U.S. patent application number 15/641269 was filed with the patent office on 2017-12-28 for device and method for producing a replicate or derivative from an array of molecules, and applications thereof.
The applicant listed for this patent is Albert-Ludwigs-Universitaet Freiburg. Invention is credited to Jochen Hoffmann, Guenter Roth, Felix von Stetten, Roland ZENGERLE.
Application Number | 20170369938 15/641269 |
Document ID | / |
Family ID | 42145127 |
Filed Date | 2017-12-28 |
United States Patent
Application |
20170369938 |
Kind Code |
A1 |
ZENGERLE; Roland ; et
al. |
December 28, 2017 |
DEVICE AND METHOD FOR PRODUCING A REPLICATE OR DERIVATIVE FROM AN
ARRAY OF MOLECULES, AND APPLICATIONS THEREOF
Abstract
A method of producing a replicate or derivative of an array of
molecules, the array having a spatial arrangement of separate
samples of molecules, includes creating, for each sample, at least
one spatially limited effective area which is separate from the
effective areas of the other samples, a surface, provided with a
binding adapter or binding properties, of a carrier bordering on
the effective areas. The molecules are amplified by means of
amplifying agents in the effective areas for creating replicates or
derivatives of the samples. The replicates or derivatives of the
samples are bound to the carrier by means of the binding adapter or
the binding properties, so that a spatial arrangement of the
replicates or derivatives of the samples on the carrier corresponds
to the spatial arrangement of the samples in the array. The carrier
having the copies of the samples is removed from the array.
Inventors: |
ZENGERLE; Roland;
(Waldkirch, DE) ; von Stetten; Felix;
(Freiburg-Tiengen, DE) ; Roth; Guenter; (Freiburg,
DE) ; Hoffmann; Jochen; (Freiburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Albert-Ludwigs-Universitaet Freiburg |
Freiburg |
|
DE |
|
|
Family ID: |
42145127 |
Appl. No.: |
15/641269 |
Filed: |
July 4, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13224645 |
Sep 2, 2011 |
9725758 |
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15641269 |
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PCT/EP2010/052849 |
Mar 5, 2010 |
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13224645 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 19/0046 20130101;
C12Q 1/6837 20130101; C12Q 2535/00 20130101; C12Q 2561/113
20130101; C12Q 2535/122 20130101; C12Q 1/6837 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B01J 19/00 20060101 B01J019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 2009 |
DE |
102009012169.2 |
Claims
1. A method of producing a replicate or derivative of an array of
molecules, the array comprising a spatial arrangement of separate
samples of molecules, the method comprising: creating, for each
sample, at least one spatially limited effective area which is
separate from the effective areas of the other samples, a surface,
provided with a binding adapter or binding properties, of a carrier
bordering on the effective areas; amplifying the molecules by means
of amplifying agents in the effective areas for creating replicates
or derivatives of the samples; binding the replicates or
derivatives of the samples to the carrier by means of the binding
adapter or the binding properties, so that a spatial arrangement of
the copies or derivatives of the samples on the carrier corresponds
to the spatial arrangement of the samples in the array; and
removing the carrier comprising the replicates or derivatives of
the samples from the array.
2. The method as claimed in claim 1, wherein producing a spatially
limited effective area comprises producing a spatially limited
amplifying agent area, or wherein the spatially limited amplifying
agent areas are defined, at least in part, by micro- or
nanostructures within an array substrate of the array or within the
carrier, or wherein the micro- or nanostructures comprise an
unordered matrix based, in particular, on a filter membrane, on a
hydrogel or on an aerogel, or wherein the micro- or nanostructures
are based on an ordered three-dimensional substrate.
3. The method as claimed in claim 1, wherein producing at least one
spatially limited amplifying agent area for each sample comprises
providing the samples in mutually associated separately recesses
within the array substrate, introducing the amplifying agent into
the recesses, and closing off the recesses by the carrier, or
wherein the spatially limited amplifying agent areas are separated,
at least in part, by phase boundaries between two liquids, a liquid
and a gas, or a physical boundary, in particular a lipid membrane,
or wherein the spatially limited amplifying agent areas are
separated, at least in part, by phase boundaries between two
liquids, a liquid and a gas, or a physical boundary, in particular
a lipid membrane, and wherein producing at least one spatially
limited amplifying agent area for each sample comprises providing
the samples in mutually separated droplets of liquid which comprise
the amplifying agent and which are fixed, in the spatial
arrangement, on an array substrate of the array, a thinner-bodied
liquid being arranged between the droplets of liquid, and arranging
the carrier in relation to the array substrate such that the
surface, provided with the binding adapter, of the carrier borders
on the droplets of liquid.
4. The method as claimed in claim 2, wherein producing at least one
spatially limited amplifying agent area comprises providing the
carrier comprising at least one recess which is associated with
each sample and comprises the binding adapter arranged therein,
introducing the amplifying agent into the recesses, and closing off
the recesses by means of the array substrate, so that the samples
are exposed to the amplifying agent area.
5. The method as claimed in claim 1, wherein producing a spatially
limited amplifying agent area for each sample comprises providing
the sample within a sequencer chip or a nanowell plate.
6. The method as claimed in claim 1, wherein the process of binding
the replicates or derivatives to the carrier is performed
simultaneously with the amplification, or wherein the spatial
limitation of the effective area is that the binding adapters are
present on the carrier, as complementary primers, in the form of a
primer array that may comprise a regular or irregular distribution
of spots, the spot size and spot density on the carrier being equal
to or smaller than that on the array.
7. The method as claimed in claim 1, wherein the spatial limitation
of the effective area is effected by applying an energy field.
8. The method as claimed in claim 1, wherein the samples are
provided in the form of molecules bound to particles.
9. The method as claimed in claim 1, wherein the array of molecules
is a non-synthetic array of biomolecules, or wherein the molecules
are single- or double-stranded oligonucleotides, polynucleotides,
DNA or synthetic molecules analogous to DNA (PNA), or. wherein the
array comprises a sequencing process for deriving the genome, a
sequencing process for deriving the transcriptome, a process of
sequencing RNA, mRNA, tRNA, siRNA, or a process of sequencing
mutations and variations, or wherein by means of amplifying and
binding to the carrier, copies are created which correspond to a
DNA, a modified DNA, expressions of a DNA, an RNA, proteins or
peptides; or wherein the amplifying agent effects a DNA
amplification, in particular a polymerase chain reaction, an
isothermal amplification, or a NASBA reaction, and the binding
adapter comprises a matching primer, or wherein the method further
comprises monitoring any changes in physical or chemical parameters
within the effective areas
10. The method as claimed in claim 1, wherein the positions of the
samples comprise further molecules or DNA sequences or cells
located thereat which are part of the sample or are immobilized and
which are needed for generating derivatives, in particular
expression vector sequences such as ori, promoters, ribosome
binding sites, start codon, endoprotease cleaving sites, fusion
sequences, reporter genes, terminators, antibiotics resistance
genes, in-vitro translation systems, or cells.
11. The method as claimed in claim 1, wherein primary, secondary
and/or tertiary derivatives are generated, from a primary array or
a replicate of the primary array, in that DNA is transcribed into
RNA, the RNA is translated into protein, or in that a binder is
enriched while using a produced protein, a produced RNA or a
produced DNA or the copy thereof from a liquid phase, or in that a
binder interacts, or wherein a derivative is generated on the solid
phase of a target array and is present there in an immobilized
manner.
12. A utilization of a replicate or derivative that was produced
while using a method of producing a replicate or derivative of an
array of molecules, the array comprising a spatial arrangement of
separate samples of molecules, the method comprising: creating, for
each sample, at least one spatially limited effective area which is
separate from the effective areas of the other samples, a surface,
provided with a binding adapter or binding properties, of a carrier
bordering on the effective areas; amplifying the molecules by means
of amplifying agents in the effective areas for creating replicates
or derivatives of the samples; binding the replicates or
derivatives of the samples to the carrier by means of the binding
adapter or the binding properties, so that a spatial arrangement of
the copies or derivatives of the samples on the carrier corresponds
to the spatial arrangement of the samples in the array; and
removing the carrier comprising the replicates or derivatives of
the samples from the array, either for associating a reaction
between a binder, in particular a protein, antibody or antigen, and
an original molecule, its replicate or its derivative, with the DNA
sequence of the original molecule, in particular for
genotype-phenotype coupling, or for associating a reaction wherein
the original molecule, its copy or its derivative catalyzes the
conversion of a substrate, with the DNA sequence of the original
molecule, in particular for genotype-phenotype coupling, or for
identifying a DNA sequence, a RNA sequence, a protein or a
catalytic, signaling (e.g. enhancing, allosteric, inhibiting . . .
