WO2006000999A1

WO2006000999A1 - Support for biological or chemical arrays having increased spot quality

Info

Publication number: WO2006000999A1
Application number: PCT/IE2005/000070
Authority: WO
Inventors: Gerasimos Mouzakitis; David Bartholomew O'leary; Thomas Cotter
Original assignee: University College Cork - National University Of Ireland, Cork
Priority date: 2004-06-24
Filing date: 2005-06-21
Publication date: 2006-01-05

Abstract

A support for supporting high-density biological or chemical arrays comprises a membrane which has a medium applied thereto to enhance spot quality and/or spot density when probe material is applied in spots to the membranes. The membrane may comprise a fibrous material which is impregnated with the medium.

Description

SUPPORT FOR BIOLOGICAL OR CHEMICAL ARRAYS HAVING INCREASED SPOT QUALITY

Technical Field

The invention relates to solid-phase hybridisation assays incorporating nucleic acids, in particular to gene arrays used for analysis of differential gene expression.

Background of Invention

The recently completed first draft of the human genome sequence indicated that the human genome was comprised of more than 30,000 genes. The interpretation of the function of each of these genes and their variants presents an enormous challenge to the scientific community. The ability to monitor different levels of switching on and off of thousands of genes simultaneously is required, in order to be able to identify different patterns. At present, the most widely utilised approach involves the use of gene arrays. Gene arrays consist of an array of hundreds or thousands of genes arrayed on a nylon membrane or a glass slide. When DNA complementary to the RNA found in a specific tissue is hybridised to such an array, the pattern of hybridisation is indicative of the extent to which each gene is switched on or off.

Historically, gene arrays evolved from Southern blot analysis, in which a single DNA fragment is hybridised to a multitude of immobilised fragments on a solid support (usually a nylon membrane) (Maskos and Southern, Nucleic Acids Res., 20, 1679-1684, 1992). This classical procedure has been reversed in gene arrays, in which a multitude of DNA fragments (called target DNA) are hybridised to individual DNA fragments (called probe DNA) immobilised on the membrane. Using such methods it is possible to analyse the differential gene expression of thousands of individual genes between different tissues, such as healthy and tumour cells, treated and untreated cell culture, and cells at different developmental stages. 0070

The data collected from a gene array analysis enable the piecing together of cellular pathways, and identification of gene/protein function(s).

In a typical gene array procedure, probe DNA fragments are applied to a nylon membrane using manual or automated methods, forming "DNA spots". The target DNA is hybridised to the immobilised probe DNA and the membrane is washed to remove un-hybridised target DNA. By comparing the intensity of each spot (i.e. the level of target DNA bound to each probe DNA spot) in different samples, differential gene expression between the samples is analysed.

Due to the nature of the gene array method, a high level of importance is placed on the quality of the probe DNA spots. To assure consistency, DNA spots must be of uniform size, circular, with clear edges. Furthermore, as the resolution of the gene array procedure is dependent on the number of probe DNA spots, there is a requirement to maximize the number of spots per unit area of membrane (spot density).

Immobilisation of DNA spots on to nylon membranes and subsequent detection has been a core technique of molecular biology laboratories for almost two decades. Despite this, there remain a number of problems with such systems. The last major advance in nylon technology was the chemical modification of nylon membranes to produce a net positive surface charge, which allows for increased binding of the negatively-charged nucleic acids. Although such positively-charged nylon membranes enhance DNA binding capacity, they do not address the issues of spot quality or spot density. Application of DNA to positively-charged nylon membranes results in diffuse, irregular spots (due to capillary action) which hampers detection and limits spot density.

There is a need for a support of this type with improved spot quality and/or maximized spot density. It will also be appreciated that in addition to gene arrays, such supports are also widely used for other types of nucleic acid applications, including RNA dot blots. A support which improves ^" spot quality and increases spot density would also be important in such non-gene array methods.

