WO2004067759A2

WO2004067759A2 - Replication of nucleic acid arrays

Info

Publication number: WO2004067759A2
Application number: PCT/GB2004/000375
Authority: WO
Inventors: Alessandra Bossi; Sergey Anatoliyovich Piletsky; Olena Volodimirivna Piletska; Philip James Warner; Anthony Peter Francis Turner
Original assignee: Cranfield University
Current assignee: Cranfield University
Priority date: 2003-01-29
Filing date: 2004-01-29
Publication date: 2004-08-12
Anticipated expiration: 2005-07-29
Also published as: WO2004067759A3; GB0516777D0; GB0302058D0; GB2413555A

Abstract

A method of producing arrays of nucleic acids corresponding to a first set of nucleic acids immobilised on a first solid support (master array) includes furnishing on said first set of nucleic acids a second set of nucleic acids, each nucleic acid of the second set having at least a portion which is complementary to at least a portion of respective nucleic acid of the first set and being hybridised to it thereby. This may involve contact with a solution containing a mixture of nucleic acids, or synthesis using the first nucleic acids as primers and templates. The second set of nucleic acids are immobilised to a second solid support, either before or while they are hybridised to the first set of nucleic acids. Separation of the first and second solid supports provides said first support bearing the master array and said second support bearing a complementary array.

Description

REPLICATION OF NUCLEIC ACID ARRAYS

Technical Field

This invention relates to methods for manufacturing nucleic acid arrays and their uses, e.g. in sequencing of nucleic acids, detecting and identifying specific nucleic acids in biological samples, for research, in pharmacology, environmental, forensic and clinical analysis. The invention is also directed to novel methods for the replication of probe arrays, to the replicated arrays, to diagnostic aids comprising nucleic acid probes and arrays useful for screening biological samples for target nucleic acids and nucleic acid variations.

Background Art

Ordered arrays of oligonucleotides immobilised on a solid support have been proposed and are finding applications in sequencing DNA fragments and for screening, detecting and identifying specific nucleic acids or modifications in nucleic acid compositions in biological samples, pharmacology and clinical analysis. In order for the device to function correctly it is important to have an array of immobilised oligonucleotides with each sequence immobilised on a predetermined area on a surface of a solid support. Nucleic acid arrays can be fabricated using in situ synthesis methods (WO 98/41531) or deposition of previously synthesised molecules (WO

95/25116 and WO 98/41531). In situ synthesis methods include different variations of solid-phase synthesis. Typically the process involves sequential repeating of three steps: a) linking a protected monomer to a suitable activated surface; (b) deprotecting the deposited monomer so that it can now react with a second protected monomer; and (c) depositing another protected monomer for linking. Different monomers may be deposited at a different time at different regions on the solid support thus creating variations in the composition of spatially separated sequences. The deposition methods involve depositing synthesised sequences at predetermined locations on a solid support, which is suitably activated. Typical procedures used for nucleic acid deposition involve loading a small volume of sample in solution on the tip of a pin or capillary and touching the pin or capillary on to the surface of the substrate. When the fluid touches the surface, some of the fluid is transferred. The pin or capillary must be washed prior to picking up the next sample for spotting onto the array. This process is repeated for each different sequence. Alternatively, the nucleic acid can be deposited using inkjet printer or by pipetting (e.g. by equipment produced by Bio-Dot Inc., Irvine, Calif, USA). Alternatively, a pre-synthesised sequence can be chemically bound to a molecule already tethered or deposited on the surface.

