Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR THE IDENTIFICATION OF CYTOSINE METHYLATION
PATTERNS IN GENOMIC DNA
The invention concerns a method for the identification of 5-methyl-
cytosine positions in genomic DNA.
The genetic information, which is obtained as a base sequence by
coi~nplete sequencing of genomic DNA, only incompletely describes the genome
of a cell. 5-Methylcytosine nucleobases, which are formed in the cell by
reversible methylation of DNA, are an epigenetic information carrier and
serve,
for example, for the regulation of promoters. The methylation state of a
genome
represents the present status of gene expression, similar to an mRNA
expression pattern.
5-Methylcytosine is the most frequent covalently modified base in the
DNA of eukaryotic cells. It plays a role, for example, in the regulation of
transcription, genomic imprinting and in tumorigenesis. The identification of
5-
methylcytosine as a component of genetic information is thus of considerable
interest. 5-Methylcytosine positions, however, cannot be identified by
sequencing, since 5-methylcytosine has the same base pairing behavior as
cytosine. Unfortunately, the epigenetic information that is carried by 5-
methylcytosines becomes completely lost in PCR [polymerase chain reaction]
amplification, and there is no method for obtaining this information by an
amplification step.
Several methods are known, which solve these problems. For the most
part, a chemical reaction or enzymatic treatment of the genomic DNA is
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conducted, as a consequence of which, cytosine nucleobases can be
distinguished from methylcytosine nucleobases. One current method is the
reaction of genomic DNA with disulfite (also denoted bisulfite or
pyrosulfite),
which leads to the conversion of cytosine bases to uracil in two steps after
alkaline hydrolysis (Shapiro, R., Cohen, B, Servis, R. Nature 227, 1047
(1970).
5-Methylcytosine remains unchanged under these conditions. The conversion of
C to U leads to a change in the base sequence, from which the original 5-
methylcytosines can now be determined by sequencing (only these [bases] will
still supply a band in the C lane).
An overview of the other known possibilities for detecting 5-
methylcytosines can be derived from the following review article together with
the
references belonging thereto: Rein, T., DePamphilis, M.L., Zorbas, H., Nucleic
Acids Res. 26, 2255 (1998).
A method for characterizing specific DNA sequences is described in DD
293,139 A5, in which the DNA molecules, whose unmethylated recognition sites
can be cleaved by an appropriate restriction endonuclease, are incubated in a
reaction mixture with a second, unmethylated DNA species (particularly
oligonucleotide duplexes, which contain the recognition site).
WO 97/46,705 A1 discloses a method for the detection of a methylated
nucleic acid containing CpG, whereby the sample containing nucleic acid is
brought into contact with a reagent, which modifies unmethylated cytosine, so
that nucleic acids containing CpG in the sample are amplified by means of CpG-
specific oligonucleotide primers, whereby the oligonucleotide primer
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differentiates between modified methylated and unmethylated nucleic acids and
detects methylated nucleic acids.
In addition, US 5,824,471 A1 describes a method for the determination of
deviations between two nucleic acid strands, whereby a multiple number of
duplexes are formed from the two strands or parts thereof and these duplexes
are contacted with a first and a second different bacteriophage resolvase and
whereby it is then established from which bacteriophage resolvase the duplex
is
cleaved, whereupon the differences are determined thereby.
However, it is not always necessary to actually determine the entire
sequence of a gene or gene segment, as is the objective in the case of
sequencing. This is particularly true if only a few 5-methylcytosine positions
are
to be found within a long base sequence in the case of a multiple number of
different samples. Here, sequencing supplies essentially redundant information
and is also very expensive. This is also true in the case when the sequence is
already known and only the methylation positions need to be shown. It is also
conceivable that in several cases in general, only the differences in the
methylation pattern between different genomic DNA samples are of interest and
that the determination of a multiple number of corresponding methylated
positions as well as sequencing can be dispensed with. For the questions posed
here, up until now, there has existed no method which supplies the desired
result
in a cost-favorable manner without sequencing each individual sample.
The sequence information per se is also continually less novel, since
genome projects, whose goal is the complete sequence of various organisms,
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are swiftly advancing. In fact, currently, even though only approximately 5%
of
the human genome has been completely sequenced, the study is progressing
rapidly, since other genome projects have been completed and in this way
sequencing resources have been freed up, so that every year another 5% is
added. It is calculated that the sequencing of the human genome will be
completed by the year 2006.
