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Partially neutral single-stranded oligonucleotide

A single-stranded oligonucleotide and nucleotide technology, applied in the field of partially neutral single-stranded oligonucleotides, can solve the problems of reducing detection sensitivity, difficult to solve FET detection and quantitative analysis, reducing Debye length, etc.

Inactive Publication Date: 2017-01-25
HELIOS BIOELECTRONICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The surface charge distribution of the antibody and the binding orientation of the bound antibody make it difficult to resolve FET detection and quantification
Additionally, the media concentration of salt in the binding buffer also reduces the Debye length and in turn reduces detection sensitivity

Method used

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  • Partially neutral single-stranded oligonucleotide
  • Partially neutral single-stranded oligonucleotide
  • Partially neutral single-stranded oligonucleotide

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0067] Example 1: Synthesis of partially neutral single-stranded oligonucleotides

[0068] Deoxycytidine (n-ac) p-methoxyphosphoramidite, thymidine p-methoxyphosphoramidite, deoxyguanosine (n-ibu) p-methoxyphosphoramidite and deoxyadenosine (n-bz) p-Methoxyphosphoramidites (all purchased from ChemGenes Corporation, USA) were used according to a given sequence based on solid-phase phosphotriester synthesis or by Applied Biosystems 3900 high-throughput DNA synthesizer (by Biosci&Tech or Mission Biotech) synthesized oligonucleotides.

[0069] The synthesized oligonucleotides were reacted with weak base in toluene at room temperature for 24 hours, and the samples were subjected to ion exchange chromatography to adjust the pH to 7. After the sample is concentrated and dried, partially neutral single-stranded oligonucleotides are obtained.

example 2

[0070] Example 2: Partially Neutral Single-Stranded Oligonucleotides Used in FET Assays

[0071] Complementary H1: 5'-CCATTGTGACTGTCCTCAAGTAGGTTGACAGAGTGTG-3' (SEQ ID No.1)

[0072] Non-complementary H5: 5'-TGATAACCAATGCAGATTTG-3' (SEQ ID No.2)

[0073] DNA probe: 5'-NH 2 -C6-CACACTCTGTCAACCTAC-3' (SEQ ID No. 3)

[0074] nDNA probe: 5'-NH 2 -C6-CACACTCTGTCAACCTAC-3' (all neutral) (SEQ ID No.4)

[0075] Partially neutral single-stranded probe 1 (hereinafter "modified 1"): 5'-NH 2 -C6-C n AC n AC n TC n TG n TCAACCTAC-3' (SEQ ID No. 5)

[0076] Partially neutral single-stranded probe 2 (hereafter referred to as "modified 2"): 5'-NH 2 -C6-C n ACA n CTC n TGT n CAACCTAC-3' (SEQ ID No. 6)

[0077] The superscript n indicates an electrically neutral nucleotide.

[0078] Surface modification for probe immobilization

[0079] Through the SiNW surface layer (SiO 2 ) for probe immobilization. First, (3-aminopropyl)triethoxysilane (APTES) was used to modify the surfac...

example 3

[0090] Example 3: Partially Neutral Single-Stranded Oligonucleotides Used in Polymerase Chain Reaction

[0091] in SYBR TM Partially neutral single-stranded oligonucleotides are used in the Green system's polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR).

[0092] Template: pUC19

[0093] Product length: 202bp

[0094] Table 2: Primer sequences

[0095] name sequence (5' to 3') SEQ ID No. FP_R TTAGCTCACTC A TTAGGCAC

7 RP_R GGGCCTCTTCGCTATTACGC 8 FP_R_m12 CTCACTC C TTAGGCAC

9 FP_n10,14 CTCACT n CATT n AGGCAC

10 FP_m12_n10,14 CTCACT n CCTT n AGGCAC

11 FP_n11,13 CTCACTC n AT n TAGGCAC

12 FP_m12_n11,13 TTAGCTCACTC n CT n TAGGCAC

13

[0096] Superscript n indicates an electrically neutral nucleotide; "_" indicates an unmatched nucleotide

[0097] Table 3: PCR program:

[0098]

[0099] Table 4: qPCR program:

[0100]

[0101] PCR resu...

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Abstract

The present invention relates to a detection unit comprising a solid surface and a partially neutral single-stranded oligonucleotide comprising a first portion. The first portion is attached to the solid surface; the length of the first portion is about 50% of the total length of the partially neutral single-stranded oligonucleotide; and the first portion comprises at least one neutral nucleotide and at least one unmodified nucleotide. The invention improves the detection sensitivity and accuracy of the detection of biomolecules.

Description

technical field [0001] The invention relates to a molecular detection technology. More specifically, the present invention relates to partially neutral single-stranded oligonucleotides. Background technique [0002] Molecular detection plays an important role in clinical diagnosis and molecular biology research. Several systems have been developed for molecular detection in order to detect and / or identify target biomolecules in a sample. Wherein, according to Watson-Crick base pairing rules, single-stranded oligonucleotides are used when detecting and / or identifying targets with specific nucleotide sequences. [0003] In detail, detection and / or identification of targets generally involves the ability to distinguish single base variants. Typically, for multiple detections and / or identifications in one operation, several single-stranded oligonucleotides are coated on a microarray substrate to hybridize a fluorescently labeled sample mixture. In such microarrays, operating...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12M1/34C12Q1/68
CPCC12Q1/004G01N27/26G01N33/68C12N15/11C12Q1/6816C12Q2565/519C12Q1/6804C12Q1/682C12Q1/6832C12Q1/6825C07H21/04G01N33/5308C12Q2565/607C12Q2565/628C12Q2525/113G01N33/536G01N33/54373G01N21/658G01N2440/00G01N27/4145C12Q1/6837C12Q1/6841G01N21/554G01N33/54393G01N33/552
Inventor 詹维康杨裕雄陈文逸
Owner HELIOS BIOELECTRONICS INC