The company Affymax uses a photo-lithographic method to produce DNA chips

(Fodor, S. P. A., et.al., Science, 1991, 251, 767-773 and U.S. Pat. No. 5,143,854). The technology utilises the methods commonly used in the electronics industry and therefore has advantages such as accurate positioning to micron accuracy, it meets clean room requirements and can use multiple photo-masks to define the array pattern. Although it is possible to produce excellent arrays using this approach, they are typically too expensive to be used as disposable elements, which is what is needed e.g. in the DNA chip. The difficulty in fabrication of such arrays and their replication creates a high price for such devices and materials, which is disadvantageous for broader application. There are four important design aspects required to fabricate a nucleic acid array. First, the array sensitivity is dependent on having reproducible spots on the substrate. The location of each type of spot must be known and the spotted area should be uniformly coated with the immobilised material. Second, since nucleic acids are expensive to produce, an optimum amount of these materials should be loaded into any transfer mechanisms. Third, it is important to avoid any cross contamination of different sequences in the array to prevent false positive signals. Finally, since the quantity of the assay sample is often limited, it is advantageous to make the spots small and closely spaced. For high throughput screening it is required to immobilise thousands of specific sequences in a distinct position on the solid surface and this is a complex task. The very critical element in creating high-density array is dimension. Thus, the smaller the size of the array elements involved in the synthesis the more economical the device will be to produce and use. Unfortunately it is also true that the smaller is the size of array elements - the more difficult is to manufacture and replicate the array.

US patents 5795714, 6323043, 6306664 and 6251595 relate to techniques for producing or replicating arrays.

It is important to note that all the array fabrication methods mentioned above suffer from a common limitation, i.e., each array and each element of each array requires a separate synthesis and fabrication protocol which is, normally laborious, time consuming and expensive. It would be extremely desirable to develop an inexpensive method for accurate replication of complementary copies of nucleic acid arrays, and this is the subject of the present invention.

Disclosure of the Invention Broadly, the invention provides a method of producing arrays of nucleic acids comprising:

(a) providing a first set of nucleic acids immobilised on a first solid support to form a master array;

(b) furnishing on said first set of nucleic acids a second set of nucleic acids, each nucleic acid of the second set having at least a portion which is complementary to at least a portion of a respective nucleic acid of the first set and being hybridised to it thereby;

(c) immobilising said second set of nucleic acids to a second solid support, either before or while they are hybridised to the first set of nucleic acids; (d) separating the first and second solid supports to provide said first support bearing the master array and said second support bearing a complementary array.

A preferred embodiment of the present invention overcomes or ameliorates some or all of the problems and disadvantages associated with current strategies and designs and provides new methods for rapidly and accurately replicating complementary copies of nucleic acid arrays. One type of embodiment of the inventions illustrated in Fig 1, is directed to methods for replicating an array of single-stranded nucleic acid probes on a solid support comprising the steps of: a) synthesising a first multiplicity of nucleic acids 10 each comprising first (10-1) and second (10-2) portions, all of the first portions 10-1 being the same (non- variable sequence) and there being a multiplicity of different second portions 10-2 (variable sequences), attached, generally through an appropriate linker, to a solid support surface 12 to constitute a master copy 14; b) immobilising a multiplicity of nucleic acid sequences 16-1 each comprising a sequence complementary to at least part of a non- variable sequence 10-1 present in the master copy 14 to another solid support 18 to provide a blank copy 20; c) bringing the two solid supports (master copy 14 and blank copy 20) in contact with each other and hybridising the nucleic acids 10-1, 16-1 present in the non- variable sequences (assembly 22); d) chemically or enzymatically extending (16-2) the nucleic acids 16-1 of the non- variable sequences of the blank copy using the non- variable sequences 10-1 of the blank copy as primers and the variable sequences 10-2 of the master copy as templates; e) optionally after denaturing the set of hybridised nucleic acids, separating the master copy and the secondary copy 24 which now resembles the replicated

(complementary) array of nucleic acids in the master copy.

The solid supports used for array preparation and replication can be porous or non- porous plastics, ceramics, glass, metals, resins, gels, membranes, silicon, silicon dioxide and/or nitride, semiconductors or possibly a two- or three-dimensional array such as a chip or microchip.

