carriers, respectively. The -sequence of the oligonucleotide is as follows:
5'-GCCTACGCCACCAGCTCCAA-3'. The solid carriers in consideration are all carriers suitable for chromatographic and electrophoretic separation. Glass and polyacrylamide shall be mentioned as separation media by way of example.
For example, porous beads are suitable as glass carriers.
The glass surface is derivatized according to common methods, the oligonucleotides are synthesized directly on the derivatized glass surfaces in an automatic DNA
synthesizer (Applied Biosystems) in accordance with the manufacturer's instructions. For the purpose of derivatization, the glass beads (10 g) are incubated in 40 ml xylene + 12 ml 3-glycidoloxypropyl trimethoxysilane having a trace of Hunig base at 80°C for about 12 hours.

After washing in methanol and ether, the beads are dried in air and in vacuo. In a second step, alkyl spacer molecules were linked to the derivatized surface. For this purpose, the beads are fed e.g. into pentaethylene glycol. After washing in methanol and ether, the beads are dried in air and in vacuo. Storage is made in argon at -20°C (Maskos U. &
Southern EM. Nucleic Acids Res. 10, 1679-1684 (1992)).
The glass beads derivatized in this way are inserted directly in the DNA synthesizer.
For linkage to polyacrylamide a methyluridine base is introduced at the 3' end in the oligonucleotide synthesis.
Hydrazine groups are introduced into the polyacrylamide gel by treatment with a 50 o aqueous hydrazine hydrate solution (1 h, room temperature). For the purpose of linkage the ribose at the 3' end of the oligonucleotide is oxidized with sodium periodate. The resulting aldehyde group is bonded to the derivatized gel (Khrapko K.R. et al., DNS Seq. 1, 375-388 (1991)).
In order to prevent rehybridization of the single DNA
strands in the course of chromatography or electrophoresis, it is useful to provide single-stranded DNA for the isolation. For this purpose, the primer in the anti-sense strand is biotinylated. The PCR product is heated and passed through a solid streptavidine phase (e.g. dynabeads). In this way, the anti-sense strand is removed. If the anti-sense strand is to be analyzed, the sense strand can also be removed by a corresponding biotinylated primer.
The wild type and amplificates mutated in codons 12 and 13, respectively, of the K-RAS gene are isolated e.g. by column chromatography or capillary electrophoresis (acrylamide gel filling of the capillaries). When the carrier is charged, buffer and temperature conditions are chosen such that the melting temperature is above that of mutant allels and below that of wild type allels. For example, SSC or SSPE in corresponding concentration (5-4x) and 3 M