) or enzymatic (e.g. lytic, phosphatase activity, kinase activity .
. . ) function of a DNA, RNA or protein, or for identifying a DNA
sequence, a RNA sequence or a peptidic sequence and for producing,
identification or preparation of a product, in particular antibody,
antigen, vaccine or antibiotic, on the basis of the DNA, RNA or
peptidic sequence, or for detecting reactions between a sample, a
replicate or derivative thereof with an interacting molecule or
particle, said detection being performed by an optical,
electrochemical or magnetic sensor, and the interacting molecule or
particle carrying a corresponding marker, or said detection being
performed, without any marker, via the change in the evanescent
field or a modified resonance frequency, or by employing optical
tweezers, or by coupling the reaction with a change in absorption,
in particular precipitation or change of color, or with the
emission of light, in particular chemiluminescence, or for
performing reactions on the replicate or derivative of the array, a
chamber or fluidic structure comprising connecting terminals being
applied over the surface of the replicate or derivative, or the
replicate or derivative being introduced into a corresponding
chamber, it being possible to incubate the chamber at a specific
temperature, and to replace liquids comprised within the chamber,
or for simultaneously performing reactions and detections on the
replicate or derivative.
13. The utilization as claimed in claim 12, wherein an identical
sequencing device that is used for detecting the sequencing is also
used for detecting the reactions or wherein the utilization is
within a device that is also employed for sequencing the array.
14. A method of sequencing a liquid-particle array, a replicate
being created from the samples comprised on the particles as
claimed in claim 8, and the replicate being sequenced in a
sequencing device.
15. A device for producing a replicate or derivative of an array of
molecules, the array comprising a spatial arrangement of separate
samples of molecules, the device comprising: a creator for
creating, for each sample, at least one spatially limited effective
area which is separate from the effective areas of the other
samples, a surface, provided with a binding adapter or binding
properties, of a carrier bordering on the effective areas; an
amplifier for amplifying the molecules by means of amplifying
agents in the effective areas for creating replicates or
derivatives of the samples, and for binding the replicates or
derivatives of the samples to the carrier by means of the binding
adapter or the binding properties, so that a spatial arrangement of
the copies or derivatives of the samples on the carrier corresponds
to the spatial arrangement of the samples; and a remover for
removing the carrier comprising the copies of the samples from the
array.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of copending
International Application No. PCT/EP2010/052849, filed Mar. 5,
2010, which is incorporated herein by reference in its entirety,
and additionally claims priority from German Application No. DE
102009012169.2-41, filed Mar. 6, 2009, which is incorporated herein
by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to methods and devices for
producing a replicate or derivative from an array of molecules,
such as biomolecules or chemically produced molecules, and, in
particular, to such methods and devices that are suitable for
producing a replicate or derivative of a microarray of said
molecules and/or molecules derived therefrom, such as of a DNA
microarray, RNA microarray or protein microarray, and to the
application of the array for identifying DNA sequences associated
with reactions involving primary sequences, copies thereof or
derivatives thereof.
[0003] A microarray is understood to mean an arrangement of many
different biomolecules on or in a surface in individual points.
Said points are also referred to as spots and typically have a
diameter ranging from 10 .mu.m to about 1000 .mu.m. One or several
identical populations of biomolecules are present within a spot.
Except for some intentional redundancies, the various spots,
however, represent different biomolecules. The biomolecules may be
deposited on the surface, may exist in a layer on the surface, may
exist within a cavity, or may exist in an immobilized manner on or
in a particle, it being possible for the particles to be arranged
as an array.
[0004] Conventionally, there have been various techniques of
producing microarrays. In accordance with one technique, the
biomolecules are synthesized direct on the surface (in-situ
synthesis), for example using light synthesis, chemical synthesis,
spot synthesis, a printing process, and the like. Such a
light-synthesis technique is employed, for example, by Affymetrix,
spot synthesis is performed by Agilent. Combimatrix produces DNA
microarrays by the means of virtual, electronically addressable
reaction compartments. In accordance with a further technique, the
(bio)molecules are at first synthesized and subsequently deposited
on the surface as an arranged array, such a technique being
employed, for example, by Agilent, Gesim and Biofluidix. Both
techniques need a high level of technical expenditure. Said
technical expenditure increases more than linearly as the number of
different biomolecules increases and as the diameters of the
deposition spots decrease. In addition, the amount of time involved
as well as the costs increase significantly when such a microarray
is to contain, e. g., twice as many substances, or if the size of
the coated structures, i. e. spots, is to be reduced. A stamping
technique for producing microarrays is described in [1].
[0005] By means of on-site synthesis on an array, it is possible to
produce millions of different DNA sequences. However, in order to
achieve a new layout or a different pattern size, it is needed to
reorganize the entire manufacturing process. This will then need
new instrument settings and, in the event of light-aided synthesis,
even new photolithography masks or reprogramming of the digital
mirror system, see [2], where utilization of digital mirrors for
creating a microarray is described. This is circumvented as far as
possible in order to keep costs down.
[0006] For lack of time alone it is not possible to transfer more
than a few tens of thousands of substances by means of synthesis in
the laboratory and by means of subsequent transmission to a
microarray, for example by means of a nanoplotter. It would take
weeks or months to produce an appreciable number of dots on a
microarray with a million different biomolecules. Whilst that time
the surface chemistry will change and the whole microarray won't
work any more.
[0007] Therefore, a method would be desirable by means of which it
is possible to copy, in a simple and inexpensive manner, existing
microarrays, that is, a regular arrangement of known biomolecules
that are complicated and expensive to produce.
[0008] Some basic ideas on this issue have already been submitted,
[3] and [4]. [3] and [4] disclose a method of replicating an
oligonucleotide array wherein one or more biotin-functionalized
oligonucleotides are hybridized into one or more oligonucleotides
and amplified on a first substrate. The biotin-functionalized and
amplified oligonucleotides are then anchored to a second substrate
with streptavidin. The biotin-functionalized oligonucleotides may
be separated from the oligonucleotides by mechanical force so as to
create a replicated array. However, such copying processes are
costly and need an additional biochemical anchoring system and in
many cases could only produce a negative copy of the original DNA
microarray.
[0009] [5] also describes copying of a DNA array by using a
streptavidin/biotin system. [6] describes how DNA can be copied
into RNA.
[0010] For about 30 years, DNA has been amplified in the
laboratory. Inter alia, polymerase chain reaction (PCR) has made
its arrival in almost all laboratories as a standard technique, and
it is the foundation of most genetic studies. However, there are
also other techniques enabling DNA to be multiplied, e. g. NASBA,
recombinase polymerase amplification, rolling-circle amplification,
and various other isothermal amplification techniques.
[0011] Not only do said techniques generally enable DNA to be
multiplied, but they also enable targeted multiplication of
individual DNA areas or subsets of the DNA. By means of
specifically selecting the start points (primers), it is also
possible to specifically multiply individual areas of the DNA. Most
DNA amplification processes take place in solution, and this is
referred to as a liquid-phase reaction. However, in the last few
years, several methods have come up which utilize an additional
solid phase for DNA amplification and in the process enrich same on
said solid phase. E.g. the primer extension reaction on slide or
solid phase [9,10] . In the following, two of the most common
methods will be described, the foundations of bridge amplification
of DNA as well as the water-in-oil emulsion PCR.
[0012] Bridge amplification of DNA: for bridge amplification, the
(partly unknown) DNA is initially extended, at both ends, with
known, so-called adapter sequences. Said extensions serve as
binding sites for complementary sequences on the surface. It is
only after binding to the surface has taken place that, later on,
amplification will occur. The DNA strand that has been copied and,
thus, newly created is now fixedly (covalently) bound to the
surface, and has a further binding site at its non-bound end. Said
further binding site may now also bind to a suitable counterpart on
the surface and start a further amplification, which in turn will
create a new DNA strand bound at one end and having the original
binding sequence at the other, free end. In this manner, more and
more new strands are generated, in an exponential manner, which are
fixedly bound at one end, and whose other end enables temporary
binding to the surface. During the amplification, the original
strand is fixedly (covalently) bound at one end, and loosely
(non-covalently) bound at the other end, and thus generates a
molecular bridge. In this respect, [11] generally describes bridge
amplification, and [12] describes utilization of bridge
amplification for sequencing.