Statements of Invention

According to the invention there is provided a support for supporting high-density biological and chemical arrays comprising a membrane which has a medium applied thereto to enhance spot quality and/or spot density when probe material is applied in spots to the membrane and a target material is hybridised to the probe material.

In one embodiment the membrane comprises a fibrous material which is impregnated with the medium.

In one embodiment the membrane is of a polymeric material. The membrane may be of a polyamide material. The membrane may be of a nylon material.

The medium may be any suitable substance that is more hydrophobic than the probe material.

In one embodiment the medium comprises an alkane of the formula

CH₃(CH₂)_nCH₃

where n is an integer from 4 to 16.

The value of n may, for example, be 8 or 10. In one embodiment the medium is oil. The oil may be a mineral oil.

In one embodiment the medium comprises a mixture of an alkane and an oil. The alkane may be dodecane or decane and the oil may be a mineral oil

The medium may contain other chemical substances: such as additives, stabilisers, preservatives, and/or anti-foaming agents, etc. Such substances may improve handling and/or stability of the medium or media-impregnated supports.

The probe material may be a nucleic acid such as RNA or DNA.

The invention also provides a method of carrying out an assay using a support of the invention. The assay may be a solid-phase hybridisation assay.

The present invention provides for methods for altering the surface characteristics of membranes, especially nylon membranes, to allow for improved quality during the application of nucleic acid solutions. The invention provides for chemicals and methods for impregnation of such membranes using at least one media, methods for application and attachment of nucleic acids, and methods for removal of the impregnation media.

In all methods for the application of nucleic acid 'spots' on such membranes, the nucleic acid is simply applied to the dry membrane. The nucleic acid spots (which are applied from an aqueous solution) are allowed to dry and the nucleic acid is bound to the nylon.

In the invention, the membrane is impregnated with media prior to application of the nucleic acids. This treatment alters the surface characteristics of the support resulting in uniform, circular nucleic acid spots. In this method the membranes are exposed to the media which permeates the structure of the membrane. The nucleic acids are applied to the media-impregnated support, dried, and processed as normal for binding. Following binding of the nucleic acids to the support, hybridisation reactions can be performed.

Most preferably the impregnation media has a higher hydrophobicity index than the nucleic acid aqueous solution. Therefore, when the aqueous nucleic acid solution is applied to the slightly hydrophobic, media-impregnated nylon membrane it forms a circular, well-defined drop; this is similar to a droplet of water on an oily film. The media serves to contain each droplet and prevents diffusion due to capillary action. The nucleic acid spots are allowed to permeate into the media-impregnated membrane, dried and processed for binding the nucleic acid to the nylon as normal. Hybridisation can then be performed as normal.

There are many methods that have been developed to deliver specific amounts of nucleic acid solutions to a support. Most preferably, the nucleic acid solution applied to the media-impregnated support is delivered using non-contact methods, including, but not limited to inkjet technology. A person knowledgeable in the art can select a suitable non-contact method for delivery of the nucleic acid solution to the media-impregnated membrane.

The impregnation media may or may not be volatile and so may or may not be removed.

If the impregnation media is composed of a multiplicity of components, at least one component may be removed before or after the application of nucleic acids to the membrane. Brief description of the drawings

The invention will be more clearly understood from the following descriptions thereof, given by way of example only, with reference to the accompanying drawings, in which:

Fig. 1 shows nucleic acid spots applied to nylon membranes impregnated with different media. Un - untreated; DE - Decane treatment; DO - Dodecane treatment; DM - Dodecane/Mineral Oil (4:1 volume/volume);

Fig. 2 is a graph showing the relative size of nucleic acid spots applied to media-impregnated nylon membranes;

Fig. 3 shows the time course of removal of Impregnation Media;

Fig. 4 is a graph showing the spot area during time course of removal of Impregnation Media;

Fig. 5 shows the removal of Impregnation Media by using high temperature; and

Fig. 6 is a graph showing the spot area during removal of Impregnation Media by using high temperature.

