U.S. patent application number 10/384148 was filed with the patent office on 2003-09-18 for method for controlling quality in the construction of oligomer arrays.
Invention is credited to Beier, Markus.
Application Number | 20030175781 10/384148 |
Document ID | / |
Family ID | 7881003 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175781 |
Kind Code |
A1 |
Beier, Markus |
September 18, 2003 |
Method for controlling quality in the construction of oligomer
arrays
Abstract
The present invention relates to a method for conrolling the
quality of oligomer assays, which is characterized in that a
phosphate unit is fused to certain array positions, said phosphate
unit being linked to a signal-generating reporter group, the degree
of oligomer synthesis is determined using the signal of the
reporter group, and the reporter group is then split off again.
Inventors: |
Beier, Markus; (Heidelberg,
DE) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Family ID: |
7881003 |
Appl. No.: |
10/384148 |
Filed: |
March 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10384148 |
Mar 6, 2003 |
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09787226 |
Jun 22, 2001 |
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6582917 |
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09787226 |
Jun 22, 2001 |
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PCT/DE99/02975 |
Sep 15, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 536/23.1 |
Current CPC
Class: |
B01J 2219/00637
20130101; B01J 2219/00722 20130101; B01J 19/0046 20130101; B01J
2219/00626 20130101; C12Q 1/6837 20130101; B01J 2219/0072 20130101;
C40B 40/06 20130101; B01J 2219/00605 20130101; B01J 2219/00659
20130101; C40B 80/00 20130101; B01J 2219/00693 20130101 |
Class at
Publication: |
435/6 ;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 15, 1998 |
DE |
DE 198 42 164.8 |
Claims
What is claimed is:
1. A cleavable label for controlling the quality of oligomer arrays
comprises a phosphate unit and a signal-generating reporter group,
wherein the signal generating reporting group is covalently bound
to the phosphate unit and is cleavable.
2. The cleavable label according to claim 1, wherein the
signal-generating reporter group is a fluorescent group.
3. The cleavable label according to claim 1, wherein the
signal-generating reporter group is covalently bound to the
phosphate via a linker.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/787,226, filed Jun. 22, 2001; which is a National Stage of
International Application PCT/DE99/02975, filed Sep. 15, 1999;
which claims the priority of DE 198 42 164.8, filed Sep. 15,
1998.
BACKGROUND OF THE INVENTION
[0002] The invention relates to a method for controlling the
quality in the construction of oligomer arrays.
[0003] For diagnostic screenings microchips are coated with
oligomers in the form of arrays (oligomer chips/biochips). A sample
can then be screened therewith for a suitable molecule, i.e. a
molecule hybridizing therewith. Such oligomer arrays on a chip may
comprise nucleic acid oligonucleotides, such as DNA, RNA or nucleic
acid biopolymers, or analog compounds thereto, which are applied to
a solid phase.
[0004] The oligomers are not always fixed quantitatively, so that
it is not always possible to obtain chips having the same coating
degree. Following the construction of such oligomer arrays it must
therefore be checked whether the synthesis was successful and/or
the degree of successful synthesis must be determined. For this
purpose, phosphate reagents provided with a permanent (fluorescent)
label have been used thus far. Since these labels could not be
split off again, they optionally impair the subsequent use of the
biochips.
[0005] A defined quality assay for the construction of oligomer
arrays on a chip surface which does not trigger any disturbing
side-effects is thus not known as yet. However, a quality control
is inevitable in the developing field of biochip technology to
ensure a reproducible production of constant quality.
BRIEF SUMMARY OF THE INVENTION
[0006] It is the object of this invention to provide a method for
controlling the quality of oligomer arrays. It shall be possible to
use this method universally and carry it out rapidly and with
little effort. In addition, the quality control shall not impair
the subsequent use as a biochip. The method shall be suitable for
all kinds of oligomer arrays.
[0007] This object is achieved by a method according to claim 1.
Advantageous embodiments follow from the subclaims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows the production of phosphoramidite
(2-cyanoethyl-2-dansylethyl-N,N-diisopropyl)phosphoramidite).