[0013] For a water-in-oil emulsion PCR, a type of bridge
amplification is employed. This involves initially extending the
DNA strands on both sides by means of adapter sequences, like for
bridge amplification. Subsequently, the extended DNA is mixed
together with an aqueous PCR mixture and solid-phase
particles--also referred to as beads--and emulsified in oil, so
that a water-and-particles-in-oil emulsion results. For this
water-in-oil emulsion, the concentrations are selected such that
ideally, precisely one DNA strand and precisely one particle will
be trapped within each droplet of water. In accordance with bridge
amplification, the surface of the particle contains sequences that
enable a DNA copy to be covalently bound thereto. In this manner,
the entire particle may be covered with copies of the original DNA
by means of amplification. This technique is used mainly in
sequencers. In this technique, only one single defined strand is
amplified, at any one time, on the solid phase or liquid phase.
[0014] In protein amplification, or protein synthesis, there is a
DNA strand that may basically be transcribed initially into RNA and
then into a protein by means of a suitable biochemical system. If
the RNA is sufficiently stable, or if there are a sufficient number
of DNA templates, a large number of proteins can be produced. This
technique corresponds to the natural process occurring within a
cell which involves creating proteins from DNA via RNA, and it is
the foundation and central paradigm of biochemistry. Since
recently, simplified biochemical systems have been available which
are capable of mastering this complex of tasks and thus enable
producing, at least in principle, a protein from a DNA strand in
the laboratory. In this respect, [7] describes a method of directly
producing a protein microarray from a DNA microarray, and [8]
describes a method of producing a protein microarray with cDNA
anchors. Alternatively, protein amplification may also be performed
using prokaryotic or eukaryotic cells which have protein-coding DNA
introduced into them.
[0015] For decoding a DNA sequence, so-called sequencing methods
are employed, an overview of relatively recent sequencing methods
being provided in [13]. In addition, sequencing methods wherein DNA
is bound to particles are described in [14].
[0016] The highly complex machines used for sequencing employ a
multiplicity of reaction steps and techniques for initially
trapping DNA that has been isolated, for multiplying it and for
subsequently reading it out building block by building block. By
means of the selected reaction chemistry and the sequencing method,
it is possible, by means of expensive bioinformatics methods, to
re-calculate the DNA sequence as a whole, and to thus obtain the
genome of the species studied.
[0017] Previous sequencing techniques comprised splitting the DNA
within a gel. This was an approach not based on solid phases and is
called Sanger Sequencing. With the sequencers of the most recent
generation, one works with a water-in-oil emulsion PCR and thus
generates millions of particles, e. g. beads, which carry many
identical copies of different DNA fragments, respectively. For
reading out the sequence, the particles are arranged, e. g., in a
so-called PicoTiterPlate.TM. having e. g. 1.3 to 3.4 million
different microcavities, and are immobilized. This already
represents a microarray as such. In this respect, please refer to
[15], where utilization of bridge amplification for sequencing is
described.
[0018] Even if a regular arrangement of biomolecules has already
been produced in this manner, it nevertheless cannot be used like a
conventional microarray with known sequences, since the individual
sequences of the biomolecules bound to the particles are not yet
known per se. However, after sequencing, the sequence of the DNA
fragment bound to a specific particle will be known per se.
[0019] Efforts have already been made to retrieve the individual
particles and reuse them as an array, for example on the part of
Scineon together with the Max-Planck-Institut fur Molekulare
Genetik [Max-Planck-Society for Molecular Genetics] in Berlin.
However, this method is costly and enables producing only one
specimen of such an array.
[0020] Soft lithography, or microcontact printing, is a stamping
technique that enables depositing molecules on a surface and to
subsequently transfer same to another surface. It also enables
integrating small cavities or microfluidics and to thus provide
complex circuits for liquids. Said circuits enable treating
surfaces in a specific manner and thus coating or modifying
extremely small structures. The material used for this purpose is a
silicone (PDMS). By means of suitable surface modification of the
PDMS, various biomolecules can be added to the surface, and thus be
transferred later on. Both DNA and RNA as well as biomolecules may
be transferred.
[0021] These transfer properties may be exploited, inter alia, for
a copying step, [16] being the first to describe how biomolecules
may be transferred using soft lithography.
[0022] DNA arrays, or DNA microarrays, are mainly used for
so-called expression analysis, sequencing and amount of genes or
SNP analysis.
[0023] In expression analysis, one wishes to study the level of
activity of specific genes. mRNA is considered to be a marker for
this. For this purpose, cells or living beings are stimulated, for
example by administering a drug, by changing environmental factors,
by putting them under stress, and the like. From the biological
material, one initially collects the mRNA, transcribes it into a
so-called complementary DNA (cDNA), and provides it with a dye. A
reference sample is provided with a different dye. Mostly, green
and red dyes are used. Equal proportions of the samples are mixed
together and then applied to the microarray. If a specific DNA
sequence is contained, in equal concentrations, in both original
samples, complementary molecules from both samples will bind, in
equal concentrations, to the respective spot of the microarray.
Reading out this spot therefore results in a secondary color. In
the case of green and red, yellow will result. If there are unequal
proportions of the same gene sequence, the corresponding spot on
the microarray will comprise a secondary color whose coloration
will represent the predominant gene product. Genes that are
switched on or off completely will have only one hue or the other.
The color pattern allows to infer the amount of mRNA and provides a
clue as to how strongly specific genes have been activated or
deactivated by the influences studied. [17] discloses application
of expression profiling for genome-wide studies by means of highly
parallel sequencings.
[0024] In SNP analyses, one investigates whether gene sequences
comprise individual mutations, i. e. sequences that are identical
except for one base pair (replacement of individual base
pairs=single-nucleotide polymorphism). The precise location of the
replaced base pair specifies whether the replacement has no or only
minor effects on the organism, or whether this is a lethal gene
defect. In the case of several serious hereditary diseases such as
Huntington's Chorea, Parkinson's disease or Alzheimer' s disease,
such severe SNPs are known. With many other SNPs, one may infer
increased risks or susceptibilities to specific diseases such as,
e. g., diabetes or rickets. For SNP analysis, the DNA is directly
collected from the biological material and marked with a dye. For
each SNP, there are four spots on the microarray, which differ, in
the same position in each case, by one base pair. From the basis of
the binding position one can specify which base is in the relevant
position in the unknown sample, see [17].
[0025] With protein arrays, production alone is considerably more
difficult, since unlike DNA, proteins have an enormously broad
spectrum of solubilities, reactivities and specificities.
Therefore, it is not trivial to bind various proteins on a surface
using the same chemical anchor. Typically, protein arrays contain
several hundred to one thousand different proteins. Protein arrays
are predominantly employed for binding studies. This comprises
placing a marked molecule onto the protein array. Such spots on the
microarray which comprise coloring are therefore potential binding
partners for the molecule studied. This technique is employed,
inter alia, for epitope mapping in order to specifically find
binding sites.
[0026] The architecture of a microarray as such is described in
[18].
[0027] The applications of microarrays are therefore far-reaching
and manifold. However, due to the lack of the needed financial
resources, or due to their cost-benefit ratio, they are restricted
considerably in use.
[0028] DNA sequences contain biochemical information and may be
multiplied by means of a biochemical replication system. Various
approaches to copying DNA sequences have already been pursued,
starting from copying the individual base pairs onto a surface, up
to the approach described in [4], [5] and [6], which set forth how
DNA can be copied, in principle, from one surface to another.
[0029] Also, an article [7] has recently been published about how a
protein copy can be made from a DNA array.
[0030] The above illustrations show that the standard technology
enables two fundamental techniques of producing microarrays, namely
directly producing substances on site, on the one hand, and
transferring the substances, after a synthesis, by means of a
microscopic dispensing or printing process, on the other hand. Each
of these techniques is technically complex and involves high
expenditure in terms of time and cost, the general rule being that
as the number of substances doubles, the time and cost needed will
also at least double. In addition, to have precise knowledge of the
biochemical information--in the case of DNA arrays, of the
sequence--prior to synthesis is essential. To obtain said
information in the case of DNA, so-called sequencers are used, as
was explained above. No direct production chain between the
sequencing and the fabrication of microarrays has so far been known
or established. This means that an unknown organism is to be
initially sequenced, whereupon the sequence is calculated from the
data of the sequencer, and an array is subsequently produced. By
means of this array immediately an expression pattern can then be
studied. In addition, it is known to produce protein arrays from
known sequences. This production chain is very protracted and
costly.
[0031] If it was possible to generate protein arrays directly as a
derivative of a DNA array by means of a simple method, the coupling
between the phenotype and genotype would be retained, and it would
be possible to perform reactions on the derivative in a spatially
resolved manner (antigen-antibodies, enzymatic reactions) and to
associate them with the underlying DNA sequence.
