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Methods of enzymatic discrimination enhancement and surface bound double-stranded DNA

a technology of enzymatic discrimination and surface bound doublestrands, which is applied in the field of methods of enzymatic discrimination enhancement and surface bound doublestranded dna, can solve the problems of large number of complex operations, large number of manual manipulations, and complex preparation, and achieve the effect of improving the quality of hybridization signals, facilitating determination or verification

Inactive Publication Date: 2006-12-28
AFFYMETRIX INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

These methods enhance the accuracy and efficiency of nucleic acid sequencing by improving hybridization signal quality and allowing for the identification of perfectly complementary sequences, while enabling the preparation of diverse nucleic acid sequences for binding affinity studies.

Problems solved by technology

Both methods, however, require a practitioner to perform a large number of complex, manual manipulations.
For example, such methods usually require the isolation of homogeneous DNA fragments, elaborate and tedious preparation of samples, preparation of a separating gel, application of samples to the gel, electrophoresing the samples on the gel, working up the finished gel, and analysis of the results of the procedure.
Unfortunately, each of these techniques produces only a relatively low density array of polymers.
For example, the technique described in Geysen, et al. is limited to producing 96 different polymers on pins spaced in the dimensions of a standard microtiter plate.

Method used

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  • Methods of enzymatic discrimination enhancement and surface bound double-stranded DNA
  • Methods of enzymatic discrimination enhancement and surface bound double-stranded DNA
  • Methods of enzymatic discrimination enhancement and surface bound double-stranded DNA

Examples

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examples

[0254] The following examples are provided to illustrate the efficacy of the inventions herein.

[0255] A. Enhanced Discrimination Using RNase A

[0256] This example illustrates the ability of RNase A to recognize and cut single-stranded RNA, including RNA in DNA:RNA hybrids that is not in a perfect double-stranded structure. RNA bulges, loops, and even single base mismatches can, for example, be recognized and cleaved by RNase A. RNase A treatment is used herein to improve the quality of RNA hybridization signals on high density oligonucleotide arrays.

Example I

[0257] The high density array of oligonucleotide probes on a glass substrate (referred to as a “chip”) is prepared using the standard VLSIPS protocols set forth above. Moreover, the pattern of oligonucleotide probes is based on the standard tiling strategy described shown in FIG. 5. Briefly, the chip used in this example consists of an overlapping set of DNA 15-mers covalently linked to a glass surface. A set of four probes f...

example iv

[0284] In this example, a chip was made using the tiling strategy (A, C, G, T -containing probes for each base in the sequence) described above that covers a 50 base region of the protease gene of HIV-1 (SF2 strain). The probes are 11-mers, linked to the glass support by three PEG linkers. The substitution position (the position being interrogated by an A, C, G, or T base in the probe) is varied between the 5′ end of the probe, and five bases in from the 5′ end (referred to as positions end, -1, -2, -3, -4 and -5). The chip is synthesized using standard VLSIPS protocols. Prior to hybridization and ligation, the chip is phosphorylated using T4 polynucleotide kinase for 5 hours at 37° C. The target is a 75-mer oligonucleotide (denoted Hpro1), labelled at the 5′ end with fluorescein, that spans the complementary 50 base region on the chip.

[0285] The chip was initially hybridized with a 10 nM solution of Hpro1 in 6XSSP-T at 22° C. for 30 minutes. After hybridization, the chip was read,...

example i

[0290] This example illustrates the general synthesis of an array of unimolecular, double-stranded oligonucleotides on a solid support.

[0291] Unimolecular double stranded DNA molecules were synthesized on a solid support using standard light-directed methods (VLSIPS™ protocols). Two hexaethylene glycol (PEG) linkers were used to covalently attach the synthesized oligonucleotides to the derivatized glass surface. Synthesis of the first (inner) strand proceeded one nucleotide at a time using repeated cycles of photo-deprotection and chemical coupling of protected nucleotides. The nucleotides each had a protecting group on the base portion of the monomer as well as a photolabile MeNPoc protecting group on the 5′ hydroxyl. Upon completion of the inner strand, another MeNPoc-protected PEG linker was covalently attached to the 5′ end of the surface-bound oligonucleotide. After addition of the internal PEG linker, the PEG is photodeprotected, and the synthesis of the second strand proceed...

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Abstract

Methods for discriminating between fully complementary hybrids and those that differ by one or more base pairs and libraries of unimolecular, double-stranded oligonucleotides on a solid support. In one embodiment, the present invention provides methods of using nuclease treatment to improve the quality of hybridization signals on high density oligonucleotide arrays. In another embodiment, the present invention provides methods of using ligation reactions to improve the quality of hybridization signals on high density oligonucleotide arrays. In yet another embodiment, the present invention provides libraries of unimolecular or intermolecular, double-stranded oligonucleotides on a solid support. These libraries are useful in pharmaceutical discovery for the screening of numerous biological samples for specific interactions between the double-stranded oligonucleotides, and peptides, proteins, drugs and RNA. In a related aspect, the present invention provides libraries of conformationally restricted probes on a solid support. The probes are restricted in their movement and flexibility using double-stranded oligonucleotides as scaffolding. The probes are also useful in various screening procedures associated with drug discovery and diagnosis. The present invention further provides methods for the preparation and screening of the above libraries.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. Ser. No. 08 / 327,522, filed Oct. 21, 1994, and U.S. Ser. No. 08 / 327,687, filed Oct. 24, 1994, each of which is incorporated by reference in its entirety for all purposes.GOVERNMENT RIGHTS [0002] Research leading to the invention was funded in part by NIH Grant No. ______, and the government may have certain rights to the invention.BACKGROUND OF THE INVENTION [0003] The relationship between structure and function of macromolecules is of fundamental importance in the understanding of biological systems. Such relationships are important to understanding, for example, the functions of enzymes, structural proteins, and signalling proteins, the ways in which cells communicate with one another, the mechanisms of cellular control and metabolic feedback, etc. [0004] Genetic information is critical in the continuation of life processes. Life is substantially informationally based, and genetic cont...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68B01J19/00C07B61/00C07H21/00C12N15/10C40B40/06G01N33/53G01N33/551
CPCC07H21/00C07B2200/11C12Q1/6827C12Q1/683C12Q1/6837C12Q1/6874C40B40/06C40B80/00G01N33/5308G01N33/551B01J19/0046B01J2219/00529B01J2219/00608B01J2219/00612B01J2219/00626B01J2219/0063B01J2219/00659B01J2219/00722C12N15/1093
Inventor LOCKHART, DAVIDCHEE, MARKVETTER, DIRKDIGGLEMANN, MARTIN
Owner AFFYMETRIX INC
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