[0009] FIG. 2 shows the coupling of the phosphoramidite to the
oligomer.
[0010] FIG. 3 shows the carrying out of the quality control.
[0011] FIG. 4 shows the splitting-off of the reporter group.
[0012] FIG. 5 shows the scheme of the method according to the
invention (1-step method).
[0013] FIG. 6 shows the scheme of the method according to the
invention (2-step method).
DETAILED DESCRIPTION OF THE INVENTION
[0014] It has been found surprisingly that a quality control
without impairing the further use of the chip can be carried out
when a phosphate unit is fused to the oligomer grid on the chip,
which is reversibly provided, or connected, with a
signal-generating reporter group. By the detection of the signal
from the reporter group attached to the oligomers at the grid
positions it is possible to control the success of the synthesis or
to compare different grid positions with one another. Having
concluded the quality control, the reporter group is removed
again.
[0015] Thus, the invention relates to a method for controlling the
quality of oligomer arrays, which is characterized in that a
phosphate unit linked to a signal-generating reporter group is
fused to oligonucleotides at certain grid positions (preferably to
all or a previously selected number), the degree of oligomer
synthesis is determined by means of the signal of the reporter
group, and the reporter group is then split off again. A general
scheme is shown in FIGS. 5 and 6.
[0016] The extent of the synthesis success can be controlled by
detecting the reporter group signals emitted from the occupied grid
positions. Having concluded the quality control, the reporter group
can be split off again and in this way that does not cause any
disturbing effects in the subsequent experiments. Only the
phosphate group remains attached to the oligomers. However, it does
not disturb the subsequent use, e.g. in a hybridization, but
optionally increases advantageously the melting temperature of the
oligomer bound to the solid phase.
[0017] According to the invention, a phosphate unit is understood
to mean a unit fused using methods of the phosphoramidite
chemistry. The phosphate units may also be produced by methods
known from the phosphomonoester, phosphodiester or H-phosphonate
chemistry.
[0018] The invention is described below by means of phosphoramidite
as a basis of the phosphate group, which is preferred according to
the invention. However, this should not be interpreted as a
limitation. According to the invention the phosphate unit is
produced by reaction of the oligomer already disposed at the solid
phase at its 5' or 3' end with the phosphoramidite after the
oxidation. Here, the phosphoramidite is reacted using an acidic
catalyst (e.g. tetrazole, tetrazole derivatives, pyridine
hydrochloride) with the oligomer strand disposed at the solid
phase. Subsequent oxidation, e.g. using iodine,
tert.-butylhydroperoxide, etc., results in a stable phosphorus(V)
compound (=phosphate unit). It proves to be advantageous that it is
possible to use in this case the steps common for the synthesis of
oligonucleotides, which can be automated without any problems on a
commercially available DNA/RNA synthesizer. No modifications of the
commonly used reagents or synthesis protocols are necessary for
this purpose.
[0019] The signal-generating reporter group may be any
signal-generating molecule which can be coupled to a
phosphoramidite via a corresponding linker. The linker takes care
that after the reaction of the phosphoramidite with the oligomer on
the solid phase and after the detection of the reporter group, the
reporter group can be split off. Signal-generating reporter groups
may be any fluorescent, coloring, radioactive, chemoluminescent
compounds. Fluorescent compounds are preferred, such as dansyl
ethanol, fluorescein or pyrene. Examples of further reporter groups
are derivatives of Cy3, Cy5, dabsyl chloride, TAMRA,
hexachlorofluorescein, suitably derivatized for coupling to the
phosphate group. According to the invention the reporter group may
already be linked to the phosphate unit when the latter is bound to
the oligomer (1-step process, see FIGS. 1 and 5) or the linker may
be bound by means of a phosphate unit to the oligomer before the
reporter group is attached using another phosphate unit (2-step
process, see FIG. 6).
[0020] The 1-step process is explained in detail in Example 1. It
is characteristic of the 1-step process that the reporter molecule
(e.g. fluorescence tag) and the cleavable linker form parts of a
single chemical molecule (e.g.