Detailed Description

The invention provides methods for altering the surface characteristics of membranes, including chemicals and methods for media impregnation of membranes, methods for application and attachment of nucleic acids and methods for removal of impregnated media. 000070

Chemicals and Methods for Media Impregnation of Membranes

The invention provides methods of impregnation of membranes with a medium to alter its surface characteristics, particularly in the application of nucleic acids.

Most preferably the impregnation media is a chemical or a multiplicity of chemicals with a hydrophobicity index higher than that of the nucleic acid aqueous solution that will be spotted on the membrane. When such a media is applied to the membrane, it confines the aqueous nucleic acid solution and prevents spreading due to capillary action, forming circular, well-defined nucleic acid spots.

The constituents of the impregnation media may or may not be volatile.

Impregnation of the membrane with the media may be performed using a wide range ' of methods, including, but not limited to immersion or spraying. A person knowledgeable in the art would be able to decide upon a suitable method. Regardless of the impregnation method used, the nylon membrane surface is evenly and homogeneously covered in the media.

Regardless of the impregnation method, the membrane is brought into contact with the impregnation media. Following a given period of time to allow for saturation of the membrane matrix, any excess impregnation media is removed. This ensures that there is a homogeneous environment on the membrane for depositing the aqueous nucleic acid solutions. Methods for Application of Nucleic Acids on to Media-Impregnated Nylon Membrane

Application of nucleic acid solutions into spots on media-impregnated membranes can be performed using a wide-variety of methods. A person knowledgeable in the art will be able to select the most appropriate method.

Most preferably the application of nucleic acids on to media-impregnated nucleic acids is performed using 'non-contact' methods, including, but not limited to, manual application by micro-pipette and inkjet technology. In such non-contact methods, only the nucleic acid solution contacts the membrane allowing for a circular, well-defined nucleic acid spot.

Application of nucleic acid solution using 'contact' methods, such as the mechanical stamping with pins, are also preferable. In such methods, a pin covered in a nucleic acid solution comes into contact with a nylon membrane. Using non-impregnated nylon membrane, the nucleic acid is transferred on to the membrane by capillary action. Such contact methods are not ideal though for media-impregnated membranes as the pins deposit the nucleic acid over a large area. Furthermore, the hydrophobic media prevents capillary action, leading to variable transfer of nucleic acids from the stamping pin.

Most preferably, a multiplicity of nucleic acid solutions is applied on each membrane.

Once the nucleic acid solution is applied on the media-impregnated membrane, they are allowed to enter the membrane matrix and dried under controlled conditions.

A person knowledgeable in the art can select any suitable method for cross-linking and immobilising the nucleic acids to the nylon membrane. 0

A person knowledgeable in the art can select any suitable method for hybridisation of nucleic acids to the immobilised nucleic and methods for detection of the hybridisation.

Methods for Removal of Impregnation Media

The impregnation media may be partially or fully removed from the membranes following the application of nucleic acids.

The constituents of the impregnation media may or may not be volatile.

If any of the constituents of the impregnation media are volatile, they may be removed following the application of the nucleic acids using a range of methods, including, but not limited to, controlled ventilation and/or vacuum. A person knowledgeable in the art can select the most appropriate method.

Some methods for cross-linking nucleic acids to a support involve subjecting the nylon to a vacuum and/or increased temperatures. Using such procedures it is possible to cross-link the nucleic acids on to the membrane while simultaneously removing the volatile constituents of the impregnation media.