Nucleic acids of the invention include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be isolated from natural sources, produced using recombinant DNA technology or artificially synthesised. They also might include polyamide nucleic acid (PNA) or any nucleic acid analogues that have the ability to hybridise with a complementary chemical structure. Although it is not limiting, the optimal length of nucleic acid sequences in both, variable and non- variable parts is 4- 300 nucleotides.

The important step in the array replication is bringing master copy and blank copy with immobilised nucleic acids into sufficiently close contact for an effective hybridisation of complementary non-variable parts of the sequences. These parts will serve as primers in the following extension step. The nucleic acids of the set in the blank copy are enzymatically and faithfully extended to form a complementary nucleic acid chain, using one or more ribonucleotides, deoxynucleotides, deoxynucleotide triphosphates or their derivatives. This is achieved most easily with a polymerase, although a revertase or ligase could be employed,. Alternatively this extension can be performed by using chemical condensing agents such as carbodiimide, and one or more nucleotides.

The secondary copy of the array, with nucleic acid sequences complementary to these of master copy array sequences, will be formed after the separation of two solid supports with immobilised nucleic acids. This separation can be done mechanically without or with denaturing of the hybridised molecules. The denaturing is performed with heat, alkali, organic solvents, proteins, enzymes, salts or combinations thereof.

The above process may be followed by the formation of a double-stranded array by hybridising the replicated array with a second set of nucleic acids complementary to the non- variable sequence of the replicated array. These sequences can be further extended chemically or enzymatically, e.g. with a DNA polymerase, revertase or ligase and one or more ribonucleotides, deoxynucleotides, deoxynucleotide triphosphates or their derivatives.

For some practical purposes it is desirable to separate the nucleic acid sequences created in the secondary copy from the solid support. To facilitate this, such double- stranded replicated arrays may comprise a restriction endonuclease site. By using a corresponding restriction endonuclease the extended nucleic acid sequences can be easily removed from the solid support and their synthesis can be repeated once more using steps described above.

Another aspect of the invention is directed to diagnostic aids and methods utilising probe arrays for the detection and identification of target nucleic acids, although this method can also provide effective separation. For diagnostic purposes it is desirable that one or more components, e.g. selected from the non-variable, extended part of sequence in secondary copy, the solid support, linker between the solid support and non-variable sequence in the secondary copy, contain a detectable label. The detectable label may be selected from enzymes, fluorescent, luminescent and chromatic chemicals, metals, polymers, electroactive compounds, compounds with high refractive index and spatial chemicals.

Another embodiment of the invention is directed towards use of the secondary copy of nucleic acid array as a new master copy for creating a new secondary copy of the nucleic acid array, which will contain sequences analogous to these, presented in the original master copy array.

Another type of embodiment of the invention, illustrated in Fig 2, is directed to methods for replicating an array of single-stranded nucleic acid probes on a solid support comprising the steps of:

a) fixing a first array of nucleic acids 210 each comprising a non- variable sequence 210-1 and a variable sequence 210-2 to a first solid support 212; b) synthesising a first set of nucleic acids 216-1 each comprising a sequence complementary to the non- variable sequence 210-1; c) hybridising the nucleic acids of the first set to the first array for forming a hybridised array 222; d) enzymatically extending (216-2) the nucleic acids 216-1 of the first set using the variable sequences 210-2 of the first array as templates; e) bringing the nucleic acid array with set of extended sequences (223) into contact with a second solid support 218 and fixing the end parts of the extended sequences to this support to create the replicated array of single-stranded nucleic acid probes ("imprinting"); f) optionally denaturing the double stranded sequences; g) separating the two solid supports with immobilised nucleic acids, thus providing a secondary copy 224 on the second solid support 218.