2-cyanoethyl-2-danyslethyl-N,N-diisopropyl phosphoramidite). It is
fused to the chip during the last step of the oligonucleotide
synthesis. Following the oxidation to form a stable phosphodiester,
the chip is checked using the detection of the reporter group for
its quality. Thereafter, the reporter group is removed e.g. by base
treatment. The phosphate protecting groups may be removed
preferably, but not necessarily, in the same step or later. A
phosphate unit is left as an attachment at one end of the
respective oligomer strand. The array can then be used in standard
experiments (e.g. in hybridizations).
[0021] In the 1-step process, the linker contains a unit which can
be split off by an acid, base or by light (hereinafter: X), e.g.
sulfonylethyl, 2-(2,2-dicarboxyethyl)propyl,
2-(2-nitrophenyl)propyl, 2-(2-nitrophenyl)ethyl. Furthermore, the
linker contains a hydroxy or amino function (hereinafter: H) for
the attachment of the signal-generating reporter group. The linker
also contains a spacer (hereinafter: M) which spatially separates
the cleavable unit (X) from the amino or hydroxyl function (H). The
linker (L) may therefore be represented by the following general
formula:
L=H-M-X-
[0022] wherein
[0023] X=cleavable unit
[0024] H=hydroxyl or amino
[0025] M=alkyl, aryl, etc.
[0026] Prior to the condensation to the oligomer array, a reagent
is formed in which the linker is connected with a phosphite amide
unit and with the reporter group. The reporter group is linked by
means of suitable reporter derivatives. Linkages to the hydroxy or
amino function (H) via an ester or amide or sulfonamide bridge are
preferred, since commercially suitable derivatized compounds (e.g.
carboxylic acids, sulfonyl chlorides) of almost all known reporter
groups are available for this. The reagent has the following
general formula: 1
[0027] L=cleavable linker unit
[0028] R.sup.1=phosphate protecting group, e.g. -cyanoethyl,
2-(4-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl
[0029] R.sup.2=isopropyl, ethyl, methyl
[0030] R.sup.3=isopropyl, ethyl, methyl
[0031] Reporter=signal-generating reporter unit
[0032] In the 1-step process, a linker system of the
2-(4-aminophenylsulfonyl)ethyl type is preferred, other cleavable
linkers with which a person skilled in the art is familiar being
also usable. The use of 2-(4-aminophenylsulfonyl)ethanol permits by
reaction of the amino function with signal-generating reporter
groups which carry a sulfonyl or carboxyl group access to almost
all known signal-generating reporter groups.
[0033] In the 2-step process (see FIG. 6), the reporter molecule
(e.g. fluorescence tag) and the cleavable linker (e.g.
sulfonylethyl linker) are parts of two different chemical
molecules.
[0034] It is preferred to use phosphate units having cleavable
units as linkers in the 2-step process. The linkers (hereinafter:
L) preferably have a protected hydroxyl or amino function
(hereinafter: H) for the attachment of the next phosphate unit.
Suitable hydroxyl or amino protecting groups (hereinafter: G) are
known to the person skilled in the art and are e.g. acid-labile
(e.g. dimethoxytrityl, monomethoxytrityl), base-labile (e.g. Fmoc)
or photo-labile (e.g. MeNPOC, NPPOC). The linker also contains a
unit cleavable by an acid, base or by light (hereinafter: X), e.g.
sulfonylethyl, 2-(2,2-dicarboxyethyl)propyl,
2-(2-nitrophenyl)propyl, 2-(2-nitrophenyl)ethyl). The linker also
contains a spacer (hereinafter: M) which spatially separates the
cleavable unit (X) from the amino or hydroxyl function (H).
Therefore, linker (L) can be represented by the following general
formula:
L=G-H-M-X-
[0035] wherein
[0036] X=cleavable unit
[0037] H=hydroxyl or amino
[0038] G=protecting group for H
[0039] M=alkyl, aryl, etc.