Matrix-assisted laser desorptionlionization mass spectrometry (MALDI) is
a new, very high-performing development for the analysis of biomolecules
(Karas, M. and Hillenkamp, F. 1988. Laser desorption ionization of proteins
with
molecular masses exceeding 10,000 daltons. Anal. Chem. 60:2299-2301 ). An
analyte molecule is embedded in a matrix absorbing in the UV. The matrix is
evaporated in vacuum by a short laser pulse and the analyte is transported
into
the gas phase unfragmented. An applied voltage accelerates the ions in a field-
free flight tube. Ions are accelerated to varying degrees based on their
different
masses. Smaller ions reach the detector sooner than larger ions. The time-of-
flight is converted to the mass of the ions. Presently, this technology can
distinguish molecules with a mass difference of 1 Da in the mass region from
1,000 to 4,000 Da. Due to the natural distribution of isotopes, most
biomolecules, however, are approximated within 5 Da. Technically, this mass-
spectrometric method can be very suitable for the analysis of biomolecules,
but
in order to distinguish them, the products that are to be analyzed must lie at
least
Da apart from one another. Therefore, 600 molecules can be distinguished in
this mass region. In the region between 4,000 and 100,000 Da, isotope
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resolution is no longer achieved, but this region can also be used. Recently,
the
application of an infrared (IR) laser coupled with the MALDI analysis of DNA
has
been described (Berkenkamp, S., Kirpkar, F. and Hillenkamp, F. 1998. Infrared
MALDI mass spectrometry of large nucleic acids. Science. 281: 260-262). It
was possible by means of this combined technique to detect DNA fragments with
a size of up to 2,500 bases.
Chemical mismatch cleavage is a method by means of which small
differences between two single strands of DNA can be indicated (Cotton,
R.G.H.,
Rodriguez, N.R. and Campbell, R.D. 1988. Reactivity of cytosine and thymine
in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its
application to the study of mutations. Proc. Natl. Acad. Sci. USA. 85: 4397-
4401;
Cotton, R.G.H. 1993. Current methods for mutations detection. Mut. Res. 285:
125-144; Saleeba, J.A., and Cotton, R.G.H. 1993. Chemical cleavage of
mismatch to detect mutations. Methods in Enzymology. 217: 286-295;
Smooker, P.M. and Cotton, R.G.H. 1993. The use of chemical reagents in the
detection of DNA mutations. Mutations Res. 288: 65-77). The chemical
reactivity of C and T relative to osmium tetroxide and of C relative to
hydroxylamine is increased, if these are not paired with their respective
complementary bases. The nucleic acid strand is broken at the modified
position
by subsequent treatment with piperidine.
Another possibility to indicate non-complementary base pairs in
heteroduplex DNA consists of the application of enzymes such as MutS, which
bind to non-complementary base pairs (Smith, J. and Modrich, P. 1996.
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Mutation detection with Mutes, Mutt, and MutS mismatch repair proteins. Proc.
Natl. Acad. Sci. USA 93: 4374-4379; Parsons, B.L. and Heflich, R.H. 1997.
Evaluation of MutS as a tool for direct measurement of point mutations in
genomic DNA. Mut. Res. 374: 277-285).
At the present time, a rapid, cost-favorable and automatable method for
finding methylated cytosines in genomic DNA is lacking. Such a method,
however, is of great interest, since different methylation patterns can be
drawn
on in a variety of ways for characterizing cell types and thus can be used for
diagnosis and classification of diseases (such as, for example, tumors) and
this
method could also be utilized, for example, for studies of cell
differentiation.
The object of the present invention is thus to create a method for a cost-
favorable parallelly-conducted dectection of epigenetic information carriers
in the
form of 5-methylcytosine bases in genomic DNA.
The object is resolved according to the invention by a method for the
identification of 5-methylcytosine positions in genomic DNA, whereby the
following method steps are conducted:
a) the genomic DNA of a cell, a cell line, a tissue or an individual is
chemically
treated in such a way that cytosine and 5-methylcytosine react differently and
a
different base-pairing behavior results for the two products in the duplex,
b) the same nucleic-acid segment is amplified by means of a polymerase
reaction,
c) the same nucleic-acid segment of at least one other cell, cell line, tissue
or
individual or any desired reference DNA is treated according to steps a) and
b),
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d) heteroduplexes are formed from the at-least two amplified products of steps
b) and c),
e) a detectable labeling is introduced into the heteroduplex by means of a
reaction, which is specific to non-complementary base pairs.