Examples

General Solutions and Methodologies

The following solutions were routinely used.

a) GAPDH nucleic acid solution Using mouse genomic DNA, in conjunction with GAPDH forward and reverse primers (see below), PCR was carried out under the conditions outlined in Table 1.1. GAPDH forward primer sequence: 5' ACC ACA ATC CAT GCC ATC AC 3' GAPDH reverse primer sequence: 5' TCC ACC ACC CTG TTG CTG TA 3'

Table 1.1

PCR reagents Stock Concentration Final Concentration 1 OX Taq polymerase buffer 1 OX IX MgCl ₂ 25mM 2.OmM dNTP (G, A, T₅ C) 1 OmM 0.2mM GAPDH forward primer 40ng/μl 40ng/20μl GAPDH reverse primer 40ng/μl 40ng/20μl Red hot Taq polymerase 5U/μl IU

PCR cycling conditions PCR amplification was performed using initial melting incubation at 95°C for 4 minutes, followed by 30 cycles at 95⁰C for 30s, 58⁰C for 30s, 72⁰C for 30s. Following amplification, products were maintained at 4⁰C.

GAPDH PCR amplification products were purified by electrophoretic separation. GAPDH PCR amplification products (5μl) were mixed with lμl of 6X loading dye (0.05% bromophenol blue, 0.05% xylene cyanol, 15% glycerol,) and then separated on a 1% (weight/volume) agarose gel. PCR amplification products were visualised using ethidium bromide staining together with UV illumination. The appropriate GAPDH PCR amplification product was excised from the gel and purified by G-25 columns (Amersham Pharmacia).

The purified GAPDH PCR amplification products were quantified using a Hybaid spectrophotometer and diluted to 100 ng/μl using sterile water. b) Preparation of GAPDH nucleic acids for spotting on nylon membranes Purified GAPDH nucleic acid were denatured by heating to 95⁰C for five minutes and immediately placed on ice for 2 minutes. Nucleic acids were diluted to a final concentration of 2.5 ng/μl in Spotting Buffer (TE and 0.01% weight/volume bromophenol blue dye). This GAPDH solution was used in the examples below.

c) Labelling of GAPDH PCR using 'Ready to go' labelling beads I μl of the purified GAPDH PCR (lOOμl/ng) was added to 19μl of TE buffer (Sambrook). 1 OuI of this mix (5ng/μl) was heated for three minutes at 95⁰C to denature the DNA and put on ice. The denatured DNA was added to 5μl of CX³³PdCTP and 35μl of sterile water and added to the 'ready to go ' labelling bead and vortexed to mix. This mixture was incubated at 37⁰C for 2 hours.

d) Hybridisation of spotted nylon with labelled probe The prehybridisation/hybridisation buffer (PerfectHyb, Sigma) was heated in an oven to 42⁰C. The spotted nylons were wetted with a 2X SSC and then each nylon membrane was put in a separate hybridisation tube (Hybaid) with 10ml of the prehybridisation/hybridisation buffer and incubated in an oven (rolling) at 42⁰C for two hours. The prehybridisation/hybridisation buffer was discarded and replaced with fresh prehybridisation/hybridisation buffer (10ml) along with the 50μl of labelled probe. This was incubated overnight rolling in an oven at 42⁰C. The prehybridisation/hybridisation buffer and the unbound labelled probe were discarded and the nylon was washed with 50ml of 2XSSC/0.1% SDS. This mixture was discarded and then two twenty-minute washes with 2xSSC/0.1% SDS at 68% were carried out at 68⁰C in a rolling incubator. This was followed by a short wash with 50ml 2XSSC at room temperature and the nylon was then wrapped in saranwrap and exposed to Storm (Molecular Dynamics ™) screen overnight. e) Visualisation of hybridised spots Using Imagequant software the image of the spots on each of the nylon membranes was stored on the Storm computer. These images were analysed using the Phoretics software package and densitometry analysis allowed comparison of spots across the different nylons. Three different parameters were examined using the Phoretics programme: spot volume, spot area and circularity.

Example 1. Chemicals and Methods for Media Impregnation of Nylon Membranes The invention provides for chemicals and methods for media impregnation of nylon membranes.