Although this approach is similar to the one described above, the difference lies in the possibility of using an activated/functionalised surface on the second solid support 218 which does not contain nucleic acids for creation of a secondary copy. One example is based on biotin-avidin (streptavidin) interactions. Thus an extended set of nucleic acids hybridised with nucleic acids of a master copy array can be labelled with biotin 250 (before or after hybridisation). When the master copy is brought into contact with a blank support, containing immobilised avidin 252 (streptavidin), an interaction between biotin and avidin (streptavidin) occurs. This binding will keep the extended set of nucleic acids immobilised in an ordered fashion at the surface of blank support. After separation, performed as described above, a new secondary copy of the nucleic acid array, complementary to the sequences of master copy array will be created. The principal difference between the present approach and the approach described in USA patent 5,795,714 lies in the important step in which a master copy is brought into contact with a support copy for the creation a secondary copy. This step ensures that the nucleic acid sequences will be fixed in ordered fashion resembling the "mirror" complementary copy of the original master copy array with the correct spatial distribution of the individual array elements. Avidin-biotin interaction is an example of an effective binding technique; others include the use of antibody-antigen interactions, metal chelation, (e.g. histidine-nickel), covalent (e.g. formation of amide bonds) and reversible covalent interactions (e.g. boronic acid - cis-diol), receptor-ligand interactions etc.

Denaturing can precede the step when two solid supports are brought into contact with each other. In this case it should be done in a way that the denatured molecules of one array element keep spatially separated from the molecules of another array element. One of the ways to achieve this is to ensure that the interaction between solid support and the nucleic acid is strong enough to minimise diffusion. Alternatively the denaturing can be performed under conditions which will slow diffusion (e.g. in a gel) or in the presence of a minimal amount of water.

Similarly to previous cases the nucleic acid sequences of the first and second set suitably have a length of 4-300 nucleotides. The important but not exclusive example describes the situation when pool of second set of nucleic acids contains sequences with length 4-8 nucleotides. These sequences can be easily chemically synthesised and array of these sequences can be particularly useful for nucleic acid sequencing. Alternatively the pool of the second set of nucleic acids may be prepared from the product of a PCR amplification reaction.

The solid supports may be selected from porous or non-porous plastics, ceramics, glass, metals, resins, gels, membranes, silicon, silicon dioxide and/or nitride, semiconductors and chips. The replicated array may be hybridised with a third set of nucleic acids (which can be also taken from the pool of sequences presented in solution of the second set of nucleic acids) complementary to the sequence of the replicated array to create a double-stranded replicated array. The double-stranded portion of the replicated array might comprise a restriction endonuclease site or a detectable label or both. The detectable label may be selected from enzymes, fluorescent, luminescent and chromatic chemicals, metals, polymers, electroactive compounds, compounds with high refractive index and radioactive compounds.

Another type of embodiment of the invention, illustrated in Fig 3, is directed towards development of a method for replicating an array of single-stranded nucleic acid probes on a solid support comprising the steps of: a) synthesising a first set of nucleic acids 310 and their immobilisation to a solid support 312 in ordered fashion - to create a master copy 314; b) synthesising or otherwise providing a second set of nucleic acids 316 and preparation of a solution 360 of these molecules; c) hybridising the nucleic acids of the first set 310 to complementary nucleic acids present in the second set 316; d) separation of the master copy with hybridised sequences (322) from non- hybridised sequences of the second set; e) bringing the master copy with hybridised sequences 322 into the contact with a second solid support 318 and fixing the end part of hybridised sequences to this support ("imprinting"); f) optionally denaturing the double stranded sequences; g) separating two solid supports 312, 318 with immobilised nucleic acids, leading to formation of a secondary copy or replicated single-stranded array 324.