According to the invention, it is preferred that for the identification of
differences in the cytosine methylation pattern between various cells, cell
lines,
tissues and individuals, only those positions are applied and indicated, in
which
the cytosine methylation is variable between different cells, cell lines,
tissues or
individuals.
It is also preferred that a disulfite (bisulfate, pyrosulfite) is utilized as
the
reagent for the selective conversion of cytosine to uracil in step a), whereby
5-
methylcytosine remains unchanged.
It is also preferred that genomic DNA of several individuals, tissues, cell
lines or cells is jointly amplified in step b).
In addition, it is preferred that genomic DNA of several individuals,
tissues, cell lines or cells is separately amplified and then jointly treated
according to step e).
It is also preferred according to the invention that erroneous base pairings
are produced by formation of heteroduplexes from the DNA of different
individuals, tissues, cell lines or cells at the positions at which a 5-
methylcytosine
was localized in the genomic DNA.
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It is also preferred that erroneous base pairings occur in step d) at those
positions at which cytosine was found in the genomic DNA by the formation of
heteroduplexes with a completely methylated reference DNA.
It is also preferred that erroneous base pairings occur in step d) at the
positions at which 5-methylcytosine was found in the genomic DNA, by formation
of heteroduplexes with a completely demethylated reference DNA.
In addition, according to the invention, it is preferred that the erroneous
base pairings lead to a specific or sufficiently selective backbone cleavage
at
these positions by means of "chemical mismatch cleavage" (chemical
modification at non-complementary positions).
It is also preferred that the DNA at the erroneous base pairings is cleaved
enzymatically specifically or sufficiently selectively.
In the method according to the invention, it is also preferred that 1 DNA
fragment is obtained in step e), the size of which provides an inference to
the
cleavage positions and thus to the position of the methylcytosines andlor the
variable methylation positions between different individuals, tissues, cell
lines or
cells.
It is preferred that the analysis of size (molecular weights) of the DNA
fragments is conducted by means of mass spectrometry.
It is particularly preferred that the fragments are analyzed by means of
matrix-assisted laser desorptionlionization time-of-flight mass spectrometry
(MALDI).
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It is also particularly preferred that the fragments are analyzed by means
of electrospray ionization mass spectrometry (ESI).
It is particularly preferred that the size of the fragments produced in step
e) is adapted to the performance capacity of the mass spectrometer.
It is most particularly preferred that several PCRs of a gene segment are
conducted and the primers are set stepwise such that the fragment size to be
expected at least in one of these PCRs falls in the mass range that can be
detected by means of mass spectrometry.
It is particularly preferred that one of the PCR primers is newly positioned
stepwise by the maximally detectable mass range of the mass spectrometer
relative to the other primer.
It is preferred according to the invention that in step b) one primer of the
PCR is provided with a chemical function, so that the PCR product can be
immobilized on a surface.
It is also preferred according to the invention that the PCR product
produced in step b) is transferred into different reaction vessels and the
surfaces
of the reaction vessels are treated chemically in such a way that the PCR
product can be bound thereon.
It is also particularly preferred that PCR products of different individuals
that are prepared in step c) are transferred into different reaction vessels
prepared as described above.
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In addition, it is preferred according to the invention that an enzyme is
used for step e), which [enzyme] forms a complex with a non-complementary
base pair.
It is very particularly preferred that this enzyme is Muts.
In addition, it is preferred that the enzyme bears a label, by means of
which a complex can be visualized.
1t is also preferred according to the invention that the label is a
fluorescence label, a chemiluminescence label, a mass label or a
photochemically cleavable mass label.
In addition, it is preferred according to the invention that an amplified DNA
sample according to step c) in claim 1, which displays a difference relative
to an
amplified DNA sample in step b) is compared in a second run of the method
itself
[with] a [similar] DNA sample according to step b) in claim 1 and with all
other
DNA samples to be investigated.
It is also preferred according to the invention that a preselection of the
gene segments to be investigated in detail by mass spectrometry will be
conducted by means of a fluorescence labeling or chemiluminescence labeling
of the immobilized DNA strand, the lack of which indicates the presence of
methylated cytosines in the investigated genomic DNA segment after conducting
steps d) and e) of claim 1 and a washing step.
It is further preferred according to the invention that a preselection of the
gene segments to be investigated in detail by mass spectrometry is conducted
by means of a more nonspecific variant according to claims 20 to 23.
