Note: Descriptions are shown in the official language in which they were submitted.
CA 02379163 2002-O1-28
Method for characterizing nucleic acid fragments
The invention concerns a method for characterizing nucleic acid
fragments.
WO-A-99129897 discloses a method and a kit for identifying nucleic acids.
Here, probes of distinguishable mass are used.
US-A 5,929,208 describes a hybridizing assay, wherein the nucleic acid to
be characterized is contacted by an oligomer array surface, whereby
complementary oligomers of the array hybridize with the DNA to be
characterized
and non-hybridized nucleic acids are removed. Here, specific positions in an
array are provided with charges, so that the binding of oligomers is
controlled at
specific sites.
Different methods are known at the present time, by which oligonucleotide
arrays can be produced. They can be roughly classified into 3 groups:
1 ) All oligomers are produced individually and in relatively large quantities
in the conventional manner in the test tube or in special automatic synthesis
devices and then individually pipetted onto the carrier. For this purpose,
usually
automatic, highly-precise micropipetting robots are used. The advantage of
this
method is that it is based for the most part on standard methods and devices
that
have already been optimized. Qualitatively superior DNA arrays with very pure
oligomers can be produced in this way, which has an extremely positive
influence
on the detection sensitivity and reliability that can be obtained with the
array.
The great disadvantage of the method is that it is very time-consuming and is
CA 02379163 2002-O1-28
thus expensive. This applies particularly to the synthesis of the individual
oligomers.
The purchase of ready-to-use oligomers does not represent a solution,
since commercially synthesized oligonucleotides in large quantities are also
extremely expensive, because the synthesis of the monomers is relatively
expensive, patent fees must be paid for several special designs, and also the
solvents used are expensive due to their required purity.
2) The oligomers are synthesized by pipetting minimal quantities directly
onto the substrate. The oligomer chain provided therein is constructed,
nucleobase by nucleobase, at each grid point. For pipetting, as in method (1 )
a
specialized micropipetting robot device is similarly used, or, e.g., a device
that
contains channels for introducing the individual synthesis building blocks to
the
respective points of the array (EP-A 0915897). The chemical synthesis method
is basically the same as for conventional oligomer synthesis in automated
synthesis equipment. The difference is that all oligomers are prepared
simultaneously, independent of their number, by a single automatic unit,
directly
at the provided determination site. The separate operating steps of oligomer
synthesis and micropipetting as in method (1 ) are now combined into a single
operating step. The expense for equipment and for manual labor is considerably
reduced when compared with method (1 ).
3) The oligomers, as in method (2), are synthesized directly on the
substrate, but the targeted binding of the correct nucleobases to the correct
grid
points is accomplished by a completely parallel photolithographic technique
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CA 02379163 2002-O1-28
originating from semiconductor manufacture, instead of sequential, precisely
targeted pipetting steps. The method is based on the fact that 5'-OH
protective
groups can be removed from oligonucleotides in a targeted manner with light of
a
specific wavelength. By suitable local irradiation patterns, oligonucleotide
ends
can thus be made reactive at precisely those grid points at which it is
desired that
a new nucleotide building block will bind in the next step. When the array
surface
is completely wetted with a nucleotide building-block solution, a nucleobase
will
thus be bound only at the previously exposed sites, and all of the unexposed
sites will remain unchanged. The local exposure patterns are produced by
positioning a photomicrograph black-and-white mask between the substrate and
the light source, which covers all grid sites which will not be made reactive.
The
lengthening of the oligomer chains on all grid points by one nucleobase is
accordingly accomplished as follows: by means of a first mask, just those grid
points are exposed, which must be extended by the first of the 4 possible
types
of nucleobases (e.g., C). Then the array is wetted with a solution of the
respective nucleotide base, whereupon only the exposed points are lengthened
by this base. Since the newly bound nucleotide building blocks are still all
made
available by means of a protective group, they can no longer react in the
following steps until their protective groups are cleaved by another exposure.
After this reaction step, the array is washed. Now, by means of a second mask,
just those grid sites are exposed, which must be extended by the second of the
4
possible nucleobases (e.g., T). The array is again wetted with a solution of
the
corresponding nucleotide building block and the exposed sites are lengthened
by
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CA 02379163 2002-O1-28
this base. The procedure is conducted in the same way for the other two
nucleobases (G and A). Consequently, four exposure steps and thus 4
photomasks are required for the lengthening of all oligomers by one
nucleobase.