[0040] The linker is connected to a phosphite amide unit. The
resulting linker phosphoramidite reagent is then fused to the
oligomer array as described above. The linker phosphoramidite
reagent comprises preferably the following general formula: 2
[0041] L=protected cleavable linker (see above)
[0042] R.sup.1=phosphate protecting group, e.g. (.beta.-cyanoethyl,
2-(4-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl
[0043] R.sup.2=isopropyl, ethyl, methyl
[0044] R.sup.3=isopropyl, ethyl, methyl
[0045] Preferred linker-phosphoramidite compounds in the 2-step
process are:
[0046]
2-[2-(4,4'-dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N-
,N-diisopropyl)phosphoramidite (Eurogentec, Liege, Belgium) 3
[0047]
[3-(4,4'-dimethoxytrityloxy)-2,2-dicarboxyethyl]propyl-(2-cyanoethy-
l)-N,N-diisopropyl)phosphoramidite 4
[0048] A preferred embodiment of the 2-step process is described in
FIG. 6. The linker-phosphoramidite reagent (e.g.
2-[2-(4,4'-dimethoxytrityloxy-
)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite)
is first fused to the oligomer array. Following oxidation to
produce a stable phosphate bond (and possible capping to prevent
uncontrolled chain extensions) and removal of the employed terminal
protecting group (e.g. the dimethoxytrityl group by acid
treatment), a reporter group (e.g. a Cy5 group derivatized with
phosphoramidite=Cy5-phosphoramidite; Pharmacia company) is fused to
the cleavable linker unit. Following oxidation to form a stable
phosphodiester bond, the array is scanned (e.g. by fluorescence
scanning) to identify the previously attached reporter group.
Splitting off the linker (e.g. by base treatment in the case of the
sulfonylethyl linker) the reporter group is removed in the same
step. The phosphate protecting groups may preferably, but not
necessarily, be removed in the same step or later. A phosphate unit
is left as an attachment at one end of the respective oligomer
strand. The array can then be used in standard experiments (e.g. in
hybridizations).
[0049] The method can be applied to any oligomer arrays of
oligonucleotide/nucleic acid biopolymers, e.g. from DNA, RNA and/or
analogs thereof. Analogs are understood to mean, e.g. phosphorus
thioates, PNA, modified DNA or RNA nucleic acids (e.g. 2'-O-methyl)
or also other nucleic acid types having a modified backbone or
sugar (e.g. pRNA, homo-DNA, alpha-para-NA) or the chimeras thereof.
The reporter group-labeled phosphoramidite can also be reacted with
the N-terminal end of peptides/proteins and be used as described
above.
[0050] The quality is controlled e.g. in a fluorescent reporter
group by evaluating the solid carrier on which the labeled oligomer
grid is disposed using a reader suitable for this dye (e.g.
fluorescence microscope, fluorescence scanner). For this purpose,
the reader determines for all oligomer positions a relative
intensity value which corresponds to the relative amount of
synthesized oligomer at this position. A comparison of the
determined relative intensities with respect to one another permits
a quantitative comparison of the respective occupation with
oligomers at all sites of the carrier and/or furnishes information
on the quality of the synthesized oligomer grid. This comparison of
the relative intensities at the various positions of the grid
permits as a sequential step (evaluation of a hybridization
experiment of the oligomer chip with probes) a safe comparison of
the intensities which result in this case. For example, when during
the quality check for a certain position the oligomer concentration
measured with respect to another is low, it is also only possible
as according to expectations to determine for this position an
intensity reduced relative to others when this chip is used in a
hybridization experiment.
[0051] Having concluded the quality control, the reporter group is
split off again. In the case of reporter groups which are bound via
a sulfonylethyl-like system to the phosphorus/phosphate groups,
this is effected by a treatment using a strong base, e.g. DBU,
ammonia, DBN, diisopropylethylamine.
[0052] The method can be automated and is therefore suitable for
mass screening or great chip charges.
[0053] The invention is explained in more detail by means of the
following examples.