The selective "fishing" approach may use a second set of nucleic acids labelled with biotin 350, and a second solid support which contains immobilised avidin 352

(streptavidin). In this case the master copy will be used for "fishing" complementary sequences from a pool of nucleic acid labelled with biotin. The hybridised molecules are fixed to second solid support in ordered way, resembling the "mirror" copy of original array by bringing master copy into the contact with second solid support. An important condition is that the second solid support should contain avidin

(streptavidin) capable of strong binding to biotin. This interaction will ensure that the bound sequences will remain in the position determined by spatial distribution of the elements of original array after the separation of solid supports. The master copy and second solid support with immobilised array of nucleic acid (secondary copy) are separated mechanically with or without a preliminary denaturing step. The denaturing may be performed by using heat, alkali, organic solvents, proteins, enzymes, salts or combinations thereof.

The denaturing can precede the step when two solid supports are brought into contact with each other. In this case it should be done in such a way that the denatured molecules of one array element keep substantially the same spatial separation from the molecules of another array element. One of the ways to achieve it is to ensure that the interaction between solid support and the nucleic acid is strong enough to minimise diffusion. Alternatively the denaturing can be performed under conditions which will slow the diffusion (e.g. in a gel) or in the presence of a minimal amount of water.

Similarly to previous cases the nucleic acid sequences of the first and second sets desirably are of length 4-300 nucleotides. Most desirably, a pool of a second set of nucleic acids contains sequences with length 4-8 nucleotides. These sequences can be easily chemically synthesised and an array of these sequences can be particularly useful for nucleic acid sequencing. Alternatively the pool of a second set of nucleic acids may be prepared from the product of a PCR amplification reaction.

The solid supports may be selected from porous or non-porous plastics, ceramics, glass, metals, resins, gels, membranes and chips.

The replicated array may be hybridised with a third set of nucleic acids (which can be also taken from the pool of sequences presented in solution of the second set of nucleic acids) complementary to the sequence of the replicated array to create a double-stranded replicated array. The double-stranded portion of the replicated array may comprise a restriction endonuclease site or a detectable label or both. The detectable label may be selected from enzymes, fluorescent, luminescent and chromatic chemicals, metals, polymers, electroactive compounds, compounds with high refractive index and radioactive compounds.

The replicated array (secondary copy) may be used as a master copy to produce a new array of nucleic acids. The main advantage of the approaches present in this invention is providing the way to produce multiple copies or complementary copies of an original nucleic acid array using standard, inexpensive and fast procedures which avoid the need to carry out individual deposition events, as is the case with the current technology.

Brief Description of the Drawings

FIG. 1. Graphic representation of the replication of a master copy array using a blank copy which contains nucleic acids. FIG. 2. Graphic representation of the replication of a master copy array using a blank support which contains avidin (streptavidin).

FIG. 3. Graphic representation of the replication of a master copy array by a "fishing" approach.

Modes for Carrying out the Invention

The present invention will now be further described with reference to the following, non-limiting examples.

Example 1. Nucleic acid hybridisation on solid surface. Freshly cut mica slides (K₂O-Al₂O₃-SiO2, muscovite) were soaked in solution of 4 M NaOH containing 10% methanol for 5 min, rinsed with water, soaked in acetone and

oven dried (70°C). Mica was derivatised for 30 min with a 5% solution of aminoprolyltrimethoxysilane in acetone, rinsed with acetone and dried. An aminated surface was treated with glutaraldehyde (0.2% in water) for 30 min on ice, then rinsed with water and dried. Polyribonucleotide solution (200 Dl, 1 mg/ml) in 50 mM sodium phosphate buffer, pH 6.5 (PB) was added to each mica slide and left for 30 min on ice. Two master copy slides were modified with poly-A and poly-G. The non- reacted species were removed by washing the slides with 0.2%SDS in 50 mM PB.

Slides were rinsed with PB and water, dried in a nitrogen stream and stored at -20°C.

Hybridisation was performed by adding 10 μl of a 550 nM solution of 4',6-diamino-

2-phenylindole dihydrochloride (DAPi) to a master copy slide, followed by 10 μl of

poly-U solution and covering it gently with a glass slide. The hybridisation process was monitored by measuring the emission fluorescence spectra in the range 400-550

nm (λ exc=358 urn) at time 0 and 15 min. Table 1 shows the results of the

hybridisation experiments.