Due to the high parallel nature in processing, this method is very rapid and
efficient, and it is also well suitable for the purpose of achieving very high
grid
densities, due to the high precision that can be obtained with
photolithography.
An overview of the prior art relative to oligomer array production can also
be taken from a special publication that appeared in January 1999 of Nature
Genetics (Nature Genetics Supplement, Volume 21, January 1999) and the
literature cited therein.
Patents, which generally relate to the use of oligomer arrays and
photolithographic mask designs, include, e.g., US-A 5,837,832; US-A 5,856,174;
WO-A 98127430 and US-A 5,856,101. There also exist several material and
method patents, which limit the use of photolabile protective groups to
nucleosides, thus, e.g., WO-A 98139348 and US-A 5,763,599.
Various methods exist for immobilizing DNA. The best-known method is
the solid binding of a DNA, which is functionalized with biotin, to a
strepavidin-
coated surface. The binding strength of this system corresponds to a covalent
chemical bond without being one. In order to be able to bind a target DNA
covalently to a chemically prepared surface, the target DNA needs to have the
corresponding functionality. DNA itself does not have a functionalization that
is
suitable. There are different methods for introducing a suitable
functionalization
into a target DNA: two functionalizations that are easy to manipulate are
primary
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CA 02379163 2002-O1-28
aliphatic amines and thiols. Such amines are converted quantitatively with N-
hydroxysuccinimide esters and thiols react quantitatively with alkyl iodides
under
suitable conditions. The one difficulty is how to introduce such a
functionalization
into a DNA. The simplest method is the introduction by a primer of a PCR. The
indicated methods utilize 5'-modified primers (NHa and SH) and a bifunctional
linker. An essential component of the immobilization onto a surface is the
condition of the surface. Systems described up to the present time primarily
comprise silicon or metal (magnetic beads). Another method for binding a
target
DNA is based on using a short recognition sequence (e.g., 20 bases) in the
target DNA for hybridizing to a surface-immobilized oligonucleotide.
Various enzymatic methods have also been described for introducing
chemically activated positions into a target DNA. Here, a 5'-NH2
functionalization
is introduced enzymatically into a target DNA. 5-Methylcytosine is the most
frequent covalently modified base in the DNA of eukaryotic cells. For example,
it
plays a role 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. In addition, in the case of a PCR amplification,
the
epigenetic information that is borne by 5-methylcytosines is completely lost.
Several methods are known for solving these problems. For the most
part, a chemical reaction or enzymatic treatment of genomic DNA is conducted,
as a consequence of which, cytosine can be distinguished from methylcytosine
CA 02379163 2002-O1-28
bases. A current method is the reaction of genomic DNA with bisulfite, which
leads to a conversion of cytosine bases to uracil after alkaline hydrolysis in
two
steps (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 methylcytosines
can still provide a band in the C lane).
An overview of the other known possibilities for detecting 5-
methylcytosines can be taken from the following review article together with
all of
the references cited therein: Rein, T., DePamphilis, M. L., Zorbas, H.,
Nucleic
Acids Res. 26, 2255 (1998).
Matrix-assisted laser desorptionlionization mass spectrometry (MALDI) is
a new, very high-performing development for the analysis of biomolecuies
(Karas, M. and Hillenkamp, F. 1998. 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
vaporized in vacuum by a short laser pulse and the analyte is transported
unfragmented into the gas phase. An applied voltage accelerates the ions in a
field-free flight tube. Based on their different masses, ions are accelerated
to
differing degrees. Smaller ions reach the detector earlier than larger ions.
The
time-of-flight is converted into the mass of the ions. Technical innovations
of
hardware have significantly improved the method. Delayed extraction (DE) is
worthy of mention. For DE, the acceleration voltage is turned on with a delay
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CA 02379163 2002-O1-28
relative to the laser pulse and in this way an improved resolution of the
signals is
achieved, since the number of collisions is reduced.
MALDI is excellently suitable for the analysis of peptides and proteins.
