Note: Descriptions are shown in the official language in which they were submitted.
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W 1545
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.
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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
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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
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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,
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- 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.
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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
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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
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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.
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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
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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
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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
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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
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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.