SUMMARY
[0032] According to an embodiment, a method of producing a
replicate or derivative of an array of molecules, the array having
a spatial arrangement of separate samples of molecules, may have
the steps of creating, for each sample, at least one spatially
limited effective area which is separate from the effective areas
of the other samples, a surface, provided with a binding adapter or
binding properties, of a carrier bordering on the effective areas;
amplifying the molecules by means of amplifying agents in the
effective areas for creating replicates or derivatives of the
samples; binding the replicates or derivatives of the samples to
the carrier by means of the binding adapter or the binding
properties, so that a spatial arrangement of the copies or
derivatives of the samples on the carrier corresponds to the
spatial arrangement of the samples in the array; and removing the
carrier having the replicates or derivatives of the samples from
the array.
[0033] According to another embodiment, a utilization of a
replicate or derivative that was produced while using a method of
producing a replicate or derivative of an array of molecules, the
array having a spatial arrangement of separate samples of
molecules, may have the steps of creating, for each sample, at
least one spatially limited effective area which is separate from
the effective areas of the other samples, a surface, provided with
a binding adapter or binding properties, of a carrier bordering on
the effective areas; amplifying the molecules by means of
amplifying agents in the effective areas for creating replicates or
derivatives of the samples; binding the replicates or derivatives
of the samples to the carrier by means of the binding adapter or
the binding properties, so that a spatial arrangement of the copies
or derivatives of the samples on the carrier corresponds to the
spatial arrangement of the samples in the array; and removing the
carrier having the replicates or derivatives of the samples from
the array, either--for associating a reaction between a binder, in
particular a protein, antibody or antigen, and an original
molecule, its replicate or its derivative, with the DNA sequence of
the original molecule, in particular for genotype-phenotype
coupling, or--for associating a reaction wherein the original
molecule, its copy or its derivative catalyzes the conversion of a
substrate, with the DNA sequence of the original molecule, in
particular for genotype-phenotype coupling, or--for identifying a
DNA sequence, a RNA sequence, a protein or a catalytic, signaling
(e.g. enhancing, allosteric, inhibiting . . . ) or enzymatic (e.g.
lytic, phosphatase activity, kinase activity . . . ) function of a
DNA, RNA or protein, or--for identifying a DNA sequence, a RNA
sequence or a peptidic sequence and for producing, identification
or preparation of a product, in particular antibody, antigen,
vaccine or antibiotic, on the basis of the DNA, RNA or peptidic
sequence, or--for detecting reactions between a sample, a replicate
or derivative thereof with an interacting molecule or particle,
said detection being performed by an optical, electrochemical or
magnetic sensor, and the interacting molecule or particle carrying
a corresponding marker, or said detection being performed, without
any marker, via the change in the evanescent field or a modified
resonance frequency, or by employing optical tweezers, or by
coupling the reaction with a change in absorption, in particular
precipitation or change of color, or with the emission of light, in
particular chemiluminescence, or--for performing reactions on the
replicate or derivative of the array, a chamber or fluidic
structure having connecting terminals being applied over the
surface of the replicate or derivative, or the replicate or
derivative being introduced into a corresponding chamber, it being
possible to incubate the chamber at a specific temperature, and to
replace liquids included within the chamber, or--for simultaneously
performing reactions and detections on the replicate or
derivative.
[0034] According to another embodiment, a device for producing a
replicate or derivative of an array of molecules, the array having
a spatial arrangement of separate samples of molecules, may have a
creator for creating, for each sample, at least one spatially
limited effective area which is separate from the effective areas
of the other samples, a surface, provided with a binding adapter or
binding properties, of a carrier bordering on the effective areas;
an amplifier for amplifying the molecules by means of amplifying
agents in the effective areas for creating replicates or
derivatives of the samples, and for binding the replicates or
derivatives of the samples to the carrier by means of the binding
adapter or the binding properties, so that a spatial arrangement of
the copies or derivatives of the samples on the carrier corresponds
to the spatial arrangement of the samples; and a remover for
removing the carrier having the copies of the samples from the
array.
[0035] Embodiments of the invention provide a method of producing a
replicate or derivative of an array of molecules, the array
comprising a spatial arrangement of separate samples of molecules,
the method comprising:
creating, for each sample, at least one spatially limited effective
area which is separate from the effective areas of the other
samples, a surface, provided with a binding adapter or binding
properties, of a carrier bordering on the effective areas;
amplifying the biochemical molecules by means of amplifying agents
in the effective areas for creating replicates or derivatives of
the samples; binding the replicates or derivatives of the samples
to the carrier by means of the binding adapter or the binding
properties, so that a spatial arrangement of the copies or
derivatives of the samples on the carrier corresponds to the
spatial arrangement of the samples in the array; and removing the
carrier comprising the copies of the samples from the array.
[0036] Embodiments of the invention provide a replicate or
derivative of an array of molecules that was produced by using a
corresponding method.
[0037] Embodiments of the invention provide a device for producing
a replicate or derivative of an array of molecules, the array
comprising a spatial arrangement of separate samples of molecules,
the device comprising:
means for creating, for each sample, at least one spatially limited
effective area which is separate from the effective areas of the
other samples, a surface, provided with a binding adapter or
binding properties, of a carrier bordering on the effective areas;
means for amplifying the biochemical molecules by means of
amplifying agents in the effective areas for creating replicates or
derivatives of the samples, and for binding the replicates or
derivatives of the samples to the carrier by means of the binding
adapter or the binding properties, so that a spatial arrangement of
the replicates or derivatives of the samples on the carrier
corresponds to the spatial arrangement of the samples in the array;
and means for removing the carrier comprising the copies of the
samples from the array.
[0038] In accordance with the invention, a spatially limited
effective area, which is separate from the effective areas of the
other samples, is made available to each sample of an array of
molecules, for example of a DNA microarray, so that the present
invention enables producing a replicate or derivative of a
corresponding array in a fast, simple and inexpensive manner while
retaining the positional information that is given by the spatial
arrangement.
[0039] In particular, embodiments of the invention relate to
corresponding methods and devices wherein the molecules are
biomolecules or synthetically produced chemical molecules. In
accordance with the invention, a replicate can be understood to
mean a 1:1 copy of the original molecules, whereas derivative can
be understood to mean a change in the original molecules, for
example descendants or subsets of the original molecules. In
accordance with the invention, samples of molecules may be
understood to mean different molecules arranged at the different
sites of an array, or different mixtures of molecules arranged
there.
[0040] Embodiments of the invention enable copying microarrays
without having any knowledge of the biochemical information, that
means also copying non-synthetical microarrays, in particular. This
enables copying out, from sequencers already, a copy of the
arrangement of DNA present there, and thus providing a DNA array
without having any knowledge of the sequences.
[0041] Thus, embodiments of the present invention enable producing
a corresponding microarray prior to, following or during a
sequencing process, even without having any knowledge of the
sequences of the individual samples. Thus, the time-consuming and
costly process step of synthetically generating an array may be
circumvented, and an array may be produced directly from the
sequencing. Embodiments of the invention thus enable multiplying a
given microarray or coating pattern in a fast manner and
independently of whether any biochemical sequences or information
of the microarray are known. Embodiments of the present invention
also enable producing different variations from one and the same
original, as may be needed for different tasks set, since different
biomolecules, for example DNA, RNA or proteins, are used for
different biochemical issues. So far, it has not been possible to
produce a suitable DNA or even protein microarray prior to, during
or following to a sequencing process.
[0042] Embodiments of the invention enable producing, by means of a
biochemical copying process, a secondary microarray directly from a
primary material, for example a primary array of particles, prior
to, during or following a sequencing process, and biochemically
mapping said secondary microarray to DNA, RNA or proteins once
again in the form of a further array, if need be. In addition,
during different copying steps, variations of the arrays may be
produced in a chemical form, which contain, for example, specific
markers or sequences, comparable to a color copy, were only the
yellow part of a picture could be copied.
[0043] Embodiments of the present invention relate to the
production of a copy of a microarray from an arrangement of DNA
sequences from a sequencing process (e. g. particle array in a
sequencer by ABI or Roche 454), which has so far been neither
described nor performed. Even though individual sub-steps, such as
biochemically copying from DNA to DNA or from DNA to protein, have
been described in standard technology, it is not known, however, to
combine individual sub-steps for producing a microarray from a
sequencing process. In particular, a production line of sequencing,
followed by production of DNA arrays by fabricating a replicate
from a DNA array from the sequencer, followed by the production of
an RNA array or protein array from the DNA array, has not been
known so far.
