GB2440209A

GB2440209A - Nucleic acid arrays replication

Info

Publication number: GB2440209A
Application number: GB0614209A
Authority: GB
Inventors: Alessandra Bossi; Olena Volodimirivna Piletska; Sergey Anatoliyovich Piletsky; Anthony Peter Francis Turner; Philip James Warner
Original assignee: Cranfield University
Current assignee: Cranfield University
Priority date: 2006-07-18
Filing date: 2006-07-18
Publication date: 2008-01-23
Also published as: GB0614209D0

Abstract

A method for replicating an array of single-stranded nucleic acid probes on a solid surface comprising: attaching nucleic acids comprising non-variable and variable sequences to a solid support (master copy), coating another solid support with covalently immobilised non-variable nucleic acids complementary to a part of a non-variable sequence on the master copy (blank copy), bringing the two solid supports in contact and hybridising the nucleic acids presented in non-variable sequences, extending the nucleic acids of the non-variable sequences using the non-variable sequences of the blank copy as primers and variable sequences of the master copy as templates, denaturing the hybridised nucleic acids and separation providing a replicated array of nucleic acid presented in the master copy.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for manufacturing nucleic acid arrays and their application 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.

2. Background.

Ordered arrays of oligonucleotides immobiised 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 inkject 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.

The present invention has certain degree of similarity with WO 01/32935 and US Pat. 5,795,714, in the sense that they all describe the way to manufacture/replicate DNA arrays. An essential difference between the present invention and the one described in WO 01/032935 is that the previous one teaches use of the second substrate (where transfer takes place) which is in fact a second microarray with pre-determined spatial positioning of the sequences associated with a distinct address on the substrate. The invention presented here does not require it and in reality the blank copy could be the substrate homogeneously coated with single nucleic acid sequence. This is essential for reducing the cost of arrays manufacturing. Yet another advantage would be the possibility to replicate nanoarrays where to achieve the spatial positioning of nanosize elements would be extremely difficult. The substantial difference between the present invention and US Pat. 5,795,714 lies in the fact that the last one describes the transfer of nucleic acid material between primary and secondary copy AFTER the denaturing of nucleic acid complexes which could seriously affect the accuracy of the array formation.

Thus 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 an 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.

3. Summary of the Invention

The present invention overcomes 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 embodiment of the invention is directed to methods for replicating an array of single-stranded nucleic acid probes on a solid support comprising the steps of: a) synthesising an array of nucleic acids (master copy) comprising a non-variable sequence and variable sequences, attached through an appropriate linker to the solid support surface; b) homogeneous coating of another solid support (blank copy) with a covalently immobilised non-variable sequence, complementary to at least part of a non-variable sequence present in the master copy; c) bringing two solid supports (master copy and blank copy) in contact with each other and hybridising the nucleic acids presented in non-variable sequences; d) chemically or enzymatically extending the nucleic acids of the non-variable sequences of blank copy using the non-variable sequences of blank copy as primers and variable sequences of the master copy as templates; e) denaturing the set of hybridised nucleic acids and separating master and secondary copy which now resembles the replicated (complementary) array of nucleic acids presented 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 andlor nitride, semiconductors or possibly a two-or three-dimensional array such as a chip or microchip. In contrast to the master copy, which has distinctive special distribution of array elements, blank copy is homogeneously coated with single nucleic acid sequence.

The advantage for this lies in the possibility of reducing cost of array fabrication. It could also essential advantage for manufacturing of nanoarrays. Thus if both, master and blank copies contain nanosize array elements it would be extremely difficult to bring them into contact in the way that separate elements on both copies will match each other. The replication of nanoarrays will be much easier if only master copy contains nanosize elements with pre-organised positioning.

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 complimentary chemical structuie. Although it is not limiting, the optimal length of nucleic acid sequences in both, variable and non-variable parts are 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 following extension step. The nucleic acids of the set in the blank copy are enzymatically and faithfully extended with a 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, e.g. 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.