For nucleic acids, the sensitivity is approximately 100 times poorer than for
peptides and decreases overproportionally with increasing fragment size. The
reason for this lies in the fact that only a single proton must be captured
for the
ionization of peptides and proteins. For nucleic acids, which have a backbone
with multiple negative charges, the ionization process is basically
inefficient due
to the matrix. For MALDI, the selection of the matrix plays an extremely
important role. Several very high-performance matrices have been found for the
desorption of peptides, which produce a very fine crystallization. In fact,
several
high-performance matrices have in the meantime been found for DNA, but the
difference in sensitivity was not reduced in this way. The difference in
sensitivity
can be reduced by modifying DNA chemically in such a way that it is similar to
a
peptide. Phosphorothioate nucleic acids, in which the usual phosphates of the
backbone are substituted by thiophosphates, can be converted into charge-
neutral DNA by simple alkylation chemistry.
The coupling of a "charge tag" to this modified DNA results in an increase
in sensitivity to the same range as is found for peptides. The possibility of
utilizing matrices that are similar to those used for the desorption of
peptides is
offered by these modifications. Another advantage of charge tagging is the
increased stability of the analysis against contaminations that greatly hinder
the
detection of unmodified substrates. PNAs and methylphosphonate
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CA 02379163 2002-O1-28
oligonucleotides have been investigated with MALDI and thus can be analyzed in
this way.
An array with many thousand target DNAs can be immobilized on a solid
phase and then all target DNAs can be investigated jointly for the presence of
a
sequence by means of a probe (nucleic acid with complementary sequence).
A correspondence of the target DNA with the probe is obtained by a
hybridization of the two segments with one another. Probes can be any nucleic
acid sequences of any length. Different methods exist for the selection of
optimal
libraries of probe sequences, which minimally overlap with one another.
Probe sequences can also be combined in a targeted manner in order find
specified target DNA sequences. Oligofingerprinting is one approach, in which
this technology is applied. A library of target DNAs is scanned with short
nucleic
acid probes. For the most part, the probes here are only 8-12 bases long. Each
probe is hybridized once to a target DNA library that has been immobilized on
a
nylon membrane. The probe is radioactively labeled and the hybridization is
evaluated on the basis of localizing the radioactivity.
The utilization of oligomer probe libraries for identifying immobilized
nucleic acids by means of mass spectrometry has been described (EP 97 12
1471.3 and EP 97 12 1470.5). Of course, no oligomer arrays are used in this
method.
Probes with multiple fluorescent labels have been used for the scanning of
an immobilized DNA array. The simple introduction of Cy3 and Cy5 dyes to the
5'0H of the respective probe has been particularly suitable for fluorescence
8
CA 02379163 2002-O1-28
labeling. The detection of fluorescence of the hybridized probes is made, for
example, by means of a confocal microscope. The dyes Cy3 and CyS, in
addition to many others, are commercially available.
The object of the present invention is to make available a method, which
overcomes the disadvantages of the prior art.
According to the invention, a method is created for characterizing a
nucleic acid fragment, whereby the following method steps are conducted:
a) a nucleic acid fragment to be characterized is immobilized on a surface;
b) an array of oligomers is prepared on a second surface, wherein the
oligomers are provided with a label;
c) the synthesized oligomers are stripped from the surface without leaving
a pregiven region on the surface;
d) the surface on which the nucleic acid to be characterized is immobilized
is contacted with the oligomer array surface, whereby complementary oligomers
of the array hybridize to the DNA to be characterized;
e) non-complementary oligomers are removed;
f) the complementary oligomers are detected by means of their label,
whereby sequence information is determined on the basis of the site on the
surface.
It is preferred according to the invention that the nucleic acid fragment to
be characterized is an amplified product of genomic DNA.
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CA 02379163 2002-O1-28
A particularly preferred form of embodiment of the method of the invention
provides that the genomic DNA is reacted with a solution of a bisulfite,
disulfite or
hydrogen sulfite prior to the amplification.
It is further preferred that the nucieic acid fragment to be characterized is
covalently bound to the surface. It is also preferred that an amino function
is
introduced into the nucleic acid fragment to be characterized and this
function
binds to a glass surface that has been derivatized by silanizing.
The method wherein the oligomers of the array are bound covalently to
the second surface is also preferred according to the invention. It is also
preferred that an amino function is introduced into the oligomers of the array
and
this function binds to a glass surface derivatized by silanizing.
It is also preferred that the oligomer array is produced by solid-phase
synthesis of the oligomers on the second surface.
