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Direct SNP detection with unamplified DNA

A technology for detecting samples and detecting oligonucleotides, which is applied in the field of direct SNP detection using non-amplified DNA, and can solve problems such as not ideal

Inactive Publication Date: 2006-12-13
内诺斯佩尔公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a major disadvantage of microarray and real-time PCR-based assays is the need for PCR, which makes them less than ideal from both a clinical and cost standpoint (see discussion of PCR for SNP identification above)

Method used

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  • Direct SNP detection with unamplified DNA
  • Direct SNP detection with unamplified DNA
  • Direct SNP detection with unamplified DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0106] One-step and two-step hybridization methods for SNP identification in non-amplified genomic DNA using nanoparticle probes

[0107] Prepared by the methods described in PCT / US97 / 12783, filed July 21, 1997, PCT / US00 / 17507, filed June 26, 2000, PCT / US01 / 01190, filed January 12, 2001 Gold nanoparticle oligonucleotide probes for the detection of target Factor II, MTHFR and Factor V sequences, which are incorporated by reference in their entirety. image 3 The use of oligonucleotide-conjugated gold nanoparticle probes for the detection of target DNA using DNA microarrays with wild-type or mutant capture probe oligonucleotides is conceptually illustrated. The oligonucleotide sequence bound to the nanoparticle is complementary to one part of the target sequence, and the capture oligonucleotide sequence bound to the glass chip is complementary to another part of the target sequence. Under hybridization conditions, the nanoparticle probe, capture probe, and target sequence bind ...

Embodiment 2

[0141] Hybridization conditions of the method of the present invention

[0142]The standard recommended protocol for efficient hybridization reactions described in the prior art (T. Maniatis, E.F. Fritsch, and J. Sambrook in "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, 1982, p324) mainly specifies the hybridization temperature ~10-20 degrees Celsius below the Tm, which is calculated based on the selected hybridization conditions, including salt and formamide concentrations. There are different methods for calculating Tm values, each based on the exact oligonucleotide sequence and buffer conditions. For example, such calculations can be performed using computer programs, commercially available or on-line, such as HYTHER developed on the Wayne State University website. TM server. The inventor will HYTHER TM All available programs on the server were used to perform these calculations, resulting in Tm values ​​for capture and detection probes (ie oli...

Embodiment 3

[0145] Preparation of Nanoparticle-Oligonucleotide Conjugated Probes

[0146] In this example, representative nanoparticle-oligonucleotide conjugated probe probes for PCR amplification of mec A and Tuf gene targets were prepared. Using the method described in PCT / US97 / 12783, filed July 21, 1997, PCT / US00 / 17507, filed June 26, 2000, PCT / US01 / 01190, filed January 12, 2001 Preparation of gold nanoparticle-oligonucleotide probes for detecting target mec A or Tuf gene sequences, the entirety of these documents is incorporated by reference.

[0147] (a) Preparation of gold nanoparticles

[0148] Such as Frens, 1973, Nature Phys.Sci., 241 : 20 and Grabar, 1995, Anal.Chem. 67 : 735, gold colloids (diameter 13nm) were reduced by using citrate to reduce HAuCl 4 to prepare. Briefly, all glassware is in aqua regia (3 parts HCl, 1 part HNO 3 ) and washed with Nanopure H 2 O rinse, then oven dry before use. HAuCl 4 and sodium citrate were purchased from Aldrich Chemical Company. h...

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Abstract

The present invention provides methods for detecting a target nucleic acid molecule in a sample that comprises nucleic acid molecules of higher biological complexity than that of amplified nucleic acid molecules. In particular, the present invention provides methods and probes for detecting a single nucleotide polymorphism (SNP) in a sample that comprises nucleic acid molecules of higher biological complexity than that of amplified nucleic acid molecules.

Description

[0001] This application is related to, and claims priority from, U.S. Provisional Application Nos. 60 / 432,772 and 60 / 433,442, filed December 12, 2002, the disclosures of which are incorporated herein by reference. technical field [0002] The present invention relates to methods for detecting target nucleic acid molecules in a sample comprising nucleic acid molecules of higher biological complexity than amplified nucleic acid molecules, for example in genomic DNA. In particular, the present invention relates to methods and probes for detecting SNPs using nanoparticle-labeled probes. The present invention also relates to methods of detecting biological organisms, in particular bacterial pathogens such as staphylococcal DNA, and detecting antibiotic resistance genes, such as the mecA gene conferring resistance to the antibiotic methicillin, in a sample. Background technique [0003] Single nucleotide polymorphisms (SNPs), or single base variations, between genomic DNA observed...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/00
Inventor 鲍奕佳萨达卡·S·马拉尤维·米勒詹姆斯·J·斯托霍夫苏珊·R·赫策尔
Owner 内诺斯佩尔公司