EXAMPLES
Example 1
[0054] Quality Control of an Oligomer Chip
[0055] a) Production of
(2-cyanoethyl-2-dansylethyl-N,N-diisopropyl)phosph- oramidite
[0056] 4.83 g 2-dansylethanol (17.3 mmol) and 5.92 ml
diisopropylethylamine (34.6 mmol) are dissolved in 30 ml anhydrous
dichloromethane and cooled in an ice bath. Then, 4.5 g
chloro-(2-cyanoethoxy)diisopropylaminophosphane (19.0 mmol) are
slowly added. The ice bath was removed. Stirring was continued
until TLC showed complete reaction. The reaction mixture was
extracted with a saturated sodium bicarbonate solution and
dichloromethane, the organic phase was dried over sodium sulfate
and evaporated. The resulting oil was purified by means of flash
chromatography (50 g SiO2; toluene/ethyl acetate; 0-20% ethyl
acetate). 5.7 g of the product were obtained as a fluorescent oil.
The yield was 69%. The reaction scheme is shown in FIG. 1.
[0057] b) Coupling of the
(2-cyanoethyl-2-dansylethyl-N,N-diisopropyl)phos- phoramidite to
the oligomer
[0058] The coupling was effected automatically by means of a DNA
synthesizer. For this purpose, the fluorescence-labeled
phosphoramidite is dissolved in analogy to a "normal" nucleoside
building block (phosphoramidite) in dry acetonitrile (0.5 molar)
and is applied by condensation to the already existing oligomers in
the solid phase. This is done by means of tetrazole. What is called
a capping step (e.g. reagent acetic anhydride with acetylating
catalyst N-methylimidazole) usually takes place after the
condensation on the DNA synthesizer to prevent uncontrolled further
growth of the oligomer chain. This step is not necessary here but
is not an impediment either, since the oligomer chain growth has in
any case been concluded by the fluorescence-labeled
phosphoramidite. The subsequent oxidation step is important which
converts the unstable phosphorus(III) group into a stable
phosphorus(V) compound. For this purpose, an
iodine/pyridine/THF/water mixture is used. The DNA chip
fluorescence-labeled in this way is now immediately available for
the qualitative checking by a suitable array reader. The reaction
scheme is shown in FIG. 2.
[0059] c) Carrying out the quality control
[0060] The quality is controlled directly after step (b). Here,
fluorescence is stimulated by irradiation of the chip with a
wavelength suitable for the dye (dansyl: 350 nm). The emission of
the fluorescence (dansyl: 510 nm) is then read by a suitable reader
(fluorescence microscope or fluorescence scanner) for each position
of the chip and quantified (typically gray-scale values). For
example, if no fluorescence is detectable at a certain grid
position although an oligomer sequence should be constructed there,
it can be concluded that the synthesis was not successful at this
position. This chip should then be discarded. By comparing the
measured intensities the amount of the oligomers synthesized at
this position can be inferred. This may be considered for the use
of the oligomer chip in hybridization experiments. In this way, a
quality profile can then be prepared for each individual chip by
the relative fluorescence intensities of all grid positions (e.g.
by way of an excel table). This is a corresponding help for the
user to evaluate his hybridization experiments. The reaction scheme
is shown in FIG. 3.
[0061] d) Splitting-off of the dansyl compound
[0062] Having concluded the quality check, the dansyl compound can
be abstracted again by treatment with a strong base, e.g. DBU or
DBN. This is usefully done by rinsing the oligomer chip in the
base. In the case of the above-mentioned dansyl dye and using 1 M
DBU in acetonitrile, this result is already obtained after 1 to 2
min. at the latest. The -cyanoethyl phosphate protecting group is
split off simultaneously with the splitting-off of the dansyl
residue. The residues (styrene derivatives) of the dansyl groups
and the cyanoethyl groups remain in the basic solution and can
simply be washed away. The DNA chip is available for the aspired
use immediately after the splitting-off and washing step (e.g.
hybridization experiments). The phosphate grouping remaining at the
oligomer additionally increases advantageously the temperature at
which the hybridization experiments may take place. The reaction
scheme is shown in FIG. 4.
* * * * *