WO2002005947A1

WO2002005947A1 - Method of preparing nucleic acid microchips

Info

Publication number: WO2002005947A1
Application number: PCT/SE2001/001256
Authority: WO
Inventors: Zicai Liang; Anil Kumar
Original assignee: Karolinska Innovations Ab
Priority date: 2000-07-18
Filing date: 2001-06-06
Publication date: 2002-01-24
Also published as: AU2001264473A1; SE0002700D0

Abstract

A method of preparing microchips having nucleic acid attached thereto is disclosed. In the method a surface of a first chip (master chip), to which surface nucleic acid is attached, and a surface of a second chip, are brought into contact with each other, whereby the nucleic acid attached to the first chip is partially transferred to the surface of the second chip, through detachment from the first chip, and immobilisation onto the second chip (copy chip).

Description

Karolinska Innovations AB

Method of preparing nucleic acid microchips

The present invention relates to a method of producing nucleic acid microchips, and microchips obtained by the method. More particularly, microchips are prepared by the duplication of a master-chip, thereby obtaining a number of master-chip copies.

DNA microchips (or DNA micro-arrays) have rapidly evolved to become one of the essential tools for life science research, ranging over monitoring gene expression, polymorphism analysis, disease screening and diagnostics, nucleic acid sequencing, and genome analysis. They are widely anticipated to be one of the major players in clinical diagnostics and drug development in the post-genome era.

Currently available methods of fabrication of DNA chips can be classified into two categories: 1) physical deposition of prefabricated DNA or oligonucleotides onto microchips; 2) on-chip synthesis of DNA molecules (mostly short oligonucleotides) by either photolithographic synthesis or by piezoelectric printing. The methods of category 1 enjoy the advantage of lower cost and higher flexibility, but suffers from the fact that it is not suitable for making very high density chip. The methods of category 2 enable the production of very high-density chips, but the enormous set-up cost for making any chips is a serious limiting factor. Furthermore, the speed of making microchips with any of the available methods is pretty slow (the making of a 5000 DNA spots/chip microchip requires hours to be completed), and subsequently, the cost of chips thus produced has posed serious restraints on the widespread application of DNA microchips.

The aim of the present invention is to provide a new method of DNA chip fabrication that has the potential to increase the current speed of manufacture, and reduce the cost for each chip thus obtained. The invention presented here discloses and claims a novel category of methods of DNA chip production that can afford higher production speed at lower cost. In particular, the present invention discloses and claims a method of producing DNA chips by direct contacting a master chip containing multiple species of DNA molecules with another surface.

Summary of the invention

A novel method of making oligonucleotide or cDNA chips is here disclosed, namely "chemical nanoprinting", which method makes it possible to obtain multiple print chips from one "master-chip", with each duplicate printed within less than a minute, i.e., about 1,000 times faster than all existing methods. High density prints can readily be produced or reproduced, when a high density master-chip is used. The prints obtained by the present method will be essentially identical to the mirror image of the master-chip used. The reproducibility of this method, when is comes to the geometric shape and the distribution of the printed pattern obtained therewith, is thus better than for any currently known alternative technique in the art. This procedure has the potential to be combined with the in situ synthesis or physical deposition methods and increase the overall throughput of DNA chip production by a factor of 10-100. Highly complex DNA chips can thereby be rapidly generated (or reproduced).

This technology allows the direct printing of chips with a wide range of density within a matter of minutes, and importantly, has the potential to make many printout chips from a single master-chip that contains sufficient oligonucleotides or DNA linked by disulphide bonds. Also, similar processes but with different chemical or physical mechanisms are conceivable, and the use of polymers other than acrylamide as the printout matrix is also possible. Even with the current loading capacity, an increase of scanner sensitivity by a factor of 10 would allow about 100-200 prints to be made from a single master-chip.

Brief description of the drawings

Fig. 1 shows a master-chip and two copies thereof, having oligonucleotides transferred from the master-chip attached to an acrylamide layer. Fig. 2 shows two master-chips, 1 and 2, before and after printing, respectively, and prints obtained from the latter master-chip at different temperatures.

In Fig. 3, a master-chip and 10 print-chips are shown, obtained from the master-chip using varying length of heating time.