[0044] As was already illustrated, it is not known from standard
technology to produce a DNA array already from an ongoing
sequencing process. Also, it has not been described so far to copy
said DNA array directly and then to reform it, by means of
selective modifications, into subsets of DNA or RNA microarrays up
to protein microarrays. Embodiments of the invention enable, for
the first time, producing a DNA array during a sequencing process
and converting it immediately to any array surfaces desired so as
to thereby enable any common microarray studies. In embodiments of
the invention, laborious synthetic production of microarrays may be
completely circumvented in this manner.
[0045] However, embodiments of the invention are not limited to
copying a microarray from a sequencing process. Rather, embodiments
of the invention enable directly copying a planar microarray, such
as, e. g., a commercially available microarray, in a simple manner
and at low cost. Embodiments of the invention enable selectively
copying out aspects of arrays and transcribing them so as to create
RNA, DNA subset, cDNA or protein arrays.
[0046] Suitable copying methods, such as suitable amplifying means
(e. g. PCR, isothermal amplification, NASBA) and respectively
matching binding adapters or binding properties of the surface
(e.g. primers, streptavidin/biotin, antigen-antibodies,
polyhistidine/nickel complexes, electrostatic/dynamic or magnetic
properties) are obvious to a person skilled in the art and need no
further explanation to be given herein. In this respect, please
also refer to the documents mentioned in the introduction, whose
teachings on this matter shall be incorporated herein by
reference.
[0047] In accordance with embodiments of the present invention, at
least one copy of the genetic information, for example of the DNA,
is thus produced from genetic information that may be referred to
as a primary array, which copy may be referred to as a secondary
array. A further array copy, which may be referred to as a tertiary
array, may again be produced from the copy. The tertiary and/or the
secondary array may either be an identical copy, a complementary
copy, a subset, or an RNA or protein array, depending on the choice
of the biochemical replication system with regard to the primary
and/or secondary array.
[0048] In embodiments of the invention, the primary array
originates from a DNA sequencer. Basically, any commercial
particle-based sequencers may be considered for this. In
alternative embodiments, however, any planar DNA microarrays may be
used as primary arrays that are to be copied. Embodiments of the
invention relate to any "copying process" from any one surface to
another, provided that each sample of the array has a spatially
limited effective area made available to it that is separate from
the other effective areas. This includes any substance libraries
ordered in a spatially resolved manner which contain biochemical
information, which includes, in addition to the planar microarrays,
also non-planar surfaces or particle arrays, for example, as are
produced in sequencers or in chemical substance libraries.
[0049] In embodiments of the invention, the amplifying agents used
may be any known amplifying agents. Previous amplifications have
aimed at multiplying specific DNA segments or sequences. With such
known amplifications, the positional information about where a
particular strand was produced typically is not retained, since
said amplifications take place in solution. In embodiments of the
invention, by contrast, the positional information from the source,
i.e. the primary array, to the copy, i.e. to the secondary array,
is entirely or partly retained in the production of the replicate
in that each sample has a spatially limited amplifying agent area,
that is separate from the amplifying agent areas of the other
samples, made available to it. This positional information is
frequently also referred to as registration. This is understood to
mean, e. g., the position of a specific DNA segment as defined by
its arrangement (row and column, or x and y positions) within a
regular grid. Due to this at least partial retention of the
positional information, the present invention enables production of
high-quality replicates. The more positional information is
retained, the better the replicate will be. A poor spatial
resolution will create, when multiple copies are made, copies that
are of increasingly poor quality and will consequently be useless
at some point.
[0050] In accordance with the invention, any known amplification
methods may generally be employed; in some embodiments, a PCR or a
bridge amplification may be used. Bridge amplification may be used
on a surface. In embodiments of the invention, the copying process
therefore represents some kind of bridge amplification which,
however, takes place from one surface to another, the positional
information, or the registration of the copied species, being
retained. In alternative embodiments, an additional binding system
may also be used for binding the copies to the carrier, for example
a streptadivin/biotin system as is described in [4], which,
however, results in increased complexity.
[0051] In embodiments of the invention, the binding adapters may be
arranged over the entire area of the carrier to which the array is
to be copied.
[0052] In embodiments of the invention, the binding adapters are
primers that are complementary to sequences of the molecules to be
copied.
[0053] In embodiments of the invention, retention of the positional
information is achieved by spatially separating or
compartmentalizing the amplifying agent areas associated with the
respective samples. In this manner, individual molecules may be
prevented from escaping from the "microcompartment", so that the
spatial resolution or registration is retained. As was already
explained, in embodiments of the invention, any amplification
techniques may be considered as the copying process, such as PCR or
isothermal amplification. During the copying process, the duplicate
is deposited onto a target surface and anchored. In addition to the
fact that the genetic information is entirely or partly retained,
the positional information is also retained. Embodiments of the
invention provide a 1:1 replicate, which generally is understood to
mean "simple" copying of the surface. In this process, a copy of
the original is produced which has the highest level of similarity
possible. In the case of microarrays, after the copying process one
will have obtained a further DNA array from a DNA array.
[0054] Embodiments of the invention enable producing a partial
replicate, i.e. a derivative, of an array. A partial replicate or a
partial copy is understood to mean a specific selection of the
information copied. For example, during the process of copying a
DNA array, only a specific type of DNA can be multiplied by
selecting primers that the carrier comprises as binding adapters.
In this manner, a subset is achieved that contains the information
desired, such as all of the DNA strands that contain a specific
sequence or a specific promoter.
[0055] Likewise, a derivative is referred to as a "conversion" of
the copy of a DNA to an RNA or cDNA, of an RNA to a DNA, cDNA or to
a protein. In this context, the biochemical information is
transformed from one type of molecule to another, and the
positional information is still retained.
[0056] In embodiments of the invention, the limited effective areas
in the form of spatially limited amplifying agent areas are created
by solid structures, whereas in alternative embodiments of the
invention, phase boundaries between liquids of different levels of
viscosity contribute to creating the spatially limited effective
areas.
[0057] In addition, embodiments of the invention relate to
applications of corresponding replicates and derivatives for
analytical purposes, with regard to their reactions or interactions
with other molecules or particles, and with regard to reaction
catalysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] Embodiments of the present invention will be explained below
in more detail with reference to the accompanying figures, in
which:
[0059] FIGS. 1a to 1d are schematic cross-sectional representations
for illustrating an embodiment of the inventive method;
[0060] FIG. 2 schematically shows a top view of a section of a
PicoTiterPlate.TM.;
[0061] FIG. 3 schematically shows a cross-sectional representation
of a sequencer chip comprising DNA particles;
[0062] FIGS. 4a to 4c schematically show cross-sectional
representations for illustrating a further embodiment of the
inventive method;
[0063] FIGS. 5a to 5d schematically show cross-sectional
representations for illustrating a further embodiment of the
inventive method;
[0064] FIGS. 6a to 6d schematically show cross-sectional
representations for illustrating a further embodiment of the
inventive method; and
[0065] FIG. 7 shows a schematic representation for illustrating a
further embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0066] With reference to FIGS. 1a to 1d, an embodiment of an
inventive method will be described below, wherein the primary array
exists in the form of a sequencer chip 10. The sequencer chip 10
comprises a plurality of microcavities 12. A schematic top view of
a section of the sequencer chip 10 comprising the microcavities 12
is shown in FIG. 2. The microcavities may have a diameter of 44
.mu.m or 29 .mu.m, for example, as is shown in FIG. 2. The
sequencer chip may be, for example, a sequencer chip (GS FLX 2005
and/or GS FLX Titanium 2008) of the 454 sequencer by Roche.
[0067] Each of the cavities 12 has a particle 14 disposed therein,
each of said particles 14 carrying millions of copies of an
individual DNA strand 16. A schematic cross-sectional
representation of the sequencer chip 10 comprising the cavities 12
which have the particles 14 comprising the DNA strands 16
introduced into them is shown in FIG. 3. Until now, the sequencer
chips have been discarded after the sequencing process and have
therefore been "waste products" of sequencing processes.
[0068] In the embodiment depicted in FIGS. 1 to 3, this chip is
used as a primary array for producing a replicate. The DNA is to be
copied out from the cavities 12. For this purpose, the cavities are
initially filled with an amplifying agent, for example a PCR mix.
