Arrays with modified oligonucleotide and polynucleotide compositions

a technology of oligonucleotide and polynucleotide, which is applied in the direction of biomass after-treatment, organic chemistry, specific use bioreactor/fermenter, etc., can solve the problems of no technique for identifying the type of binding partner, probe sequence on the array, etc., and achieve the effect of speeding up the cycle time for regeneration

Inactive Publication Date: 2004-06-24
LAKEWOOD AMEDEX
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  • Abstract
  • Description
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Benefits of technology

0105] After detection of the test sample is complete, the array may be regenerated by removal and/or degradation of the test sample. For example, a two hour incubation of the sample-bound array in an acid solution at pH 1.5, 39.degree. C., results in complete loss of a full-length unmodified 14-mer oligonucleotide. Under these conditions the bound array oligonucleotides of the invention maintain full length structural integrity. Following the acid incubation, a variety of wash conditions may be used to clear the test sample from the probe array. For example, increased temperature incubation of a low salt wash solution would result in the dissociation of short test fragments from the array. Alternatively, a chemical denaturant (e.g., ur

Problems solved by technology

Despite the wide variety of array technologies currently in preparation or available on the market, there is currently no technique for distinguishing the type

Method used

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  • Arrays with modified oligonucleotide and polynucleotide compositions

Examples

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example 1

Synthesis, and Purification of Modified Nucleic Acids

[0124] Oligonucleotides having 2'-5' linkages and 3'-methoxy substitutions were synthesized using commercial phosphoramidites on commercially purchased DNA synthesizers from 1 mM scales using standard phosphoramidite chemistry and methods that are well known in the art, such as, for example, those disclosed in Stec et al., J. Am. Chem. soc. 106:6077-6089 (1984), Stec et al., J. Org. Chem. 50(20):3908-3913 (1985), Stec et al., J. Chromatog. 326:263-280 (1985), LaPlanche et al., Nuc. Acid. Res. 14(22):9081-9093 (1986), and Fasman, Practical Handbook of Biochemistry and Molecular Biology, 1989, CRC Press, Boca Raton, Fla., herein incorporated by reference.

[0125] Oligonucleotides were deprotected following phosphoramidite manufacturer's protocols. Unpurified oligonucleotides were either dried down under vacuum or precipitated and then dried. Sodium salts of oligonucleotides were prepared- using the commercially available DNA-Mate (Bar...

example 2

Specificity of Different Modified Oligonucleotides

[0131] The binding specificity of 20-mer modified oligonucleotides (5'-ggt ggt tcc tcc tca gtc gg-3'; SEQ ID NO:1) to its RNA or DNA complement (5'-ccg act gag aag gaa cca cc-3'; SEQ ID NO:2) were tested in a solution having 10 mM NaPO.sub.4 with three different NaCl concentrations: no salt, 100 mM NaCl, and 1M NaCl. The RNA or DNA test molecules were labeled with a fluorescent label, Oregon Green 488, to allow detection of these molecules upon hybridization to the test sequences. Following hybridization, the level of binding of each of these molecules was determined by the level of Oregon Green 488 that was detectable on the controlled pore glass (CPG) beads having the specific modified oligonucleotides.

[0132] Results were as follows:

1TABLE 1 Binding of Polymers Having Different Backbones with RNA and DNA Sample numbers are shown in parenthesis. Binding Partners No salt 100 mM NaCl 1000 mM NaCl 2'-O-Me DNA (1) 133207 (2) 86762 (3) 8...

example 3

Isoation and Detection using a Bead Array

[0134] Magnetic beads are coated with 2'-5'linked 3'-methoxy substituted oligonucleotides having sequences complementary to transcripts of genes known to be involved in breast cancer, such as HER-2, p53, ras and the like. Each bead is coated with multiple copies of an oligonucleotide having the same gene-specific sequence. Approximately 100 ng of each oligonucleotide are added to 1 .mu.g of coated magnetic beads and incubated for 10 minutes. Magnetic beads were collected using a cobalt magnet and washed in PBS, followed by resuspension in 1 ml PBS, and the beads combined for the creation of the bead array. All incubations are performed with gentle agitation or vortex mixing at room temperature.

[0135] Cells taken from a breast tumor are assayed by exposing 1 ml samples of homogenized tumor tissue and 1 ml of homogenized normal breast tissue, diluted 1:10 in PBS, with 1 to 2 .mu.g of the bead array for 1 hour at room temperature and washed thre...

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Abstract

The present invention provides arrays having associated modified oligonucleotides that selectively bind to DNA or RNA, methods of making such arrays, assays for using such arrays, and the like. In one embodiment, the arrays of the invention exhibit an increased binding affinity with complementary nucleic acids, and in particular with complementary RNA. In another embodiment, the associated nucleic acids of the array of the invention exhibit substantial acid resistance, allowing the arrays to be treated with low pH solutions. In another embodiment, the modified associated nucleic acids of the array of the invention exhibit substantial resistance to nuclease degradation.

Description

[0001] This application is a continuation-in-part of our earlier filed application Ser. No. 09 / 408,088, filed Sep. 29, 1999, which is a continuation-in-part of application Ser. No. 09 / 223,498, filed Dec. 30, 1998, to each of which we claim priority under 35 U.S.C. .sctn. 120 and which are each incorporated herein by reference in their entirety.[0002] The field of this invention is arrays having associated modified oligonucleotides and / or polynucleotides, methods of producing such arrays, and uses thereof.[0003] Arrays of binding agents, such as oligonucleotides and polynucleotides, have become an increasingly important tool in the biotechnology industry and related fields. These arrays, in which a plurality of binding agents are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like. One important use of arrays is in the analysis of differenti...

Claims

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Application Information

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IPC IPC(8): C07B61/00C07H21/00C07H21/04C12M1/34C12M3/00C12Q1/68
CPCC07H21/00C07B61/00
Inventor DALE, RODERIC M.K.
Owner LAKEWOOD AMEDEX
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