In this example, a manual method for media impregnation is utilised. Nylon membranes are cut into approximately two centimetre square pieces. Each nylon membrane piece is treated by complete immersion in media impregnation for five minutes at room temperature. Following this incubation, each nylon membrane is lightly blotted between two pieces of Whatmann 3MM paper to remove excess impregnation media from the surface of the nylon membrane.

To assess the effects of different chemicals on the nucleic acid spotting characteristics of nylon, membranes were treated with one of the following types of media: decane (DE), dodecane (DO), or dodecane/mineral oil (DM) (4:1 mixture volume/volume). Untreated (Un) nylon membranes were used as baseline controls. Immediately after each treatment, 2 μl of GAPDH solution (containing bromophenol blue dye, see above) was applied to each membrane in quadruplicate (Fig. 1). Due to the presence of the dye, the area of each nucleic acid spot was easily measured by densitometry.

For each nylon treatment (DE, DO, DM), the average area of the four spots was calculated and expressed as a percentage of the average spot size in the untreated membrane (Fig. 2). Under these conditions, all three treatments decreased nucleic acid area to between 50% to 55% of the area in the untreated membrane. Dodecane treatment (DO) resulted in the smallest spots, approximately 51.5% of the area of nucleic acids spots on untreated membrane. It is important to note that either a single or a multiplicity of chemicals can be utilised to alter the spotting characteristics of the nylon membrane.

Example 2. Methods for Application of Nucleic Acids onto Media-Impregnated Nylon Membranes. The invention also provides methods for application of nucleic acids onto media- impregnated membranes.

In this example, DNA nucleic acids were applied to media-impregnated membranes using four different methods. To determine spot size and quality, a radioactively labelled probe was produced, hybridised to the nucleic acids on each membrane and visualised (as described under "General Solutions and Methodologies" sections c, d, e).

For each method of application, the average spot volume (intensity) and area as well as the average reduction in spot area relative to the untreated nylon were measured (Table 2.2).

Non-contact manual spotting Non-contact manual spotting involved spotting 2μl of the purified GAPDH PCR (lOOng/ul) diluted in TE to a concentration of 2.5ng/μl. The spotting was performed by using a Gilson PlO to take up 2ul of the sample DNA into the gilson tip and then expressing the drop so that it hung from the tip. Then holding the Gilson PlO at a 45° angle to the nylon membrane, gently touching the drop on the surface of the membrane. This is a non-contact method as only the sample comes in contact with the membrane. Non-contact robotic spotting Non-contact robotic spotting was carried out commercially (GeSiM, Gesellschaft fur Silizium Microsysteme). A non-contact piezoelectric system was used to apply two spot sizes; 0.0378μl and 0.0756μl. Spots consisted of GAPDH PCR diluted in TE to a concentration of lOOng/μl. Ten spots of each size were applied to untreated nylon membranes and nylon membranes treated with various immersion media.

Contact manual spotting Contact, manual spotting of nucleic acids was carried out using a replicator (V &P Scientific, Inc). The pins of the replicator corresponded to the wells of a 96 well plate. The volume that adheres to the pin tips is 20 nl. Sample was deposited in the wells of the 96 well plate and the pins were dipped in the well and then printed on the nylon membrane. Replicator spotting involved applying 20nl of the purified GAPDH PCR diluted in TE at a concentration of 50ng/μl onto each membrane in a 3 well by 3 well grid to give a total of nine spots per membrane.

Contact robotic spotting Contact, robotic spotting was performed using a robotic spotter (Q-pix, Genetix). This worked in a similar way to the replicator used for manual contact spotting. Contact robotic spotting with the Q-pix involved spotting 500nl of the purified GAPDH PCR diluted in TE at a concentration of 50ng/μl. Samples put in the wells of a 96 well plate and the computer was programmed to dip the robotically controlled pins into the wells of the 96 well plate and print on a nylon membrane. The volume that adheres to the pin tips is 500nl. Table 2.2 Spot volume, area, and percentage reduction of nucleic acids applied to impregnated membranes using different methods.