In Fig. 4, the hybridisation signals of the 10 print chips and master chip after printing, shown in Fig. 4, are compared.

Fig. 5 shows a high density master-chip and print chip obtained therefrom.

In Fig. 6, enlarged portions of the chips from Fig. 5 are shown.

Detailed description of the invention

The method of the present invention is based on partial detachment of oligonucleotides bonded to a first chip, the so called master-chip, and subsequent binding of said detached oligonucleotides to another substrate, when said substrate is brought into physical contact with the master-chip. The nature of the detachment and bonding is believed to be essentially chemical.

There have been several reports about using disulphide linkage to tether DNA or oligonucleotides onto a glass surface. Theoretically, such -S-S- linkage can also be incorporated between oligonucleotides and the solid support during DNA synthesis.

The present inventors have discovered that acrylamide has a limited reactivity towards the disulphide bond and can cause the detachment of oligonucleotides from the solid support, and simultaneous conjugation of these oligonucleotides with the acrylamide.

It has now surprisingly been found that one characteristic of the interaction between the acrylic group and the disulphide bonds is that the reaction only results in partial displacement of the oligonucleotides from the original chip (in the following referred to as the master-chip).

This fact has been demonstrated by overlaying an oligonucleotide chip with a layer of acrylamide : bis-acrylamide (20: 1), and triggering the polymerization by means of TEMED and ammonium persulfate, and then heating the chip-gel complex at 95-100°C for 1 min. Thereafter, when staining both the original chip and the polyacrylamide layer, which was bound to a glass slide coated with a Bind Silane (Amersham Pharmacia Biotech.), the phenomenon, that there is always a proportion of the oligonucleotides that becomes transferred from the oligonucleotide chip to the acrylamide layer, can be observed. As shown in Fig. 1, a strong hybridisation signal emerges on the acrylamide layer. This was initially attributed to the stripping of non-covalently attached oligonucleotides from the master-chip in the presence of acrylamide molecules.

Three lines of experimental data, however, seem to rule out this possibility: a) The oligonucleotides were bound to the glass support through a (3-mercaptopropyl) trimethyoxysilane and extensively washed; b) The experimental data suggest that more than 98% of the oligonucleotides on the original chip were tethered on the glass by end S-S linkage and non-specific binding accounts for less that 2% of the total signal. The proportion of non- specifically "stuck" oligonucleotides simply can not account for the significant level of signal changes on neither the original chip nor the polyacrylamide layer and this strongly argues that these dramatic signal changes were due to the detachment of the covalently bound oligonucleotides; c) Cy3 labelled oligonucleotides were spotted on an amino-silane-coated surface. It is known that the binding of oligonucleotide on amino surface is predominantly by electrostatic interaction (non-covalent bond). When the gel lifting procedure was repeated on these chips, no significant amount of Cy3 oligonucleotides over to the acrylamide gel was obtained. This provides another hint that the oligonucleotide transfer from chip to gel is not the simple stripping of non-covalent binding oligonucleotides.

The interaction between acrylic groups and thiol or disulphide groups has also been confirmed experimentally, according to the following scheme. For visualisation purposes an acrylic labelled oligonucleotide, Lac-acrylic (acrylic-5'-TCATGGTCATAGCTGTTTCC-3'), and a thiol labelled oligonucleotide (Lac-thio, thiol-5'- TCATGGTCATAGCTGTTTCC-3'), were utilised to react with a (3-mercaptopropyl) trimethoxysilane in solution. The immobilisation of Lac-acrylic was compared to that of Lac-thio by hybridization with a Cy3, and significant levels of covalent immobilisation of Lac-acrylic oligonucleotides was observed. The covalent nature of the linkage of Lac-acrylic to (3-mercaptopropyl) trimethoxysilane was further confirmed by repeated stripping by boiling in water. This result indicates that the acrylic group does have a limited level of reactivity towards thiol or disulphide groups. The reason why the term thiol or disulphide groups is used is that thiol groups in mercaptosilane molecules could undergo spontaneous oxidation and form disulphide bonds. It is not a major focus for the purpose of this disclosure to distinguish the two states of the mercaptosilane, but the present inventors, without wishing to be bound to any specific theory, suspect that the reactivity of acrylic groups is towards disulphide bonds.