Subsequently, as is shown in FIG. 1b, a carrier 20 is deposited
which seals the cavities 12 and which carries binding adapters
matching the amplifying agent, schematically shown as spots 22 in
FIG. 1b. Once the cavities 12 have been closed off by the lid 20, a
spatially limited amplifying agent area 24 has thus been produced
for each sample, i. e. each particle 14 having DNA strands 16 bound
to it, which amplifying agent area 24 is separated from the
amplifying agent areas 24 of the other samples. The binding
adapters 22 border on these amplifying agent areas 24. For example,
the binding adapters 22 are primers matching the PCR mix. Said
primers are binding sites for DNA polymerase. FIG. 1b shows the
state after the polymerase step wherein biochemical copies are made
of the particle's DNA. These copies are depicted as dashed lines 18
in FIG. 1b. For example, by means of the selection of the primers,
a mixture of enzymes used as an amplifying agent may produce, in
this step, a complementary DNA, i. e. a negative copy.
[0069] Subsequently, the copies 18 of the DNA that have been made
are released from the particles 14, which may be performed, for
example, by heating the sequencer chip and, thus, the cavities
arranged therein. Thereafter, the released copies 18 add to the
binding adapters 22, which may be promoted, for example, by cooling
the sequencer chip. The result of the copies 18 adding to the
binding adapter 22 and, thus, to the carrier 20 is depicted in FIG.
1c. In this step of copying the copies to the carrier 20, the
positional information, or the registration, is retained, since a
spatially limited amplifying agent area 24 is provided for each
sample, and since the amplifying agent areas 24 are separate from
one another.
[0070] Subsequently, the carrier 20 with the DNA copies 18 bound to
it is removed from the sequencer chip 10 and represents a replicate
of the DNA particle 14, 16 arranged within the cavities 12 of the
sequencer chip 10. The particles 14 comprising the DNA strands 16
remain within the cavities 12, so that said cavity may again serve
as a primary array for a new copying process with a new carrier. In
this manner, basically any number of copies may be produced. The
carrier 20 having the DNA copies 18 bound to it may be employed,
for example, as a biochip in a transcriptome analysis, in detection
of binding of proteins onto DNA, RNA onto DNA, or even RNA onto
RNA.
[0071] So as to once again prepare, after the copying process, the
primary array (the sequencer chip 10) for a further copying cycle,
the amplifying agent within the cavities, e.g. the PCR mixture, may
be replaced or removed. To avoid contaminations a washing step,
which may also contain enzymes (like the uracil-N-glycosylase,
which digest special DNA products), removes the PCR products and
allows as such more copies without contaminating the original
master.
[0072] FIGS. 4a to 4c depict a further embodiment of an inventive
method, wherein a conventional planar microarray serves as a
primary array. The planar microarray is arranged on an array
substrate 30 and contains the desired DNA samples. The DNA samples
have a two-dimensional spatial arrangement. For the process of
copying the DNA samples, a microstructure 34 comprising cavities 36
is provided. At least one cavity 36 is provided for each DNA sample
32. To obtain a relatively high resolution, a plurality of, in each
case, relatively small cavities 36 may be provided, in alternative
embodiments, for each DNA sample 32. The cavities 36 have binding
adapters 38 arranged therein.
[0073] The cavities, or microcavities, 36 are filled with an
amplifying agent, for example a polymerase mixture. The
microstructure 34 is then deposited onto the microarray 30, so that
the cavities 36 are closed off by the array substrate 30 (a small
distance would also work but would enhance contaminations between
cavities), and such that the DNA samples are arranged within the
cavities 36 associated with them, respectively. In this manner, a
spatially limited amplifying agent area 35, which is separate from
the amplifying agent areas 35 of the other samples, is produced,
again, for each DNA sample. The binding adapters 38 may again be
formed, again, by a primer matching a polymerase mixture.
[0074] After producing the amplifying agent areas 35 thus closed
off, an amplification like the polymerase step takes place once
again, wherein the DNA samples 32 are multiplied and copied into
the cavities 36. The copied DNA is anchored at the binding adapters
38, as is schematically shown by the DNA 42 in FIG. 4b. To this
end, again, the temperature of the cavities having the DNA samples
arranged therein may be controlled accordingly. Finally, the DNA
substrate 30 having the DNA samples 32 located thereat is removed
from the microstructure 34, so that the microstructure 34 with the
copied DNA 42 represents a replicate of the original microarray.
The microstructure 34, which is thus loaded with the copied DNA 42,
may now be used as a template for further copying steps which may
be performed, for example, analogously with the method as was
described above with reference to FIGS. 1a to 1d. In this context,
the microstructure 34 may be configured such that after the copying
process it will have the same properties as a sequencer chip, for
example the sequencer chip from the 454 sequencer by Roche.
[0075] An embodiment may comprise a combination of the methods in
accordance with the above-described embodiments. Initially, a
sequencer chip may be used for producing, by a copying process in
accordance with the above FIGS. 1a to 1d, a planar carrier
comprising a microarray of the entire DNA. Subsequently, this
carrier is copied once again in accordance with the embodiment
described with reference to FIGS. 4a to 4c. The microcavities thus
occupied by DNA (microcavities 36 in FIGS. 4a to 4c) may now be
used for producing further copies of the DNA, for example in
accordance with the method of FIGS. 1a to 1d. Alternatively, the
microcavities occupied by DNA may be used for producing modified
copies in the form of complementary DNA, subgroups of DNA,
shortened, extended or modified DNA, or even RNA up to proteins. In
this manner, any areas comprising DNA, RNA or proteins or peptides
may be produced.
[0076] A further embodiment of an inventive method will now be
described with reference to FIGS. 5a to 5d. In this embodiment, DNA
is multiplied into individual particles 50 by means of a
water-in-oil emulsion PCR. One type of DNA is anchored per particle
(like in the preparation of the beads for the 454 sequencing or in
the ABI SOLID sequencer). Said particles are placed on a surface of
a carrier 52. More specifically, the particles 50 are located in
respective droplets of liquid 54, for example droplets of water.
The droplets of water are separated from one another by an oil film
56. The droplets of water thus contribute to defining spatially
limited, mutually separated amplifying agent areas 54' (FIG. 5c).
The droplets of water 54 may be attached to the respective
positions on the surface of the carrier 52 by means of hydrophilic
coating, so that they will be arranged on the carrier 52 in a
defined spatial arrangement. For example, the carrier 52 may
comprise a regular pattern of hydrophilic dots which corresponds to
the arrangement of the samples of biochemical molecules. The
droplets of water 54 have an amplifying agent introduced therein in
each case. Thus, for each sample in the form of the particle 50
comprising the DNA bound thereto, a spatially limited amplifying
agent area is provided which is separated from the amplifying agent
areas of the other samples by the phase boundaries between the
liquids, e.g. water and oil. As is shown in FIG. 5b, a carrier 60
comprising binding adapters 62 is provided. The carrier 60
comprising the binding adapters 62 is pressed onto the oil film 56,
so that the oil, which is thinner-bodied than water, is displaced,
and such that the carrier 60 does not come into contact with the
droplets of water 54 with that surface which comprises the binding
adapters 62. Said droplets of water 54 may be easily compressed in
the process, as is depicted in FIG. 5c.
[0077] Subsequently, the DNA that is bound to the particles within
the droplets 54 is amplified by means of the amplifying agent so as
to create DNA copies. Said DNA copies 64 are bound to the binding
adapters 62 and removed from the substrate 52 together with the
carrier 60. The carrier 60 having the copied DNA samples 64 bound
thereto thus represents a replicate of the original array.
[0078] In alternative embodiments, binding adapters may be provided
on the substrate 52 rather than on the carrier 60, so that the DNA
is copied to the substrate 52, whereas the carrier 60 merely serves
as a counter support. This embodiment, too, therefore enables
producing a planar microarray as a copy of a particle array while
using a water-in-oil emulsion PCR. Thus, fast production of a DNA
array is possible. Also, RNA copies, protein copies or modified DNA
copies may be produced.
[0079] In accordance with the invention, a spatially limited
effective area is produced for each sample of the array to be
copied, i.e. for which a replicate or derivative is to be created.
The spatial creation of the effective area may be performed in
various ways. In embodiments of the invention, a spatially closed
cavity is provided for each sample. In embodiments, a spatial
demarcation may be provided which facilitates diffusion in specific
directions, and impedes diffusion in other directions, such as an
arrangement of columns or trenches, for example. In embodiments, a
porous material, a diffusion-defining material or molecular
structures which prefer or restrict diffusion in specific
directions may be used, such as hydrogels, aerogels or polymer
surfaces. In embodiments, ordered or unordered nano- or molecular
structures such as polymer branches, dendrimers, particle arrays,
filter membranes, lipid membranes (spherical or planar) may be used
so as to implement spatially limited effective areas.