Table 1. Fluorescent emission of glass slides with hybridised nucleic acids

The results clearly indicate that hybridisation takes place between complementary sequences, one of which has been immobilised on the solid support.

Example 2. Hybridisation of nucleic acids covalently attached to polymeric membranes.

The monomer mixture used for membrane preparation contained oligourethaneacrylate (OUA), triethyleneglycol-dimethacrylate (TRJ ) and methacrylic acid (MAA) (13.5% : 76.5% : 10%, v/v). Monomers were mixed with dimethyl formamide (DMF) (2 : 1) and 1% of azo-bis-dicyclohexanecarbonitrile (ABCN). The solution was placed between glass slides, treated with dichlorodimethyl-silane, separated by Teflon film (~ 60 micron thick) and polymerisation initiated with UV light for 5 minutes. A transparent membrane was obtained which was washed out with methanol and water.

The membrane was modified with poly-deoxyribonucleic acids (∞380 bps) using protocol: 1) 30 min treatment with 0.1M N-hydroxysuccinimide (NHS) and 0.4 M N-ethyl-

N'-dimethylaminopropylcarbodiimide (EDC) mixed 1:1, followed by brief rinsing and drying

2) 1 hour incubation with 200 μL of poly-deoxyribonucleic acid (0.2 mg/ml).

3) Washing with 0.2% SDS buffer, washing with buffer and drying in nitrogen.

Hybridisation was performed by adding 5 μl of a 550 nM solution of DAPI to a

master copy membrane, and covering it gently with a blank membrane. The hybridisation process was monitored by collecting the emission fluorescence spectra

in the range 400-550 nm (λ 6X0=358 nm) at time 0, 15, 30 min, 1 hour, overnight (17 hours) and 22 hours.

Data on fluorescent emission during hybridisation are reported in Table 2.

Table 2. Fluorescent emission of polymeric membranes with hybridised DNA.

The results clearly indicate that hybridisation takes place between complementary sequences, both of which have been immobilised on the solid support.

Example 3. Hybridisation of the nucleic acids covaiently attached to the gels.

A 6.7% solution of monomers in water containing MAA, bisacrylamide (BIS) and acrylamide (AA) (10 : 5 : 85) was cast in a cassette made of glass slides treated with

γ-methacryloxypropyl-trimethoxysilane (bottom part) and dichloro-dimethylsilane

(top part). Polymerisation was initiated with 40% w:w ammonium persulfate (5 μl/ml)

and tefraethylendiaminomethane (TEMED) (5 μl/ml) at room temperature and

continued for 2 hours.

The gels were modified with poly-deoxyribonucleic acids («380 bps) using the

protocol: 1. 30 min treatment with 0.1 M N-hydroxysuccinimide (NHS) and 0.4 MN- ethyl-N'-dimethylaminopropylcarbodiimide (EDC) mixed 1:1, followed by brief rinsing and drying 2. 1 hour treatment with 50 μL of poly-deoxynucleic acids 0.2 mg/ml or 50 μl of polyribonucleic acid (1 mg/ml). 3. Washing with 0.2% SDS buffer, washing with buffer.

The calibration made for fluorescent labelled DNA indicates that the quantity of hybridised poly-nucleotides was approximately 285-770 ng/ cm² gel. Hybridisation was performed by adding 10 μL of a 100 μM solution of D API to a

master copy gel and covering it gently with a blank gel. The hybridisation process was monitored by collecting the emission fluorescence spectra in the range 400-600

nm (λ exc=358 nm) at time 15 and 80 min. Relative peak areas for the hybridisation process are reported in Table 3.

Table 3. Relative fluorescence of DNA hybridised on gels.

The results clearly indicate that hybridisation takes place between complementary sequences immobilised to different type of surfaces.