It is particularly preferred that the solid-phase synthesis of the
oligonucleotides on the second surface is conducted in a closed synthesis
chamber, in which the synthesis reagents are introduced selectively. It is
particularly preferred here that the synthesis of the oligomers is conducted
by
selective introduction of the synthesis reagents at the respective sites at
which
the oligomers are synthesized.
It is further preferred according to the invention that photolithographic
methods and photolabile protective groups are used for the oligomer synthesis.
In addition, it is also preferred that electronically controllable and/or
changeable masks are used for the photolithographic process. Here, it is
CA 02379163 2002-O1-28
particularly preferred that a minor array that can be selectively turned on is
used
for producing an exposure pattern for the photolithographic process.
It is further preferred according to the invention that the synthesis of
oligomers is conducted in an array of cavities, which are also used, if
needed, as
chambers for the hybridization.
A preferred variant of the method according to the invention is also that
the nucleic acid fragments to be characterized are immobilized on an array of
cavities, which are used also, if needed, as chambers for the hybridization.
It is preferred that chemical groups that effect a change in mass andlor
fluorescence are used as the labels of the oligomers. In particular, It is
preferred
in the method of the invention'that the hybridized oligomers are detected by
means of mass spectrometry, preferably by means of matrix-assisted laser
desorptionlionization mass spectrometry (MALDI).
Another subject of the present invention is a kit for conducting the method
according to one of the preceding claims, comprising reagents andlor reference
nucleic acid fragments andlor reference DNA andlor treated surfaces andlor
photolithographic masks andlor oligomers.
The present invention thus describes a method for characterizing nucleic
acids. In a particularly preferred variant, it serves for identifying cytosine
methylation patterns in amplified products of genomic DNA.
The invention thus concerns a method for the characterizing of nucleic
acids. For this purpose, the nucleic acid is immobilized on a surface. An
array of
labeled oligomers, preferably oligonucleotides, is prepared on a second
surface
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CA 02379163 2002-O1-28
by means of solid-phase synthesis, and then the oligomers are cleaved from the
surface, without the oligomers leaving a pregiven region on the second
surface.
The two surfaces are contacted after the addition of a small quantity of a
buffer
solution and the synthesized oligomers hybridize at those sites at which their
synthesis also has occurred, to the nucleic acids immobilized on the first
surface.
At those sites at which a hybridization has occurred, the oligomers can
subsequently be recorded by means of their label. The pattern of hybridized
oligomers, which is obtained in this way, is used for determining sequence
information in the nucleic acid to be characterized. In a particularly
preferred
variant, the method serves for identifying cytosine methylation patterns in
genomic DNA. For this purpose, the latter is treated and amplified with a
bisulfate
solution before it is introduced onto the first surface.
For the characterizing of an amplified genomic DNA sample, oligomer
arrays are used, in which the DNA sample is fluorescently labeled and is
hybridized to such an array of immobilized oligomers (prior art). It is also
possible to prepare an array of amplified products and to hybridize probes
thereto. However, only a few probes can be used simultaneously for this
purpose, since they can differ only in a limited manner, due to, e.g., their
fluorescence. A patent that deals with this problem by means of mass
spectrometry is mentioned in the prior art.
This invention describes the inverse variant, of immobilizing the DNA to be
identified on the surface, and nevertheless to utilize the advantages of
oligomer
array technology. It offers numerous advantages. First of all, the
hybridization
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CA 02379163 2002-O1-28
can be conducted substantially more rapidly, since in the variant described
here,
short oligonucleotides can diffuse over an extremely short distance to the
immobilized target DNA and these can also be present in a large excess.
Secondly, the chip with the costly immobilized target DNA can be reused as
frequently as necessary for its complete characterization after the removal of
the
oligomers. It is also in many cases simpler to introduce labels on the
oligomers
rather than on the amplified DNA which is present frequently in substantialy
smaller quantities. This is true particularly if detection is produced by
means of
mass spectrometry, since in this case, the oligomers can be detected directly.
This variant also permits the use of oligomer libraries, whose components can
all
be distinguished by means of their mass.
Another substantial advantage of the present invention is that the
oligomers no longer necessarily hybridize to the target DNA in the direct
vicinity
of the surface, as is otherwise necessarily the case in arrays of immobilized
oligomers. This frequently causes problems, particularly with respect to
hybridizing efficiency. However, the general advantages of oligomer-array
synthesis, as described in the prior art, remain in the case of the present
invention.