Another embodiment of the invention is directed to 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 with a DNA polymerase, revertase or ligase and one or more nbonucleotides, 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. Due to this the next embodiment of the present invention involves development of such double-stranded replicated arrays which 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 embodiment 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 a diagnostic purposes it is desirable that some or few of the following components: 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 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.

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

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

Example 1. Nucleic acid hybridisation on solid surface.

Freshly cut mica slides (K2OAl2O3SiO2, muscovite) were soaked in solution of 4 M NaOH containing 10% methanol for 5 mm, rinsed with water, soaked in acetone and oven dried (70 C). Mica was derivatised for 30 mm with a 5% solution of aminoprolyltrimethoxysilane in acetone, rinsed with acetone and dried. An aminated surface was treated with gluthardialdehyde (0.2% in water) for 30 mm on ice, then rinsed with water and dried. Poly-ribonucleotide solution (200 t1, 1 mg/mI) in 50 mM sodium phosphate buffer, pH 6.5 (PB) was added to each mica slide and left for 30 miii 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 nitrogen stream and stored at -20 C.

Hybridisation was performed by adding 10 gil of a 550 nM solution of 4',6-diamino-2-phenylindole dihydrochloride (DAPJ) to a master copy slide, 10 gil 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 (A exc=358 nm) at time 0 and 15 mm. Table 1 shows the results of the hybridisation experiments.

Table 1. Fluorescent emission of glass slides with hybndised nucleic acids Slides hybridised Time = 0 miii Time = 15 mm Poly-A slide + 794000 851000 Poly-U ________________ ________________ Poly-G slide + 720000 683400 poly-U ____________ ____________ C) 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 the nucleic acids covalently attached to the polymeric membranes.

The monomer mixture used for membrane preparation contained oligourethaneacrylate (OUA) and triethyleneglycol-dimethacrylate (TRIM) and methacrylic acid (MAA) (13.5%: 76.5%: 10%, v/v). Monomers were mixed with dimethyl formaniide (DMF) (2: 1) and 1% of azo-bis-dicyclohexanecarbonitrjle (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 mm treatment with 0.1M N-hydroxysuccinimide (NHS) and 0.4 M N-ethyl-N'-dimethylammopropylcarbodjimide (EDC) mixed 1:1, followed by brief rinsing and drying 2) 1 hour incubation with 200 pL of poly-deoxyribonucleic acid (0.2 mg/mI).

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

Hybridisation was performed by adding 5 lii of a 550 nM solution of DAPI to a master copy membrane, covering it gently with a blank membrane. The hybridisation process was monitored by collecting the emission fluorescence spectra in the range 400-550 nm (?. exc=358 nm) at time 0, 15, 30 mm, 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.

Hybridisation Peak Area, AU Lfoly-T / poly-A 51.69 control 27.51 The results clearly indicate that hybridisation takes place between complementary sequences, both of which have been immobilised on the solid support.

Example 3. Hybndisation of the nucleic acids covalently 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 y-methaclyloxypropyltrimethoxysjIane (bottom part) and dichioro-dimethylsilane (top part). A polymerisation was initiated with 40% w:w ammonium persulfate (5 j.tl/ml) and tetraethylendianjinomethane (TEMED) (5 p.1/mI) at room temperature and continued for 2 hours.

The gels were modified with poly-deoxyribonucleic acids (380 bps) using the protocol 1. 30 mm treatment with 0.1 M N- hydroxysuccinjmjde (NHS) and 0.4 M N-ethyl-N'-dimethylaminopropylcarbodiimjde (EDC) mixed 1:1, followed by brief rinsing and drying 2. 1 hour treatment with 50 jiL of poly-deoxynucleic acids 0.2 mg/mI or 50 j.il of polyribonucleic acid (1 mg/mI).

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 ngf cm2 gel.

Hybridisation was performed by adding 10 j.iL of a 100 iM solution of DAPI to a master copy gel, covering it gently with a blank gel. The bybridisation process was monitored by collecting the emission fluorescence spectra in the range 400-600 nm (?. exc=358 inn) at time 15 and 80 mm. Relative peak areas for the hybridisation process are reported in

Table 3.

Table 3. Relative fluorescence of DNA hybridised on gels.