tetramethylammonium chloride, respectively, are in consideration as buffers. A constant temperature in the separation means must be ensured by a heating block (e. g.
58°C when 20meric oligonucleotides and 3 M
tetramethylammonium chloride solution are used).
The fluorescence-labeled amplificates are detected by means of laser-induced fluorescence using e.g. an argon lif detector. For the sensitive detection of the DNA it is also possible to add a second PCR.
EXAMPLE 2: Detection of point mutations in the 5°h exon of the p53 gene The DNA is isolated as described in Example 1. The following primers are used for the amplification of the 5t'' exon:
Sense: 5'-TTTCCACTCTGTCTCCTTCC-3';
Anti-sense: AACCAGCCCTGTCGTCTCTC-3'.
Since the primers are present in introns, the entire sequence of exon 5 can be analyzed. The anti-sense primer is labeled with a biotinylated oligonucleotide at the 5' end, the sense primer is labeled with a fluorescence-labeled oligonucleotide at the 5' end. PCR is carried out as described above. The biotinylated DNA strand is separated by bonding to immobilized streptavidine.
(a) Separation by means of separation means arranged in parallel The separation means consists of columns having a defined separation medium, e.g. glass beads, polyacrylamide (figure 1). A defined 20meric oligonucleotide is covalently bonded to the separation medium in a certain column, as described above. The oligonucleotide is complementary to a given section of the DNA of exon 5 of the p53 gene. The oligonucleotides bonded to the separation medium in the various columns cover the entire sequence of the 5t'' exon as a whole (see figure 1). The columns are disposed in a heating block to ensure a constant temperature. The temperature for the separation of wild type and mutated allel is 58°C. The columns are equilibrated with 3 M
tetramethylammonium chloride solution. The PCR product to be investigated, which contains wild type and mutated allels, is also dissolved in this solution.
Furthermore, the PCR product is single-stranded (sense strand) and fluorescence-labeled. It is applied in aliquots to the columns. The mutated allel has a mismatch with respect to the oligonucleotide in the third separation chamber from the left. The mutated allel is present in shortage as compared to the wild type allels. The wild type allels are bonded in all of the columns. The mutated allel passes the third column from left. It is detected via the fluorescence signal.
Columns or detector can be movable. Prior to the charge of a defined column, the detector is connected with the outlet of the column. After the expiration of the reaction, the detection is connected with the next column. By this, the fluorescence signal of a certain column and the mutation can thus be attributed to a defined section of the DNA sequence. This enables the well-calculated detection of a mutation after a second PCR; e.g. by allel-specific oligonucleotide hybridization or DNA sequencing.
(b) Separation by means of separation chambers arranged in series A column-chromatographic separation of separation chambers arranged in series is described. As described under item (a), the individual columns are filled with a separating fluid to which one defined oligonucleotide is bonded per column. The oligonucleotides bonded in the various columns in turn cover the entire sequence of exon 5. The columns are temperature-controlled. In the case of series arrangement, a valve is disposed between the individual columns. Charge and valve position are illustrated by way of diagram in figure 2a. The oligonucleotides bonded to the separating matrix are given on the right. Fluorescence labeling, isolation of the single-stranded DNA as well as columns and sample solution, respectively, are as described under item (a). Before the sample is charged, the valve between columns 1 and 2 closes the inlet to column 2 and opens the inlet to detector or collecting tank (position I). The temperature in column 1 is 58°C. The single-stranded PCR product passes through column 1.
Wild type DNA is bonded. If a point mutation is present in the DNA section of the mutated allel, which is complementary to the bonded oligonucleotide, the mutated allel will not be bonded and supplied to the detector and collecting tank, respectively. If the point mutation is present in another DNA section, the mutated allel will also be bonded. After the conclusion of the reaction, the valve position between columns 1 and 2 is changed such that the inlet to column 2 is released and the outlet to detector/collecting tank is closed. The valve between columns 2 and 3 closes the inlet to column 3 and opens the inlet to detector/collecting tank (valve positions II). the temperature of column 2 is kept at 58°C. Column 1 is heated to a temperature which is above the temperature of the wild type. Then, buffer is pumped through the system. The bonded DNA is eluted from column 1. In column 2, the wild type DNA hybridizes with the oligonucleotide shown on the right-hand side. If a point mutation is present in the DNA section of the mutated allel, which is complementary to the bonded oligonucleotide, the mutated allel will not be bonded and supplied to the detector and collecting tank, respectively. If the point mutation is present in another DNA section, the mutated allel will also be bonded. Thereafter, the valve position between columns 2 and 3 is changed such that the inlet to column 3 opens and the inlet to detector/collecting tank is closed (III). The sample DNA is eluted by heating and fed into column 3. The procedure is continued correspondingly until all columns have been passed through. A separation means is shown in figure 2b, which enables the detection of point mutations throughout exon 5 of the p53 gene. The bonded oligonucleotides shown on the left-hand side cover the entire region of the sense strand of exon 5 of the p53 gene.

Claims (9)

Claims

1. A method for detecting mutated allels in an excess of wild type allels, comprising the separation of the wild type allels by means of a separation process using a carrier to which one or several oligonucleotides complementary to the wild type allel are bonded.

2. The method according to claim 1, wherein the separation process is chromatography or electrophoresis.

3. The method according to claim 1 or 2, wherein the separation process is capillary electrophoresis.

4. The method according to any one of claims 1 to 3, wherein the mutated allel has one or several point mutations, deletions, inversions, insertions and/or substitutions of small or relatively great gene regions.

5. The method according to any one of claims 1 to 4, wherein several separation means are arranged in parallel or in series for the separation process.

6. The method according to any one of claims 1 to 5, wherein sense and/or anti-sense oligonucleotides which are complementary to the wild type allel are used.

7. The method according to any one of claims 1 to 6, wherein the oligonucleotides comprise 16 to 20 base pairs.

8. Use of the method according to any one of claims 1 to 7 for the diagnostics of genetic modifications.

9. Use according to claim 8, wherein cancer diagnostics is concerned.