The method according to the invention for characterizing nucleic acids is
comprised of the following steps:
1. A nucleic acid to be characterized is immobilized on a surface.
2. An array of oligomers, which are provided with a detectable label, is
prepared on a second surface.
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CA 02379163 2002-O1-28
3. The synthesized oligomers are stripped from the surface, without
leaving a pregiven region on the surface.
4. The surface on which the nucleic acid to be characterized has been
immobilized is contacted with the oligomer-array surface.
5. Complementary oligomers of the array hybridize to the DNA to be
characterized and non-complementary oligomers are removed by washing steps.
6. The complementary oligomers are detected by means of their label and
sequence information is determined on the basis of their site on the surface.
The nucleic acid to be characterized can be genomic DNA, preferably
amplified fragments of a genomic DNA sample, or also RNA. In a particularly
preferred variant of the method, the genomic DNA sample can be pretreated with
a bisulfite solution in order to effect a conversion of cytosine to uracil,
while
methylcytosine is not converted under these conditions. It is possible by
means
of the preceding step to use the method for the identification of methylation
patterns in genomic DNA. The genomic DNA sample is preferably amplified in
order to improve the sensitivity of the method. This can be produced in a
particular preferred manner by means of PCR.
In the first step of the method, the DNA to be characterized is now
immobilized on a surface. This surface may be comprised of glass, quartz
glass,
or also, for example, silicon. In a preferred variant of the method, the
surface is
chemically activated, so that one or more covalent bonds of the nucleic acid
to be
characterized can be created with the functionalized surface. This activation
of
the surface is preferably conducted by silanization. First of all, an
activation with
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CA 02379163 2002-O1-28
epoxy functions is considered, to which the DNA to be characterized either
binds
directly or, however, preferably by means of a primary amino function
introduced
in the PCR by means of a primer. Secondly, the surface can be amino-
functionalized by means of silanization. The DNA to be characterized may then
be coupled with this amino function by means of a linker molecule. The DNA to
be characterized in this case preferably bears amino or mercapto functions
preferably added in the PCR. For example, SIAB, DMS or PITC are used as
bifunctional linker molecules.
With respect to the pretreatment of the surface and the linker procedure,
the same holds true for the second step of the method, the preparation of the
oligomer arrays on the second surface. The oligomer arrays are efficiently
produced, as in the prior art, but with the difference that they can be
cleaved
again from the solid phase. This cleavage can be produced either
photochemically or by the action of an acid or base, preferably a base. The
surface can be derivatized for the production of the oligomer arrays both with
primary alcohol or amino functions. The primary alcohol functions are
introduced
according to the prior art by means of epoxidation of the surface and
subsequent
reaction with an oligoethylene glycol, but are preferably introduced by the
amino
functionalization of glass surfaces that has been recently proposed by Beier
et al.
(Nucleic Acids Research, 1999, pp. 1970-77). In order to be able to conduct a
photochemical cleavage, a photocleavable linker must first be preferably
introduced after the amino or OH functionalization. In another embodiment of
the
method, the modified protective group strategy proposed by Beier and
Pfleiderer
CA 02379163 2002-O1-28
can be applied for the cleavage of oligomers after synthesis, whereby the base
DBU is used for the cleavage of oligomers from the surface (DE-A 196 25 397).
In a particularly preferred variant of the method, the oligomer arrays are
synthesized in a closed synthesis chamber, into which synthesis reagents can
be
selectively introduced. The monomers are guided each time directly to the
respective sites of synthesis, preferably by a pin tool or by means of a piezo
pipetting robot, whereby the synthesis chamber is opened for this step.
Alternatively, synthesis can be conducted by means of photolithographic
methods, as also described in the prior art. By means of a mask, light is
conducted only to those regions in which a chain lengthening of the oligomer
is to
be produced by one specific monomer each time (see the prior art). The use of
dynamic masks, which can be changed by electronic control, is particularly
preferred. These masks can be either an LCD display, a microseal array or a
fiberoptics bundle that can be turned on. Also, light can be selectively
introduced
to points on the surface by means of arrays of tiltable micromirrors, as are
used
in modern projectors. Alternatively, viscoelastic micromirrors are used.
Photolithographic methods require the use of photolabile protective groups on
the monomers, whereas conventional protective groups can be used when the
monomers are introduced by means of, e.g., a pipetting system.