Hybridisation Re!. Peak Area. Re!. Peak Area, L Time 15' Time 80' LPo1y-u / poly-A 1.34 2.13 [c9ntrol 1 1 The results clearly indicate that hybridisation takes place between complementary sequences immobilised to different type of surfaces.

Example 4. PCR with immobilised primers.

BAA and BAS primers (3 d) were immobilised onto mica slides, prepared as described for Example 1. Amplified dsDNA was boiled at 95 C for 5 mm, then aliquots of 5 gI 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 MM DAPJ solution and brought in the contact again. The fluorescent emission measured presented in Table S. Table 5. Fluorescence of the DNA-immobiljsed slides before and after PCR.

PCR Sample I Sample 2 Hybridised slide 299 372 after PCR Hybridised slide 210 195 before PCR The results show that PCR results in an enhanced signal in both cases.

The accompanying drawings which are incorporated in and constitute a part of this application, illustrate the principle of the invention.

Claims

We claim: 1. A method for replicating an array of single-stranded

nucleic acid probes on a solid support comprising the steps of: a) synthesising an array of nucleic acids (master copy) comprising a non-variable sequence and variable sequences, attached through the appropriate linker to the solid support surface; b) homogeneous coating of another solid support (blank copy) with a covalently immobilised non-variable sequence, complementary to at least part of a non-variable sequence present in the master copy; c) bringing two solid supports (master copy and blank copy) in contact with each other and hybridising the nucleic acids presented in non-variable sequences; d) chemically or enzymatically extending the nucleic acids of the non-variable sequences of blank copy using the non-variable sequences of blank copy as primers and variable sequences of the master copy as templates (formation of secondary copy); e) denaturing the set of hybridised nucleic acids and separation of master and secondary copy which now comprises the replicated (complementary) array of nucleic acids presented in master copy.

2. The method of claim I wherein the variable part of nucleic acids of the array in master copy has a length 4-300 nucleotides and the non-variable part of nucleic acids has a length between about 4-300 nucleotides in length.

3. The method of claim I wherein the solid supports are selected from the group consisting of porous or non-porous plastics, ceramics, glass, metals, resins, gels, membranes, silicon, silicon dioxide andlor nitride, semiconductors and chips.

4. The method of claim I wherein the nucleic acids of the set are chemically extended with a condensing agent such as, e.g. carbodlimide and one or more nucleotides.

5. The method of claim I wherein the nucleic acids of the set are enzymatically extended with a DNA polymerase, revertase or ligase and one or more ribonucleotides, deoxynucleotides, deoxynucleotide triphosphates or their derivatives.

6. The method of claim 1 wherein denaturation is performed with heat, alkali, organic solvents, proteins, enzymes, salts or combinations thereof.

7. The method of claim 1 where master and secondary copies are separated mechanically without denaturing.

8. The method of claim I further comprising the step of hybridising the replicated array with a second set of nucleic acids complementary to the non-variable sequence of the replicated array to create a double-stranded replicated array.

9. The method of claim 1 further comprising the steps:

C

a) hybridising the replicated array with a new set of nucleic acids complementary to the non-variable sequence of the replicated array; b) chemically or enzymatically extending the nucleic acids of the non-variable sequences of the replicated array using the variable sequences of nucleic acid array as templates to create a double-stranded replicated array.

10, The method of claim I wherein the solid supports are two-dimensional or three-dimensional matrixes.

ii. The method of claim I wherein the replicated array (secondary copy) is used as a master copy to produce a new array of nucleic acids.

12. The method of claim 8 and claim 9 wherein a double-stranded portion of the double-stranded replicated array comprises a restriction endonuclease site.

13. The method of claim I wherein one or more of the following components contain a detectable label: 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 14. The method of claims 13 wherein the detectable label is selected from the group consisting of enzymes, fluorescent, luminescent and chromatic chemicals, metals, t6 polymers, eleciroactive compounds, compounds with high refractive index and spatial chemicals.

15. The methods of claims 1, 6, 9, 11, 15 where all processes or part of them are

performed in electrical or magnetic field.