Example 3. Methods for Removal of Impregnation Media The invention also provides methods for removal of the impregnation media following application of the nucleic acid spots. Removal of impregnation media may be desirable to ensure that it does not interfere with any subsequent treatment of the nylon membrane and/or the applied nucleic acids.

Although a person knowledgeable in the art would be aware of the best method for removal of the impregnation media, in this Example volatile media is removed by either incubation at room temperature or at elevated temperatures.

Example 3a. Removal of Volatile Media by Incubation at Room-Temperature Nylon membranes were treated and spotted with a nucleic acid/dye solution at different times post-immersion. Removal of media was monitored by measuring the area of the nucleic acid/dye spots at each time point. Untreated nylon membranes were used as baseline controls.

Nylon membranes were treated with decane (DE), dodecane (DO), or dodecane/mineral oil (DM, 4:1 ratio, volume/volume) as described in Example 1, above. Untreated membranes were used as controls.

Immediately after each treatment, 2 μl of GAPDH solution (containing bromophenol blue dye, see above) was applied to each membrane in duplicate (Fig. 3). Due to the presence of the dye, the area of each nucleic acid spot was easily measured by densitometry. This nucleic acid spotting procedure was repeated at different times post-treatment (Fig. 3).

For each nylon treatment (DE, DO, DM), the average area of the two spots was calculated and expressed as a percentage of the average spot size in the untreated membrane for that time point (Fig. 4). Note that past 0.5 hrs post-immersion, the area of nucleic acids spots in the decane-treated membrane increases to almost the area of the untreated membrane. These results are consistent with removal of volatile media from the membrane by evaporation. Similarly, past 8 hours the spot area in the dodecane-treated membranes increases to untreated levels.

Example 3b. Removal of Volatile Media by Incubation at High Temperature A common procedure in the preparation of nylon membranes for nucleic acid hybridisations is the covalent linking of the applied nucleic acids by incubation at high temperature. It is therefore possible to utilise high temperatures to covalent link the applied nucleic acids to the nylon membrane while simultaneously removing a volatile impregnation media.

To demonstrate that the high temperature incubation procedure used for covalent linking of nucleic acids to nylon membranes can also be used to remove volatile media, duplicate membranes were treated with decane (DE), dodecane (DO), or dodecane/mineral oil (DM, 4:1 ratio, volume/volume) as described in Example 1, above. Untreated membranes were used as controls. In one set of membranes, 2 μl of GAPDH solution (containing bromophenol blue dye, see above) was applied to each membrane in quadruplicate (Fig. 5, top panel). The other set of membranes was incubated at 80°C for two hours and the same nucleic acid solution applied in quadruplicate (Fig. 5, bottom panel).

Due to the presence of the dye, the area of each nucleic acid spot was easily measured by densitometry. For each nylon treatment (DE, DO, DM), the average area of the four spots was calculated and expressed as a percentage of the average spot size in the untreated membrane (Fig. 6). With high temperature incubation, the spot area in the untreated membrane did not significantly change, indicating that the incubation procedure itself does not affect the nylon spotting characteristics. Conversely, incubation of the nylon membranes treated with volatile media (decane and dodecane) resulted in significantly larger spots, demonstrating that the media was removed during this procedure. Interestingly, with nylon membranes treated with the dodecane/mineral oil solution, the high temperature incubation resulted in an increase in relative spot area from 54% to 60% (in comparison to the untreated control membrane). This result indicates that even though the non-volatile mineral oil was applied to the nylon membrane in the presence of a solvent, it is still able to decrease the nucleic acid spot area. Therefore, this Example also demonstrates that partial removal of the impregnation media may be preferable before or after application of the nucleic acid solution. For example, an impregnation solution may be applied to the nylon membranes and at least one component removed prior to application of nucleic acids.