[0080] In embodiments, physical fields such as electrical or
magnetic fields which also create an advantageous direction of
diffusion (electrophoreses, optical tweezers, magnetophoresis,
surface acoustic waves, thermophoresis, . . . ) or a diffusion
barrier and, thus, build a spatial separation may be used so as to
create spatially limited effective areas. For example, a magnetic
liquid and "hardening" magnetic fields may be used, or a laser
light grid which separates the individual areas.
[0081] In embodiments, activation and/or deactivation may take
place within or outside the effective areas in order to create
spatially limited effective areas, e.g. by means of electrical
fields, charge, a change in the pH, deactivation/activation by
means of light, pressure and the like. For example, light
activation of the polymerase or creation of activated nucleotides
by light may be performed within a limited area. No reaction will
then take place in the dark areas.
[0082] In further embodiments, surface structures may be used which
provide a spatially limited effective area with specific physical
effects. For example, hydrophobic/hydrophilic areas (e.g. oil and
water) or polymers may be mentioned in this context which may swell
and harden in specific areas due to electrical fields, and which
thus may also define spatially limited effective areas.
[0083] A further embodiment wherein a spatially limited effective
area is defined by a three-dimensional structure will now be
described with reference to FIGS. 6a to 6d. As is shown in FIG. 6a,
samples 100 of molecules that are part of an array are arranged on
elevations 102 of an array substrate 104. Between the elevations
102, depressions 103 are formed within the array substrate 104. A
carrier 106 comprising binding adapters 108 in the form of a
solid-phase primer is placed in the vicinity of the array substrate
104, as is shown in FIG. 6b. Due to the spatial vicinity of the
array substrate 104 and of the carrier 106, spatially limited
effective areas arise, in the area of the elevations 102, between
the opposite surfaces of the elevations 102 and of the carrier 106.
By contrast, the spacing between opposite surfaces of the
depressions 102 and of the carrier 106 is sufficiently large so
that here, no effective area forms.
[0084] In the effective areas, the contact between the solid-phase
primer and the samples 100 enables hybridizing, so that an
amplification may start, as is shown in FIG. 6c. Material for the
amplification may be additionally supplied from the depressions, as
is indicated in FIG. 6c by arrows 112. In this manner, replicates
114 of the samples 100 bound to the carrier 106 are produced, and,
thus, a replicate of the array formed by the samples 100 is
produced.
[0085] Starting from the state depicted in FIG. 6c, the array
substrate 104 and the carrier 106 may now be separated, the samples
100 remaining at the array substrate 104, and the replicates 114
being removed with the carrier 106. Further copies may then be made
either of the array located on the array substrate 104, or of the
replicate located on the carrier 106.
[0086] In the embodiment described with reference to FIGS. 6a to
6d, an amplification and a transfer to the carrier take place
essentially simultaneously. If transfer and amplification take
place separately from each other, one step may be performed, in
alternative embodiments, over a large surface area, and the other
may be performed in a spatially defined manner.
[0087] In the embodiment described with reference to FIGS. 6a to
6d, the spatially limited effective areas are thus produced by the
structures described as well as by the presence of a primer. In
this context, the reaction corresponds to a bridge amplification.
The surfaces are brought into physical contact with one another.
Due to the spatial proximity, an "effective area", wherein the DNA
is copied to the other surface, forms at the elevations, i.e. the
peaks of the columns. Subsequently, said peaks may even be removed,
since the amplification is then a classical bridge amplification
which defines its own effective area, as it were. However, the
start of the reaction comes about only due to the initial condition
of the spatial effective area. This reaction might be referred to
as an edge or peak amplification. The spatial edge or peak starts
off the reaction. The empty space next to the edge supplies the
reaction with any materials needed.
[0088] In an alternative embodiment, the array to be copied may be
arranged, in deviation from FIGS. 6a to 6d, on a planar substrate,
whereas the elevations are formed on the carrier to which the array
is copied. Again, alternatively, elevations may be formed both on
the array substrate and on the carrier.
[0089] An embodiment referring to how spatially limited effective
areas may be produced by energy fields, for example magnetic or
electrical fields, is depicted in FIG. 7. FIG. 7 merely shows,
schematically, an array substrate 120 and a carrier 122. Molecule
samples on the array substrate 120 that are to be copied as well as
binding adapters on the carrier 122 are not depicted for
simplicity's sake. In the area of respective samples of molecules,
field generation means 124 are arranged which are configured to
generate energy fields 126 in an amplifying agent arranged between
the array substrate 120 and the carrier 122. In this manner,
spatially limited effective areas 128 are created wherein the
amplifying agent is activated, whereas this is not the case in the
remaining areas.
[0090] Embodiments of inventive methods have been illustrated
above. Embodiments of corresponding devices or means for
implementing the inventive method steps result from the description
or are obvious to a person skilled in the art. Therefore, there is
no need to further illustrate that an inventive device may comprise
suitable handling means for positioning the physical entities, e.g.
the various arrays, carriers or substrates, as needed. In addition,
it is not needed to further explain that suitable fluidic means may
be provided so as to supply the respective liquids or agents at the
needed positions. In addition, it is obvious to a person skilled in
the art that a corresponding controller may be provided to control
the device to perform the inventive methods. Means for creating an
environment needed for performing the methods, for example
temperature sensors, may also be provided.
[0091] Embodiments of the invention are suited, in particular, to
create a replicate or derivative of arrays wherein the molecules
are single- or double-stranded oligonucleotides, polynucleotides,
DNA or synthetic molecules analogous to DNA (PNA). In embodiments
of the invention, a spatially planar arrangement, such as a
microarray, a spatial arrangement of particles, for example within
a sequencer chip, a spatial arrangement of cavities, for example
within a PicoTiterPlate.TM., or a spatial arrangement of different
phases, for example of individual droplets of liquid, may serve as
the primary array. In addition, particle-based assays, such as by
the companies of Illumina or Applied Biosystems (SOLID), for
example, may also be regarded as such types of arrays. In
embodiments, the biochemical molecules, for example the oligo- or
polynucleotides, may be copied from a sequencing process for
deriving the genome, from a sequencing process for deriving the
transcriptome, from a process of sequencing RNA (such as mRNA,
tRNA, siRNA or RNA in general), or from a process of sequencing
mutations and variations. The copies produced may be, in
embodiments of the invention, DNA, modified DNA (extended,
shortened, artificial, inserts, deletion, mutation . . . ), DNA
constructs (expression vectors, siRNA), artificial molecules (PNA,
modified peptides), expressions, RNA or proteins, in each case for
producing an array.
[0092] In embodiments of the invention, oligo- or polynucleotides
may be copied from a sequencing process for generating an array or
a structured surface. In embodiments, oligo- or polynucleotides may
be copied from an arrangement of particles for producing an array
or for coating a surface. In embodiments of the invention, oligo-
or polynucleotides may be copied from a surface for creating a
copy, for creating a complementary copy, or for physicochemically
modifying the surface.
[0093] In embodiments of the present invention, oligo- or
polynucleotides may be copied to a further surface for the purpose
of chemical or biochemical modification for an application on the
basis of the new surface properties, or for biochemical process
chains for producing chemical substances. In embodiments of the
invention, a particle array that may, but need not, be produced,
for example, by means of a water-in-oil emulsion PCR may be copied,
without having to be sequenced, for producing an array of a DNA
library, for producing an array comprising various DNA mutants, for
further copying said arrays to RNA or proteins, or for using the
copies in cellular experiments.
[0094] Embodiments of the invention may be employed in numerous
fields of application. Examples of such fields of application are
sequencing, transcript analysis, measuring DNA, RNA or protein
activity, expression studies, display techniques while employing
phage displays, ribosome displays or cell displays, and metabolite
studies. In addition, the invention may be applied in interaction
studies, for example in the following: DNA/DNA; DNA/RNA;
DNA/protein; RNA/protein; RNA/cell; protein/protein; kinase
activity; protease activity; phosphatase activity; DNA-binding
proteins; epitope mapping; determination of pathogens; and
determination of substances or inhibitors. The invention may enable
this analysis which is partially not possible today with a large
number of interaction partners on the array side.
[0095] In addition, the present invention may be applied in the
field of vaccine development, one example being as follows. Let us
assume that a new virus/bacterium appears. A cell sample or a blood
sample is taken from the first living being that survives. The cell
sample is infected with the virus, and the mRNA is isolated. Said
mRNA is then sequenced, and the DNA obtained is copied out from it.