In order to accomplish the printing, or the transfer of the oligonucleotides from the master- chip to the print chip, the oligonucleotides must be brought into intimate contact with the surface layer of the print chip. It is presently believed that, in order to be able to do this, said surface layer should preferably be in a reasonably liquid state. An alternative surface material of the chip to be printed could be a rubber material, which is compressible, and therefore not necessarily has to be in a liquid state.

Accordingly, the present inventors have found that, by loading the master chip with a sufficiently high quantity of oligonucleotides, the oligonucleotides can be "printed" onto acrylamide monolayers through this partial detachment process, caused by the interaction between the acrylic groups and the disulphide groups. For the purpose of the present invention, it is preferred that the following criteria be fulfilled: a) the master-chip should preferably be able to harbour sufficient amount of oligonucleotides for multiple printing, i.e., more than 2 prints; b) the downloading of oligonucleotides should be able to be performed in a controlled manner, so that each "printout" will be configured to have the same intensity; c) the printing should preferably be able to be done at high resolution, so that high density chips can be reproduced by this mechanism.

1 nM dots (50 pi x 1 nM/0.01mm², equals to 0.05 pmoles/cm²) can be routinely detected with a laser scanner from Genetic Microsystems, and a surface density of 5 pmoles/cm will give satisfactory hybridisation signals. Good deposition methods, such as, for example, photolithographic synthesis or by piezoelectric printing can generate oligonucleotide chips with surface density of 50 pmoles/cm² and in situ synthesis can produce oligonucleotides at a density of 1,000-2,000 pmoles/cm . These numbers suggest that current state of art technology would easily produce chips that contain sufficient oligonucleotides for printing purposes and in ideal case, these chips would enable 100-200 printouts to be reproduced from them.

Example 1

A hand-spotted master chip, as shown in Fig. 1, was used to make prints on two acrylamide- coated chips (print 1 and print 2) at a temperature of 99°C. Then all three chips were hybridised to a complementary probe, labelled with Cy3. As can be seen from Fig. 1, oligonucleotides immobilised on glass surface via disulphide bond can be transferred to acrylamide coated chips with great spatial precision.

Example 2

This Example was designed for quantitative control of downloading. The contribution of contact time and temperature to the downloading process was analysed. Seven acrylamide monolayer prints, 1 to 7, were sequentially made from a single chip, i.e. master-chip 2, at room temperature and allowed the gel, and the master-chip to remain in contact for 20 sec, 40 sec, 1, 2, 3, 4 and 5 min., respectively. With reference to Fig. 2, the hybridisation results indicated that there was no significant transfer of oligonucleotides from the master-chip to any of the printouts 1 to 7, with the exception that the first printout monolayer seems to have received a little more oligonucleotides than the other printouts, 2 to 7 (all similar or less than print 7). The somewhat higher DNA transfer of print 1, might be due to oligonucleotides that were non-covalently attached on the glass surface of the master-chip.

When making the eighth print, the chip-gel complex was put in water and the water brought to boil in a microwave (60 sec. in total), before separating the gel monolayer from the master chip 2. In contrast to prints made at room temperature, this eighth print shows a strong signal when hybridised to the fluorescent probe, indicating significantly higher levels of oligonucleotides. This result was further confirmed by the making of a ninth print from the same master-chip in the same way as for the eighth print, also shown in Fig. 2. The hybridisation signal visualised on the ninth print appears to be at similar levels as the eighth print. These results suggest that heat treatment is essential to trigger the "downloading" of oligonucleotides from master-chip to the printout acrylamide monolayers. The transfer of disulphide bond-tethered oligonucleotides from glass surface to acrylamide surface can be controlled by modulating the temperature.

Chip 2 after 9 printings, and another master-chip, chipl, were also hybridised to Cy3 labelled probes, and shown in Fig. 2.

Example 3

As mentioned above, theoretically, about 100-200 prints could be made from best slides generated from the state of the art technology. In order to prove the feasibility on this aspect, multiple copies on acrylamide coated chips were made sequentially from S-S linked oligonucleotide chips at a temperature of 99°C, with the following printing regime: print 1, 10 sec; print 2, 15 sec; print 3, 20 sec; print 4, 30 sec; print 5, 45 sec; print 6, 1 min.; print 7, 2 min.; print 8, 3 min.; print 9, 4 min.; and print 10, 10 min. Then the prints and the master- chips were all evaluated by hybridisation with a Cy3 labelled probe, as shown in Fig. 3. Thus, multiple print-chips can be manufactured by printing from a single glass chip.