Definition of Terms

Nucleic acids If the nucleic acid is RNA it can be total RNA or mRNA (poly-A enriched). It can be a single RNA species, but most preferably it refers to a plurality of RNA species.

If the nucleic acid is DNA it can be: genomic DNA, or fragments of genomic DNA, cDNA, an EST, a plasmid vector containing heterologous DNA nucleic acids, or an oligonucleotide. Furthermore, the DNA nucleic can be produced by purification, by an enzymatic process, or by synthetic chemistry. The DNA nucleic acid can be a single species or a plurality of DNA nucleic acid species.

It is important to note that by nucleic acid, a single or a plurality of nucleic acid species is referred to.

Solid-Phase Hybridisation Assay A solid-phase hybridisation assay is defined as an assay in which one nucleic acid is immobilised onto a solid support and hybridised to a non-immobilised nucleic acid. This type of assay includes (but is not limited to): Southern blot analysis, Northern blot analysis, DNA or RNA dot blots, and gene arrays. Membrane A sheet of filamentous or fibrous or porous material that can be used in a solid phase hybridisation assays. The membrane can be of a polymeric material such as a polyamide, especially nylon.

Media Media can be an individual or a multiplicity of chemicals. The media may or may not be volatile. The media may impregnate the membrane to suppress capillary action. Examples of suitable media are the higher alkanes such as decane or dodecane. Suitable oils such as mineral oil may also be used.

Probe and Target nucleic acids For purposes of clarity, target nucleic acid is defined as the non-immobilised nucleic acids in a solid phase hybridisation assay. The probe nucleic acids are DNA as defined above (see Nucleic acids).

Similarly, probe nucleic acids are defined as the immobilised nucleic acids in a solid phase hybridisation assay. The target nucleic acids are most preferably DNA (as defined above in Nucleic acids definition) but may also be RNA.

The invention is not limited to the embodiments hereinbefore described which may be varied in detail .

Claims

1. A support for supporting high-density biological or chemical arrays comprising a membrane which has a medium applied thereto to enhance spot quality and/or spot density when probe material is applied in spots to the membrane.

2. A support as claimed in claim 1 wherein the membrane comprises a fibrous material which is impregnated with the medium.

3. A support as claimed in claim 1 or 2 wherein the membrane is of a polymeric material.

4. A support as claimed in any of claims 1 to 3 wherein the membrane is of a polyamide material.

5. A support as claimed in any of claims 1 to 4 wherein the membrane is of a nylon material.

6. A support as claimed in any of claims 1 to 5 wherein the medium is more hydrophobic than the probe material to be applied to the medium.

7. A support as claimed in any preceding claim wherein the medium comprises an alkane of the formula

CH₃(CH₂)_nCH₃

where n is an integer from 4 to 16.

8. A support as claimed in claim 7 wherein n is 8.

9. A support as claimed in claim 7 wherein n is 10.

10. A support as claimed in any preceding claim wherein the medium is an oil.

11. A support as claimed in claim 10 wherein the oil is a mineral oil.

12. A support as claimed in any preceding claim wherein the medium comprises a mixture of an alkane and an oil.

13. A support as claimed in claim 12 wherein the alkane is dodecane or decane.

14. A support as claimed in claim 12 or 13 wherein the oil is a mineral oil.

15. A support as claimed in any preceding claim wherein the medium contains at least one of an additive, a stabiliser, a preservative or an anti-foaming agent.

16. A support as claimed in any preceding claim wherein the probe material is a nucleic acid.

17. A support as claimed in claim 16 wherein the probe material is DNA.

18. A support as claimed in claim 16 wherein the probe material is RNA.

19. A support substantially as claimed in any preceding claim, with reference to the accompanying drawings.

20. A method of carrying out an assay using a support as claimed in any preceding claim.

21. A method as claimed in claim 20 wherein the assay is a solid-phase hybridisation assay.