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Another subject of the present invention is a kit for conducting the method
according to the invention, comprising DNA of at least two individuals,
tissues,
cell lines or cells that are as different as possible as well as reagents, in
order to
indicate the variable methylation positions.
The [kit] according to the invention also comprises completely methylated
andlor demethylated DNA and reagents, which are necessary for the detection
of methylated cytosines in any DNA sample.
The method according to the inventions serves for the identification of 5-
methylcytosine positions in genomic DNA, which can be of the most varied
origin. The genomic DNA is first treated chemically in such a way that a
difference is produced in the reaction of cytosine bases and of to 5-
methylcytosine bases. Possible reagents here include, e.g., disulfite (also
denoted bisulfite or pyrosulfite), hydrazine and permanganate. In a preferred
variant of the method, the genomic DNA is treated with disulfite in the
presence
of hydroquinone or hydroquinone derivatives, whereby the cytosine bases are
converted to uracil selectively after subsequent alkaline hydrolysis. 5-
Methylcytosine remains unchanged under these conditions. After a purification
process, which serves for separating the excess disulfite, a specific segment
of
the pretreated genomic DNA is now amplified in a polymerase reaction. In a
preferred variant of the method, the polymerase chain reaction is used here.
Then, the same segment of another genomic DNA sample is amplified to the
same extent. The two amplified products are combined, whereby
heteroduplexes are partially formed. In a preferred variant of the method,
this is
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produced in such a way that one of the PCR primers bears a function suitable
for
immobilization and that only one strand of the amplified product of the first
sample is immobilized and then a hybridization is performed with the amplified
product of the second sample. In another preferred variant, a multiple number
of
different amplified products of the same nucleic-acid segment are hybridized
in
this manner with the immobilized single strand of the amplified product of the
first
sample, which was first distributed in many wells of a microtiter plate. Now
one
hybridization experiment can be conducted in each well.
After the hybridization, a method is conducted, which leaves behind a
detectable label at those positions in which an erroneous base pairing occurs
in
the heteroduplex. In a preferred variant of the method, this is conducted by
chemical mismatch cleavage, which leads to a break of the backbone at the
positions where an erroneous base pairing has occurred. The fragments
obtained in this way can be analyzed by any method that can indicate the size
of
DNA fragments. Such a method should ideally permit conclusions on any
position in the amplified nucleic-acid segment of the sample, at which an
erroneous pairing has occurred in the heteroduplex. Erroneous base pairings in
the heteroduplex are present particularly if, in the DNA of one sample,
cytosine
was present at this position, which was converted to uracil, but in the other
sample, 5-methylcytosine was present, which remained unchanged in the
chemical pretreatment. The method can be utilized also for the comparison of
two or more genomic DNA samples; in this case, the analysis of the fragments
supplies only the differences in the methylation pattern between the two
samples
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in the respective amplified nucleic-acid segment. However, it is also possible
to
utilize a DNA as a reference which has been completely methylated or
demethylated enzymatically at C. In this case, the analysis of the fragments
supplies all 5-methylcytosine positions in the respective amplified nucleic-
acid
segment.
In a particularly preferred variant of the method, mass spectrometry is
applied to the analysis of fragments. After a preliminary purification, the
fragments can be analyzed in the MALDI mass spectrometer. Alternatively, the
solutions can be analyzed by electrospray ionization mass spectrometry (ESI).
It
may be necessary to investigate the nucleic-acid segment in question in
several
substeps by newly positioning a primer stepwise in several PCRs, each time
depending on the performance capability of the method and the instrument
utilized, and thus various amplified products of the substeps result ("primer
walking")
In a variant of the method, the erroneous base pairings - alternatively to
the analysis of fragments after a backbone cleavage in the heteroduples - may
also be detected by means of an enzyme, which forms a complex with a non-
complementary base pair. In a preferred variant, this enzyme is MutS, which
bears a label, e.g., a fluorescence, chemiluminescence or mass label.
In another variant of the method, the presence of erroneous base
pairings, i.e., in this case also the presence of relevant information in the
amplified nucleic-acid segment, is detected by a fluorescence or
chemiluminescence labeling. In a preferred variant of the method, an
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immobilized DNA strand of the amplified product of sample 1 is provided with a
fluorescence label on the end that does not serve for the immobilization.