In Fig. 4, the hybridisation signal of the 9 print-chips and the master chip (after printing) are compared. As can be seen, using S-S linked oligonucleotide chips of this Example as the master-chips, up to 10 prints on acrylamide monolayers can be made with similar levels of intensity, i.e., oligonucleotide transfer, with the exception of print 1, suggesting that 10 sec. may not be enough for the printing complex to be heated to a critical temperature.

Example 4

The resolution of the printing was studied by introducing an automated arrayer (Genetic Microsystems) into the process. A temperature of 95°C, and a contact time of 1 min. were used. This arrayer can deposit DNA samples at a density of 1,000 spot/cm² (10,000/chip) with a distance of 375 μm between spots (average volume of liquid delivery is 50 pl/spot). A master chip with 100 μm spots (300 μm pitch) was printed with an acrylamide coated chip. The master and print chip were then hybridised to a Cy3 labelled probe. master chip with 100 μm spots (300 μm pitch) was printed with an acrylamide coated chip. The master and print chip were then hybridised to a Cy3 labelled probe.

With reference to Fig. 5, corresponding regions of the master and print chips were enlarged, and the size of 400 spots on the print chip was compared with that of the master-chip. As evidenced by Fig. 5, only minimal resolution loss can be observed on the print chip. However, when the spots were sufficiently enlarged, a fuzzy edge around the spots on the print chip could be observed, which can amount to an increase of a few μm in diameter per spot. Since the actual oligonucleotide transfer occurs after the polymerisation of the acrylamide is, it is believed that this increase of the spot size is the result of the locomotion of individual acrylamide fibers in the subsequent handling. As long as the gaps between spots are significantly larger than 20 μm (with a spot density of 1000- 50,000 spots/cm² (10, 000- 400,000/chip), the present inventors believe that the printing resolution is high enough to generate printout chips with acceptable quality. When the spot density reaches even higher, there seems to be the likelihood that spots will start to smear each other on the printout chips. In case of the acrylamide, however, the locomotion of polyacrylamide fibers would be closely related to the degree of crosslinking of the acrylamide gel, and better polymerisation scheme may be able to diminish this "diffusing" effect, thus allowing the skilled person to make prints from chips of much higher spot density.

Other polymers and corresponding chemistry could also be used in the method of the present invention, as long as a sufficient degree of contact between the oligonucleotides and the polymer surface is guarantied, in order to allow for the detachment and binding to take place.

Although the method only has been described herein for the temperatures T, and 95 - 100°C, other, conveniently used temperatures, or temperatures which are less harsh to the specific oligonucleotides used, can readily be established by the person skilled in the art by merely performing routine experiments.

Claims

1. Method of preparing microchips having nucleic acid attached thereto, characterise d in that a surface of a first chip (master chip), to which surface nucleic acid is attached, and a surface of a second chip, are brought into contact with each other, whereby the nucleic acid attached to said surface of the first chip is partially transferred to said surface of the second chip, through detachment from the first chip, and immobilisation onto the second chip (copy chip).

2. The method according to claim 1, characterised in that high density master chips are obtainable.

3. The method according to claim lor2, characterised in that the temperature is comprised within ambient to about 100 °C.

4. The method according to any of the previous claims, characterised in that the time for obtaining the second chip is in the range 10 seconds to 20 minutes.

5. The method according to any of the previous claims, characterised in that 2 - 200 copy chips are obtained from one and the same master chip.

6. The method according to any of the previous claims, characterised in that the nucleic acids are attached to the first chip by means of disulphide bonds.

7. The method according to any of the previous claims, characterised in that the surface of the second chip is comprised of a liquid material that can be subsequently solidified into a solid surface, preferably, the surface of the second chip is comprised of an acrylamide solution containing ammonium persulfate and TEMED (N,N,N',N'-tetramethylethylene- diamine).

8. Use of a microchip having nucleic acid(s) attached thereto in the method according to any of the claims 1 to 7.

9. Microchip obtained by means of the method of any of the claims 1 to 7.