It is also possible to synthesize several different oligomers or oligomer
libraries to one or more sites on the second surface, as long as they can all
be
distinguished after hybridizing by means of the detection method used.
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The oligomer arrays may be comprised of oligonucleotides andlor PNAs
(peptide nucleic acids). Little is changed with respect to the principal
procedure
for synthesis, and the synthesis methods corresponding to the prior art are
applied each time. However, if libraries are used at one or more points of the
array, it is preferred to use PNA, since the resulting components of the
library
can be more easily distinguished by mass in this case. In the array synthesis,
the oligomers are provided with a labeling, which can be preferably either a
fluorescent dye or, however, a specific mass. The mass can be either that of
the
oligomer itself or, however, that of the oligomer plus a mass labeling in the
form
of a functional group.
In the third step of the process, the cleavage of the oligomers from the
surface is now conducted. Care is to be taken that the cleavage must not lead
to
a lateral movement of these oligomers on the surface, because the order of the
array would be disrupted. In order to avoid this, in a particularly preferred
variant
of the method, the synthesis of the oligomer array is already conducted in
such a
way that the synthesis of any one type of oligomer occurs in a cavity assigned
to
it. The oligomer does not leave this cavity even after its, e.g.,
photochemical
cleavage from surface.
Finally, in the fourth step of the method, the stripped and labeled
oligomers that are still present, however, in the original two-dimensional
arrangement are hybridized to the nucleic acids to be characterized. This is
done in a particularly preferred variant of the method by placing hybridizing
buffer
in the cavities of the second surface, in which the oligomers are found, and
then
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CA 02379163 2002-O1-28
the first surface with the nucleic acids to be characterized is contacted with
the
second surface. Alternatively, the first surface exclusively or both surfaces
can
also contain cavities, which are then filled each time with hybridizing
buffer. This
can be done, for example, preferably with a pipetting robot or a pin-tool
robot.
The grid of cavities preferably corresponds to the grid of oligomers in the
array
on the second surface.
After hybridizing the complementary oligomers to the nucleic acids to be
characterized, the two surfaces are separated from one another and the non-
complementary oligomers are removed from the first surface by washing steps.
In the sixth step of the method, the complementary oligomers are detected
by means of their labelings. It is known from the array synthesis, which
oligomers may have hybridized at which point on the first surface. The
labelings
are fluorescent dyes in a particularly preferred variant of the method and Cy3
or
Cy5 again are particularly preferred. Now, the fluorescence at each site on
the
surface can be detected in a fluorescence scanner and the identity of the
hybridized oligomers can be determined with the spatial information present.
Complete or partial sequence information on the DNA to be characterized can in
turn be obtained from this.
In a particularly preferred variant of the method, the identity of the
hybridized oligomers provides information on cytosine methylations in the DNA
to
be characterized, if the latter has been pretreated with a bisulfite solution
prior to
step 1 of this method.
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In another preferred variant of the method, the hybridized oligomers are
detected by means of mass spectrometry, preferably matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS). For this purpose, the
first
surface with the hybridized oligomers is first coated with a matrix and then
the
respective points are entered directly in the mass spectrometer. The software
of
the mass spectrometer permits the assignment of the mass spectra obtained to
the respective points on the surface. The hybridized oligomers are clearly
identified by means of their masses. In a preferred variant, the oligomers in
the
array synthesis are provided with mass labels. These involve functional
groups,
the objective of which is only to increase the mass of the oligomer by a
specific
amount, in order to reliably distinguish it from oligomers of another
sequence.
Thus several different oligomers or even oligomer libraries can be synthesized
even at one point on the second surface beforehand, and their masses will be
reliably distinguished at least one point of the array each time. In a
particularly
preferred variant of the method, the identity of the hybridized oligomers
again
provides information on cytosine methylations in the DNA to be characterized,
if
the latter has been treated with a bisulfite solution prior to step 1 of this
method.
In a particularly preferred variant of the method, this information is entered
into a
database and correlated with the phenotype belonging to the DNA to be
characterized.
