Method for the light-controlled synthesis of biochips

Details Method for the light-controlled synthesis of biochips Method for the light-controlled synthesis of biochips

2001-11-28

2004-01-06

Riley, Jezia (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S094000, C536S025300, C536S026100, C536S027400, C536S028500, C536S055300, C514S789000

Reexamination Certificate

active

06673544

ABSTRACT:

This application is a National Stage of International Application PCT/DE99/04051, filed Dec. 17, 1999; which claims the priority of DE 198 58 440.7, filed Dec. 17, 1998.
FIELD OF THE INVENTION
The present invention relates to a method for the light-controlled (photolithgaphic) synthesis of biochips.
BACKGROUND OF THE INVENTION
The field concerning the synthesis of oligonucleotides and DNA or RNA chips has been booming for several years, and efficient methods are required in this connection.
Synthetic DNA fragments (oligonucleotides) are nowadays prepared almost exclusively in accordance with to the phosphite amide method. This preparation is made with the aid of what is called DNA synthesizers. After inputting the target sequence, these machines automatically synthesize the desired DNA oligomer. For this purpose, the DNA synthesizer has a computer-controlled programming determining in which order certain reagents are pumped onto the synthesis column where the actual synthesis of the DNA oligomer takes place in the solid phase on glass beads of e.g. controlled pore glass (CPG). The reagents include inter alia the 4 (protected) phosphite amides for the 4 bases (adenine, cytidine, guanosine, thymine), the catalyst (usually tetrazole), an oxidizing agent (usually iodine), capping reagent A & B (usually acetic anhydride, N-methylimidazole), and detritylation reagent (usually trichloroacetic acid). A reaction cycle consists of the following sequence:
(1) detritylation
cleavage of the temporary 5′-O-dimethoxytrityl
protecting groups by trichloroacetic acid;
(2) condensation
the phosphite amide corresponding to the target
sequence is condensed onto the released 5′-hydroxyl
function; this is done with the aid of the acidic catalyst
(tetrazole);
(3) oxidation
the unstable phosphite triester linkage (P-III) is
converted by oxidation with iodine into a stable
phosphorus triester (P-V);
(4) capping
the 5′-hydroxyl functions which did not react in the
preceding step shall be trapped by an acetylation step
(acetic anhydride + N-methylimidazole); this shall
prevent uncontrolled growth of the DNA strand.
Steps (1) to (4) are then repeated until the target sequence has been obtained.
The initiator nucleoside, which carries preferably a 5′-O-dimethoxytrityl protecting group, is usually found on the support already, so that the detritylation step is started. As soon as the target sequence has been obtained, the phosphate protecting groups and the protecting groups of the exocyclic amino functions of the heterobases have to be cleaved. This is usually done by means of ammonia. The cleavage of the oligomer from the support also takes place simultaneously with ammonia, so that the fully synthesized oligomer is then in the ammonia cleavage solution. Having evaporated the ammonia solution, the target oligomer is obtained.
The step decisive for the quality of the synthesis is the condensation, since all of the other steps (detritylation, oxidation, capping) proceed quantitatively. The condensation is carried out in the absolute absence of humidity. Condensation yields of up to 99% can be achieved.
The synthesis of nucleic acid chips on support surfaces is carried out according to an analogous method, so that, in principle, the below described chip synthesis methods are also suitable for the preparation of oligonucleotides if they are removed from the support surface.
Some time ago, another method was developed for the nucleic acid-chip synthesis: the light-controlled chip synthesis. The phosphite amides (A, C, G, T) used in the light-controlled synthesis have the same chemical structure as in the “normal” DNA synthesis on a commercial DNA synthesizer, the only difference being that the 5′-acid-labile dimethoxytrityl protecting group is replaced by a photolabile protecting group which may be at position 5′ or 3′. This leads to the following reaction course on the DNA chip synthesizer:
(1) Irradiation
cleavage of the temporary photolabile protecting group
by irradiation using light of corresponding wavelength;
(2) condensation
the phosphite amide corresponding to the target
sequence is condensed onto the released hydroxyl
function; this is done with the aid of the acidic catalyst
(tetrazole);
(3) oxidation
the unstable phosphite triester linkage (P-III) is
converted by oxidation with iodine into a stable
phosphorus triester (P-V);
(4) capping
the hydroxyl functions which did not react in the
preceding step shall be trapped by an acetylation step
(acetic anhydride + N-methylimidazole);
this shall prevent uncontrolled growth of the
DNA strand.
Steps (1) to (4) are then repeated until the target sequence has been obtained.
The initiator nucleoside is usually not found on the support, so that the condensation step is started with here. As soon as the target sequence has been obtained, the phosphate protecting groups and the protecting groups of the exocyclic amino functions have to be split off as well.
This is done by means of ammonia but under milder conditions (2 h) so that the DNA strands synthesized on the chip are not split off the support. This becomes possible by using more labile protecting groups than employed for the common synthesis of CPG materials as protecting groups of the exocyclic amino functions. The DNA chip is then simply removed from the ammoniacal solution, washed with water and can immediately be used for hybridization experiments.
The quality of the DNA chip synthesis is determined in the light-controlled method not only by condensation alone but, above all, by the efficiency of the cleavage of the photolabile protecting groups which only in rare cases is as effective as the cleavage of the dimethoxytrityl protecting group by means of acid. Since yields of up to 95-99% can usually be achieved in the condensation step, the quality of the DNA chip is determined more or less by the efficiency of the photoprotecting group cleavage.
WO 96/18634 and WO 97/44345 are special photolabile protecting groups of the 2-(2-nitrophenyl)ethyl type which shall be suitable for the preparation of oligonucleotides on a DNA chip. However, the inventors have already found out that it is very difficult to split off the photolabile protecting groups shown in these applications by common methods and that the preparation of DNA or RNA chips is not very efficient. Moreover, it turned out that due to their inferior quality chips prepared in such a way cannot be detected by means of fluorescence, which is current standard to detect DNA chips. Probe labeling by means of radiography would be required for the detection of these chips. This is, however, not usable for commercial exploitation.
The object of the present invention consists in providing a method by means of which high-quality biochips, in particular DNA or RNA chips, can be produced.
This object is achieved by the subject matters defined in the claims.
SUMMARY OF THE INVENTION
The present invention is achieved by a method for the light-controlled biochip synthesis in which photolabile protecting groups of the 2-(2-nitrophenyl)ethyl type are used. The irradiation step common in the light-controlled chip synthesis is carried out in the presence of a base.


REFERENCES:
patent: 5539082 (1996-07-01), Nielsen et al.
patent: 5744305 (1998-04-01), Fodor et al.
patent: 5763599 (1998-06-01), Pfleiderer et al.
patent: 5981734 (1999-11-01), Mirzabekov et al.
patent: 6153744 (2000-11-01), Pfleiderer et al.
patent: 36 06395 (1987-03-01), None
patent: WO 96/18634 (1996-06-01), None
patent: WO 97/44345 (1997-11-01), None
Chemical Abstracts 129, p. (1998).
Fodor, et al., “Light-Directed, Spatially Addressable Parallel Chemical Synthesis,”Research Articlepp. 767-773 (1991).
Falbe and Regitz, Römpp Chemie Lexikon pp. 419, 3060.
Giegrich, et al. “New Photolabile Protecting Groups in Nucleoside and Nucleotide Chemistry—Synthesis, Cleavage Mechanisms and Applications,”Nucleosides and Nucleotides,17(9): 1987-1996 (1998).
Pfister, et al. “The 2-(4-Nitrophenyl)ethylsolfonyl (Npes) Group: A New type of Protection in Nucleoside

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