Subsequently, the DNA array is transcribed into a protein array. In
this manner, this array will contain proteins of the cell and
proteins that are modified due to the virus attack. The blood
sample is placed onto the array, and the antibodies contained
therein bind to the proteins. Only antibodies will bind to the
viral proteins, since the antibodies per se do not bind to proteins
of the same body. The bound antibodies may then be identified by
means of a dying step. Thus, the DNA and protein sequences of the
virus can be determined. In this manner, one has gained knowledge,
within a very short period, about epitopes and binding proteins of
the antibodies. With this information, therefore, both passive and
active vaccines can be produced immediately. In this manner, in the
event of an epidemic, the time taken before a vaccine can be
produced may be reduced dramatically.
[0096] Embodiments of the present invention therefore enable a
complete work cycle wherein the array of DNA sequences (primary
array) that is produced during a sequencing process is to be
transferred to a surface, and wherein, thus, a copy of this DNA
(secondary array) is to be produced. In addition, in embodiments,
the primary or secondary array additionally is to be modeled as a
further copy in the form of RNA or protein (tertiary array). In
embodiments, each array of biochemical molecules, such as DNA, may
be regarded as a primary array. Also, by suitably selecting the
copying technique, an identical or selective copy of the original
may be produced. Therefore, embodiments of the invention relate to
mapping--even prior to, during or after gene sequencing--the array
used in the process, and to optionally reforming it into a gene,
cDNA, RNA or even protein array in further copying steps.
[0097] Embodiments of the invention are advantageous in that
molecular information may be replicated, in a spatially resolved
manner, any number of times even during a sequencing process. Only
one original is needed as a master for this purpose. No information
needs to exist about the nature of the original and the data
contained therein. The copying process is therefore independent of
the information included. In addition, embodiments of the invention
allow producing microarrays or copying biochemical surface
structurings without employing in-situ syntheses or
printing/dispensing units. The copying process takes place at a
molecular level and uses well-established biochemical systems.
Since the positional information is retained, the copying process
allows highly parallel processing of the biochemical information.
This enables connecting different types of microarrays at a
molecular level and circumvents the time-consuming and costly
production of microarrays after gaining knowledge of a
sequence.
[0098] In embodiments of the invention, micro- or nanostructures
which contribute to defining spatially limited amplifying agent
areas comprise an unordered matrix based, in particular, on a
filter membrane, on a hydrogel or on an aerogel. In embodiments of
the invention, the micro- or nanostructures are based on an ordered
three-dimensional substrate.
[0099] In embodiments of the invention, the spatially limited
amplifying agent areas are separated, at least in part, by phase
boundaries between two fluids, a fluid and a gas, or a physical
boundary, in particular a lipid membrane.
[0100] In embodiments of the invention, the process of binding the
replicates or derivatives to the carrier may also be performed
simultaneously with the amplification, or be part of the
amplification, in that an immobilized binding adapter acts as a
primer for the amplification. In addition, derivatives may be bound
to the carrier via an immobilized capture molecule, or in that they
remain coupled to the system used for producing them, and in that
said system is immobilized on the carrier. This system may consist
of enzymes, ribosomes or cells, for example.
[0101] In embodiments of the invention, the spatial limitation of
the effective area consists in that the binding adapters are
present on the carrier, as complementary primers, in the form of a
primer array that may comprise a regular or irregular distribution
of spots, the spot size and spot density on the carrier being equal
to or smaller than that on the array.
[0102] In embodiments of the invention, the amplifying agent is
configured to effect a DNA amplification, in particular a
polymerase chain reaction, an isothermal amplification, e.g a NASBA
reaction, and the binding adapter comprises a matching primer.
[0103] In embodiments of the invention, primary, secondary and/or
tertiary derivatives are generated, from a primary array or a
replicate of the primary array, in that DNA is transcribed into
RNA, the RNA is translated into protein, or in that a binder is
enriched while using a produced protein, a produced RNA or a
produced DNA or the copy thereof from a liquid phase, or in that a
binder interacts.
[0104] In embodiments of the invention, a derivative is generated
on the solid phase of a target array, and is present there in an
immobilized manner. In embodiments of the invention, the positions
of the samples have further molecules or DNA sequences or cells
located thereat which are part of the sample or are immobilized and
which are needed for generating derivatives, in particular
expression vector sequences such as ori, promoters, ribosome
binding sites, start codon, endoprotease cleaving sites, fusion
sequences, reporter genes, terminators, antibiotics resistance
genes, in-vitro translation systems, or cells.
[0105] Embodiments of the invention relate to a replicate or
derivative of an array of molecules that was produced while
employing an inventive method, and to applications of such a
replicate or derivative. In embodiments of the invention, such a
replicate or derivative is used for associating a reaction between
a binder, in particular a protein, antibody or antigen, and an
original molecule, its replicate or its derivative, with the DNA
sequence of the original molecule, in particular for
genotype-phenotype coupling. In embodiments of the invention, such
a replicate or derivative is used for associating a reaction
wherein the original molecule, its copy or its derivative catalyzes
the conversion of a substrate, with the DNA sequence of the
original molecule, in particular for genotype-phenotype coupling.
Embodiments of the invention relate to a DNA sequence identified by
such an utilization, and to products or preparations produced on
the basis of such a DNA sequence, in particular antibodies,
antigens, vaccines or antibiotics.
[0106] In embodiments of the invention, a replicate or derivative
that was produced in accordance with an inventive method is used
for detecting reactions between a sample, a replicate or derivative
thereof with an interacting molecule or particle, said detection
being performed by an optical, electrochemical or magnetic sensor,
and the interacting molecule or particle carrying a corresponding
marker, or said detection being performed, without any marker, via
the change in the evanescent field or a modified resonance
frequency, or by employing optical tweezers, or by coupling the
reaction with a change in absorption, in particular precipitation
or change of color, or with the emission of light, in particular
chemiluminescence. In embodiments of the invention, an identical
sequencing device that is used for detecting the sequencing is also
used for detecting the reactions.
[0107] Embodiments of the invention relate to utilization of a
corresponding replicate or derivative for performing reactions on
the replicate or derivative of the array, a chamber or fluidic
structure comprising connecting terminals being applied over the
surface of the replicate or derivative, or the replicate or
derivative being introduced into a corresponding chamber, it being
possible to incubate the chamber at a specific temperature, and to
replace liquids contained within the chamber. Such utilization may
also take place in a device that is also used for sequencing the
array.
[0108] Embodiments of the invention relate to utilization of a
corresponding replicate or derivative for simultaneously performing
reactions and detections on the replicate or derivative.
Embodiments of the invention relate to a method of sequencing a
liquid-particle array, a replicate being created from samples
contained on particles, and the replicate being sequenced in a
sequencing device.
[0109] In embodiments of the invention, the progress of the
reaction may be read out, during the amplification or binding
process, by using standard methods. This enables applications in
the fields of enzyme, binding and reaction kinetics. For example,
an enzyme that binds CO.sub.2 may be produced. Said enzyme might
then be immediately identified by at a change in the pH value.
Similarly, other enzymatic or catalytic activities or binding
properties might be identified. These include, as it were, any
biochemical measuring techniques measuring the mere presence of a
molecule up to its mode of action. Embodiments of the invention
therefore comprise monitoring any changes in physical or chemical
parameters using well-known detection methods within the individual
effective areas during the application, which enables a level of
insight into the operating mechanisms of both the amplifying agent
and the primary array as well as its derivatives that has hitherto
not existed.
[0110] Embodiments of the invention provide for the utilization of
a replicate or derivative of an array of molecules that was
produced while using a method according to the invention for
identifying a DNA sequence, a RNA sequence, a protein or a
catalytic, signaling (e.g. enhancing, allosteric, inhibiting . . .
) or enzymatic (e.g. lytic, phosphatase activity, kinase activity .
. . ) function of a DNA, RNA or protein.
[0111] Embodiments of the invention provide for the utilization of
a replicate or derivative of an array of molecules that was
produced while using a method according to the invention for
identifying a DNA sequence, a RNA sequence or peptidic sequence and
for producing, identification or preparation of a product, in
particular antibody, antigen, vaccine or antibiotic, on the basis
of the DNA, RNA or peptidic sequence.
[0112] While this invention has been described in terms of several
embodiments, there are alterations, permutations, and equivalents
which fall within the scope of this invention. It should also be
noted that there are many alternative ways of implementing the
methods and compositions of the present invention. It is therefore
intended that the following appended claims be interpreted as
including all such alterations, permutations and equivalents as
fall within the true spirit and scope of the present invention.
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* * * * *
References