The following examples explain the invention:
Example 1:
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Synthesis of DNA with 5'-DMT and photolabile protective groups
DNA oligomers are synthesized according to the established
phosphoramidite method, here on modified glass surfaces. Acid-labile DMT
groups or photocleavable groups, which are known in and of themselves, may be
used as 5' protective groups. For cleaving these groups, in the case of DMT, a
solution of trichloroacetic acid in dichloromethane is used, while in the case
of
the photolabile groups, light with a suitable wavelength, preferably between
320
and 365 nm, is used. The protective groups on the exocyclic amines and the
phosphates are cleaved according to standard procedures with concentrated
ammonia.
Example 2:
Synthesis of PNA oligomer arrays
PNA oligomers are synthesized (as an oligomer array) by spotting the
individual components onto the chip. After spotting a monomer or a reagent,
the
chip is washed with dimethylformamide, in order to remove unreacted chemicals.
Thus, e.g., the PNA sequence 5'-AGC CAG CTC ACT ACC TAG-3' is
constructed from the C-terminal (3') up to the N-terminal (5'). Synthesis is
produced by binding the acid function of the N-protected monomer G (Fmoc, 5
equivalents) in a DMF solution together with 5 equivalents of D1PEA, 7.5
equivalents of lutidine and 4.5 equivalents of HATU, onto the modified glass
surface. The 5'-amino groups of monomers A, C, G and T are protected with an
Fmoc group, and the exocyclic amino groups on monomers A, C and G are
CA 02379163 2002-O1-28
protected with a Bhoc group. PNA monomers and chemicals can be obtained
commercially (e.g. Perkin Elmer).
Then, washing is conducted with DMF, unreacted NH2 groups are capped
with a mixture of acetanhydridellutidine in DMF, washed with DMF, and the
protected 5'-amino function is deprotected with 20°~ piperidine in DMF.
After
washing with DMF, the A monomer is spotted onto the bound G monomer. The
complete PNA sequence is synthesized by the subsequent synthesis cycles.
The Bhoc groups of the exocyclic amino groups of bases A, C and G are
removed at the end of the synthesis with trifluoroacetic acid in the presence
of m-
cresol.
Example 3:
Synthesis of DNA on chips, which bear a photocleavable linker
in a first step, the glass substrates are chemically modified, so that a
targeted binding of oligonucleotide building blocks can occur, as in the prior
art.
By introducing various types of oligonucleotides onto a substrate,
oligonucleotide
arrays can be produced in the usual manner. For this purpose, the substrates
are silanized, whereby the silane bears a functionalized (for example OH- or
NH2) alkyl chain. Then the surface is provided with a photolabile linker, for
example, 4[4-1-(Fmoc-aminomethyl)-2-methoxy-5-nitrophenoxybutanoic acid
(Novabiochem). This linker permits a cleavage of the oligonucleotides upon
irradiation of light of 365-nm wavelength. The oligonucleotides, for example,
the
sequence 5'-AGC CAG CTC ACT ACC TAG-3', are synthesized as was
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described in Example 1 for DMT on the deprotected amino function of the
linker.
Protective groups of the exocyclic amines, for example, benzoyl or isobutyryl,
are
removed with concentrated ammonia. The DNA is cleaved photochemically from
the entire chip and is then washed from the surface.
Example 4:
Synthesis of PNA on chips, which bear a photocleavable linker
The modification of the surface of the glass substrate is conducted as
described in Example 3 with a spotted photolabile linker. The PNA sequence,
for
example, 5'-AGC CAG CTC ACT ACC TAG-3', is synthesized by spotting the
components as described in Example 2 onto the deprotected amino function of
the photolabile linker. The Bhoc protective groups are removed with
trifluoroacetic acid and the synthesized PNA strand is cleaved with light of
365-
nm wavelength.
The PNA synthesis on photocleavable linkers may also be conducted over
a large surface on the entire chip, whereby the components are rinsed over the
chip. Deprotection is carried out as described.
Example 5:
Synthesis of DNA on chips, which bear a non-photocleavable linker
In the first step, the glass substrates are modified chemically, so that a
targeted binding of oligonucleotide building blocks, which is known in and of
itself, can be conducted. For this purpose, the substrates are silanized,
whereby
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the silane bears a functionalized (for example, OH- or NH2-) alkyl chain. The
non-photocleavable linker is synthesized by coupling 5'-protected T, A, C or G
monomers with succinic anhydride and dimethylaminopyridine. For example,
conventional DMT groups are used as protective groups on the monomers for a
large-surface DNA synthesis or photolabile groups are used in the case of a
DNA
array. The pretreated monomer is coupled to the substrate and the synthesis,
for
example, of the sequence 5'-AGC CAG CTC ACT ACC TAG-3', is conducted as
described in Example 1. The 5'-protective groups are cleaved either with
trichloroacetic acid or by irradiation of light (365 nm). The DNA is dissolved
from
the substrate by the action of ammonia vapors and the array is transferred to
a
nitrocellulose membrane by blotting.