Heteroduplexes are formed with the amplified product of sample 2, and these
are
subjected to a chemical mismatch cleavage. If a backbone cleavage occurs at
the immobilized strand, then the fluorescence label disappears after a
denaturing
washing step, and if the strand is not cleaved, then the label remains. Only
the
amplified products that have been cleaved are subsequently investigated in
more detail by mass spectrometry.
Examples
Example 1
Method for finding all methylated cytosine positions
The genomic DNA to be investigated that derives from a cell line, or as
much as possible from only one cell, is divided into two reaction vessels and
one-half of this is either completely methylated or demethylated enzymatically
at
the cytosine. The enzyme is thermally inactivated and then both parts are
again
combined and treated with disulfite and then alkali. After a purification,
amplification is conducted by means of PCR.
The chemical mismatch cleavage that is specific for the C mismatch and
that is now conducted leads to a cleavage at the positions of a corresponding
heteroduplex, at which an originally methylated C was found, if a complete
demethylation of one-half of the genomic sample was conducted. On the other
hand, a cleavage occurs at all originally non-methylated positions, if a
complete
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methylation of one-half of the genomic sample was previously conducted. For
reliability, both methylation as well as demethylation may be conducted as a
reference, but in this case, these procedures must be performed separately in
two PCRs.
Variant 1: The above method is conducted in such a way that a primer is
introduced in the PCR, which is functionalized such that a simple and specific
immobilization is made possible after,PCR. The immobilization is conducted
onto beads or unto the surface of a microtiter plate. This permits the simple
separation of components of the polymerase and mismatch cleavage reactions.
After the chemical mismatch cleavage reaction, the duplex is thermally
denatured and the solution is pipetted off. The DNA fragments from this
solution
are introduced onto a reversed-phase material and purified.
In the mass spectrometer, the fragments produce a "ladder" of peaks from
which the methylated positions can be inferred. Theoretically, two peaks per
CpG always occur at CpG postions on the basis of the symmetrical methylation;
these peaks originate from the sense and anti-sense strands.
Variant 2: The reactions are conducted in solution and a purification is
conducted after the individual reaction steps, if necessary, each time by
means
of a reversed-phase material.
Variant 3: Several individuals or cell types are parallelly investigated. A
reference DNA is completely demethylated and then treated with disulfite. It
is
amplified by means of PCR after purification. A primer is again used, which
bears a function suitable for the immobilization. The solution is distributed
onto
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the wells of a microtiter plate and immobilized. Then a hybridization is
conducted against the PCR products from the samples also treated with
disulfite,
each time, one [product] per well (see also the described example with 97
individuals).
Variant 4: If the mass spectrometer cannot cover the [entire]
measurement range, which would be necessary for the analysis of the total PCR
product to determine methylations, the region of interest can also be found
stepwise by conducting several PCRs and each time placing one of the primers
closer to the other by the respective measurement range of the mass
spectrometer. Thus, for example, only that region is detected, which lies
between the primer to be shifted of the PCR in question and the next PCR. The
method can be combined with the other variants.
Example 2
Method for finding positions with variable cytosine methylation
DNA of various individuals or cell lines is pooled and a treatment with
disulfite is conducted as described above. After alkaline hydrolysis of the
bisulfite adducts and purification of the product DNA, the latter is amplified
by
means of PCR. It is then purified again and after several minutes of
reannealing
at 25°C with Os04, the PCR product is cleaved at the positions with a C
mismatch (chemical mismatch cleavage). A C ~ A mismatch then always
occurs, if a methylated cytosine has been present only in several individuals
prior
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to the bisulfate treatment. In this process, possible SNPs (single nucleotide
polymorphisms), as it were, also lead to the cleavage of the DNA. The latter
must be distinguished from the methylated positions to be found, which is
assured by employing the above-described method for finding all methylated
cytosines.
The DNA product is now investigated by mass spectometry, as described
above. If the initially generated PCR product is longer than can be detected
with
the currently available technology relative to mass spectrometry, then it is
possible that the fragments produced by the chemical mismatch cleavage cannot
be detected. In order to get around this, several PCRs can be conducted
iteratively, i.e., one primer will always be kept constant, while the other
primer will
be positioned closer to the other primer continually in several steps, each
time by
the detection limit of the mass spectrometer (primer walking).