Example 4. Replication of a DNA master copy by "fishing" approach.

Poly-dT (ca. 380 bp) was physically adsorbed onto mica slides. Blank slides were prepared by immobilisation of streptavidin (0.1 mg/ml) on mica slides treated with aminopropyl-trimethoxysilane and glutaraldehyde. After incubation (1 hour, 4 °C), the slides were washed with phosphate buffer and water, and dried before use. Hybridisation was performed by adding respectively: 5 μl of biotinylated-oligo-dA-

fluorescein-isothiocyanate (FITC), 5 μl of biotinylated-oligo-dC-6-

(Tetramethylrhodamine-5(6)-carboxamido) hexanoate (Tamra), or 10 μl of their

mixture (1 : 1) to the master copy (all oligos were 40 bp). Incubation (1 hour, 4°C) was followed by washing with 125 mM sodium citrate, pH 7.0 containing 0.1% Tween-20 (SSC buffer) and drying. The transfer of DNA to the blank slide was performed by bringing master copy and slide coated with streptavidin into close contact for 1 hour. The amount of transferred biotinylated oligonucleotide was monitored by measuring the fluorescent spectra of FITC and Tamra. Table 4 reports the fluorescence peak areas measured on the copy-slide.

Table 4. Relative fluorescence of the probes after the transfer.

The results show that complementary sequence can be transferred to the blank slide.

Example 5. PCR with immobilised primers.

BAA and BAS primers (3 μl) were immobilised onto mica slides, prepared as

described for Example 1. Amplified dsDNA was boiled at 95 °C for 5 min, then

aliquots of 5 μl were spotted onto the primer-modified surface and allowed to hybridise at 42 °C. Deoxynucleotides and Taq polymerase were added to a BAS primer slide hybridised with its complementary sequence and covered with a BAA primer slide. The sandwiched surfaces underwent 4 PCR cycles (hybridisation 42 C 2 minutes, elongation 72 °C 2 minutes, de-annealing 94 °C 1 minute), after which the slides were taken apart, washed with buffer and dried. Slides then were covered with

100 μM DAPI solution and brought in the contact again. The fluorescent emission measured presented in Table 5. Table 5. Fluorescence of the DNA-immobilised slides before and after PCR.

The results show that PCR results in an enhanced signal in both cases.

Claims

1. A method of producing arrays of nucleic acids comprising:

(b) furnishing on said first set of nucleic acids a second set of nucleic acids, each nucleic acid of the second set having at least a portion which is complementary to at least a portion of a respective nucleic acid of the first set and being hybridised to it thereby; (c) immobilising said second set of nucleic acids to a second solid support, either before or while they are hybridised to the first set of nucleic acids;

(d) separating the first and second solid supports to provide said first support bearing the master array and said second support bearing a complementary array.

2. The method of claim 1 wherein the solid supports comprise materials selected from porous or non-porous plastics, ceramics, glass, metals, resins, gels, membranes, silicon, silicon dioxide, silicon nitride, semiconductors and chips.

3. The method of claim 1 or 2 wherein the solid supports are two-dimensional or three-dimensional matrixes.

4. The method of any preceding claim wherein the first set of nucleic acids are each immobilised to the first solid support via a linker.

5. The method of any preceding claim wherein separation step (d) is preceded by denaturation of the hybridised nucleic acids.

6. The method of claim 5 wherein denaturation is performed with heat, alkali, organic solvents, proteins, enzymes, salts or combinations thereof.

7. The method of any of claims 1-4 wherein separation step (d) is performed mechanically without prior denaturation of the nucleic acids.

8. The method of any preceding claim further comprising a step (e) of converting the complementary array into a double-stranded replicated array.

9. The method of any of claims 1 -7 wherein the complementary array on the second support is used as a master array to produce a new array of nucleic acids by steps corresponding to steps (b), (c) and (d).