Example 6:
Blotting of DNA oligomer arrays
In order to be able to hybridize the oligomer arrays of Example 5 which
were cleaved from the modified glass surface to the sample DNA, the arrays are
blotted onto nitrocellulose (Amersham Pharmacia Biotech, Hybond series,
nitrocellulose on nylon or PVDF), analogously to the established method of
"Southern blots". The arrangement of the oligomers in the array is not
substantially changed, as long as the distance between the points in the array
is
sufficient. For this purpose, the glass carrier or slide is placed by its
loaded side
onto a nitrocellulose substrate, which has been premoistened with a
hybridizing
buffer (SSC + SDS, Denhardt's reagent). By exercising pressure, there is a
transfer of the oligomers to the nitrocellulose. The sample DNA, with which
the
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oligomers then specifically hybridize, is already immobilized on the
nitrocellulose.
Non-hybridized oligomers are washed away. Detection is effected by means of a
fluorescent label introduced on the oligomers (see Example 9).
Example 7:
Synthesis of PNA on chips, which bear a non-photocleavable linker
The substrate surface is silanized, whereby the silane bears a
functionalized (for example, NH2- or glycidoalkyl) alkyl chain. Then the amine
is
coupled with a carboxylic acid of a non-photocleavable tinker, for example, a
protected hydroxyalkyl carboxylic acid or epoxide under acidic conditions with
ethylene glycols of different chain lengths. The PNA synthesis, for example,
of
the sequence 5'-AGC CAG CTC ACT ACC TAG-3' is conducted as described in
Example 2. The coupling of monomer G with the terminal alcohol leads to the
formation of an ester. The PNA synthesis may also be conducted with a
photocleavable protective group on the amino group. Cleavage is produced with
light of 365-nm wavelength instead of with 20% piperidine in DMF. The PNA is
stripped from the substrate by the action of ammonia vapors and is contacted
with the sample DNA by blotting onto nitrocellulose.
Example 8:
Blotting of PNA oligomer arrays
The method for identifying PNA oligomers is similar to that for DNA
identification; a PNA hybridizing buffer is used. This method is analogous to
the
established methods of "Western blots" for proteins.
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Example 9:
Coupling of fluorescent dye to PNA oligomers
In order to couple a fluorescent dye, preferably Cy3 or CyS, to PNA
oligomers, the N-terminal (the 5'-amino group) is reacted with the N-
hydroxysuccinimide derivative of the dye with the formation of an amide. For
this
purpose, the dye derivative is dissolved in DMF or DMSO and contacted with the
immobilized oligomers in the presence of triethylammonium hydrogen carbonate
buffer. After 2 h, the reaction is terminated, and the excess or hydrolyzed
dye is
removed by washing steps with water and methanol.
Example 10:
Immobilizing of PCR products onto modified glass surfaces
In order to be able to immobilize PCR products onto glass substrates, both
the PCR products as well as the glass surface are modified. An aminopropyl
derivative, which supplies a free amino group, is coupled to the 3' end of the
PCR product. The glass surface is derivatized with 3'-glycidoxyalkyl
trimethoxysilane and catalytic quantities of diisopropylethylamine and bears
an
epoxide on the surface. The immobilization is conducted by spotting a solution
of
PCR products in 0.1 M potassium hydroxide and incubation for 6 hours at
37°C
and 100°r6 air humidity. Then unbound PCR products are removed by
washing
with water.
Example 11:
Immobilizing the sample DNA on nitrocellulose or PVDF membranes
CA 02379163 2002-O1-28
Sample DNA is immobilized on nitrocellulose or PVDF membranes
according to the instructions of the manufacturer. Particularly suitable are
membranes which have already been introduced onto a glass surface in a
microscope slide format (e.g., of Schleicher and Schull) and thus can be
simply
analyzed in conventional fluorescence scanners after the hybridization. After
immobilizing, the membranes are moistened with hybridizing buffer.
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