Example 3
Method with 97 individuals
A genomic segment of an individual (reference individual) is treated with
disulfite and in this way, the cytosines are converted into uracils after
subsequent
alkaline hydrolysis of the bisulfate adduct. The methylcytosines remain
unaffected in this reaction sequence. The product is purified and amplified by
means of PCR. One of the PCR primers is provided on the 5' end with a
chemical modification, which serves for immobilization. The product of this
PCR
is placed in the 96 wells of a microtiter plate and the PCR products are
induced
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to bind to the surface. Since only one primer is provided with the chemical
modification for such binding, only one DNA strand binds to the surface. The
plate is washed to eliminate the reagents of the binding chemistry and the
complementary strands. In this way, the plate containing the reference DNA
piece is prepared. The same genomic segment in each of the 96 other
individuals is treated analagously with disulfite and then amplified. Each
time
two normal unmodified primers of the same sequence as for the reference
individual are used for this PCR. The 96 PCR products are placed in the 96
wells of the prepared plate. By heating and slow cooling, the complementary
strands of the 96 individuals are hybridized to the reference DNA (formation
of
the heteroduplex). The 96 individuals and reagents of the previous reactions
(are] to be eliminated. An Os04 solution is added to each of the 96 wells,
incubated, and then a backbone cleavage is induced with piperidine in a
heteroduplex with a non-complementary base pair, one base of which is C. This
will happen only if a methylcytosine is present instead of a cytosine in one
strand
of the heteroduplex, i.e. in one of the individuals. In this case, only the
cytosine
of one individual was converted to a uracil prior to the PCR, whereby a
mismatch
results in the heteroduplex with the counter-strand of another individual. The
assay thus does not directly produce all methylated cytosines of a genomic
segment, but only those that are variable between different individuals,
tissues,
cell lines or individual cells.
The heteroduplex is melted apart by heating and the solution is
transferred to a mass spectrometer.
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Example 4
Transfer of the solution to the mass spectrometer
One good variant is to take up the solution after melting the heteroduplex
in a pipette tip, which is furnished with a reversed-phase material. The DNA
products bind thereon by forming hydrophobic interactions by means of their
trialkylammonium counter-ions and can thus be purified of the reagents of the
chemical mismatch cleavage in several washing steps. The DNA products can
be dissolved again from the reversed-phase material with 30% acetonitrile.
This
makes it possible to obtain the products directly on a prepared matrix on a
MALDI target. After a single drying, the target is introduced into the mass
spectrometer and the products [areJ analyzed.
Example 5
Preselection by means of fluorescence labeling of gene segments relevant for
methylation detection.
The genomic DNA to be investigated is immobilized on beads or an
appropriately coated microtiter plate after the bisulfate reaction with
subsequent
PCR amplification as described above, in which one of the primers again bears
a function that can serve for the subsequent immobilization. Completely
demethylated DNA, treated like the sample DNA, is used as reference DNA and
forms a heteroduplex with the immobilized sample DNA. Then an individual
fluorescence-labeled base is attached enzymatically, for example, with
terminal
transferase to the 3' ends of the product. The subsequently conducted
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"chemical mismatch cleavage" reaction in the case where a CIA mismatch is
found in the product, leads to a cleavage of the immobilized strand, so that
all
fluorescence labelings are removed in the subsequent washing step after the
thermal dehybridization. Thus a methylation was present within the amplified
product, so that fluorescence no longer occurs in the microtiter plate or on
the
bead. The fluorescence only remains, if no mismatch is present, and thus no 5-
methylcytosines are present in the gene segment in question. In the method, it
must be considered that, e.g., SNPs may yield a false positive signal.
Accordingly, this method may be utilized also for simple, f(uorescence-
based detection of 5-methylcytosines in small gene segments, e.g. promoters.
However, information may only be found of whether or not methylations are
present in the region in question, but not how many and at which positions. Of
course, this is compensated by a relatively small experimental expenditure and
a
good capability for conducting parallel experiments.
Then, analagously to one of the above-described examples, the precise
position of methylcytosines can be determined by mass spectrometry in the gene
segments classified as relevant.
Example 6
Preselection of heteroduplexes with mismatches
The heteroduplexes immobilized in one microtiter plate are first combined
with a solution of MutS, to which a fluorescent dye is bound. Only the
vessels, in
which MutS has attached to erroneous base pairing positions, which is
indicated
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by the fact that the fluorescence can still be detected after several washing
steps, are subsequently subjected to the chemical mismatch cleavage and
analyzed in the mass spectrometer. In this way, time in the mass spectrometer
and costs for purification are spared, since analysis of samples without
detectable epigenetic information is avoided.