10. The method of any preceding claim wherein the complementary array comprises nucleic acids which, when made double-stranded if necessary, each include a restriction endonuclease site.

11. A method according to any preceding claim wherein the second solid support and the second nucleic acids are adapted for specific mutual binding; and wherein step (b) comprises a step of bringing the master array into contact with a solution containing a set of nucleic acids comprising the second nucleic acids under conditions permitting hybridisation of complementary portions; and this is followed by step (c) which comprises bringing the master array with hybridised second nucleic acids into contact with the second solid support under conditions such that the second nucleic acids bind to the second solid support.

12. A method according to claim 11 comprising the steps of: a) fixing a first array of nucleic acids each comprising a non-variable sequence and a variable sequences to a first solid support; b) synthesising a first set of nucleic acids each comprising a sequence complementary to the non- variable sequence; c) hybridising the nucleic acids of the first set to the first array; d) enzymatically extending the nucleic acids of the first set using the variable sequences of the first array as templates; e) bringing the nucleic acid array with set of extended sequences into contact with the second solid support and fixing the end parts of the extended sequences to this support to create the replicated array of single-stranded nucleic acid probes; f) optionally denaturing the double stranded sequences; g) separating two solid supports with immobilised nucleic acids.

13. A method of claim 12 where denaturing precedes the step when first and second solid support are brought into contact.

14. A method of claim 12 or 13 where the extended set of nucleic acids contains biotin, the second solid support contains immobilised avidin (streptavidin), and fixation of the extended set of nucleic acids to the solid support involves the formation of biotin-avidin (streptavidin) bonds.

15. A method of claim 12 or 13 where the fixation of the extended set of nucleic acids to solid support involves antigen-antibody interaction, metal chelation, formation of amide bonds, receptor-ligand interactions or reversible covalent interaction.

16. A method according to claim 11 comprising the steps of: a) synthesising a first set of nucleic acids and their immobilisation to the first solid support to create the master array; b) synthesising a second set of nucleic acids and preparation of a solution of these molecules; c) hybridising nucleic acids of the master array to complementary copies present in the solution; d) separation of the master array with hybridised sequences from the solution; e) bringing the master copy with hybridised sequences into the contact with the second solid support and fixing the end part of hybridised sequences to this support; f) optionally denaturing the double stranded sequences; g) separating the two solid supports with immobilised nucleic acids.

17. The method of claim 16 wherein the nucleic acids of the master array have a length of 4-300 nucleotides and the nucleic acids in the second set have a length of

4-300 nucleotides.

18. The method of claim 16 or 17 wherein the nucleic acids of the second set are chemically synthesised or are product of PCR amplification.

19. The method of claim 16, 17 or 18 further comprising the step of hybridising the replicated array with a third set of nucleic acids, optionally provided by the pool of sequences present in solution of the second set of nucleic acids, complementary to the sequence of the replicated array to create a double-stranded replicated array.

20. The method of any of claims 16-19 wherein the nucleic acid sequence in the secondary copy or the solid support contain a detectable label, optionally selected from enzymes, fluorescent, luminescent and chromatic chemicals, metals, polymers, electroactive compounds, compounds with high refractive index and spatial

chemicals.

21. A method according to any of claims 1-15 wherein the nucleic acids of the first set each comprise a common first portion and a variable second portion; and said second set of nucleic acids each comprise a portion complementary to at least part of said common first portion; and step (b) comprises: (i) bringing the second set of nucleic acids into contact with the master array under conditions permitting hybridisation of complementary portions; and (ii) extending the nucleic acids of the second set using the nucleic acids of the first set to which they are hybridised as templates so that the extended nucleic acids include portions complementary to at least parts of the variable second portions of the nucleic acids of the first set.

22. The method of claim 22 wherein the detectable label is selected from the group consisting of enzymes, fluorescent, luminescent and chromatic chemicals, metals, polymers, electroactive compounds, compounds with high refractive index and spatial chemicals.