One step sample preparation and detection of nucleic acids in complex biological samples

A sample and nucleic acid technology, applied in the 0-24 field, can solve problems such as labor consumption, time-consuming, skin corrosion, etc.

Inactive Publication Date: 2002-05-22
EXIQON AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, phenol or phenol / chloroform mixtures are corrosive to human skin and are considered hazardous waste that must be handled with care and discarded correctly
Moreover, the extraction method is time-consuming and labor-intensive

Method used

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  • One step sample preparation and detection of nucleic acids in complex biological samples
  • One step sample preparation and detection of nucleic acids in complex biological samples
  • One step sample preparation and detection of nucleic acids in complex biological samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Example 1 GnHCl enables and promotes LNA hybridization in phosphate buffer

[0111] To investigate the role of strong chaotropic agents such as guanidine hydrochloride (GnHCl) in hybridization, the following experiments were performed.

[0112] LNA-modified oligomers with 5' anthraquinones (see Table 1-1) are covalently immobilized on the wells of a microtiter plate by UV (ultraviolet) irradiation, and are analyzed by hybridization with complementary target DNA oligomers used as capture probes. The hybridized material is detected by a 5' biotinylated DNA detection probe contained in the hybridization mixture.

[0113] name

EQ number

SEQ ID

NO

sequence

feature

C8

EQ-3133

4

5’-AQ-tac atg tta tgc ttt

GAC met C met GT GTg-3'

5'Anthraquinone modification

LNA

C11

EQ-3131

5

5’-AQ-tac atg tta tgc ttt

AAG AC met C met GTG

TG...

Embodiment 2

[0122] Hybridization specificity (competition experiment) in embodiment 2GnHCl

[0123] To investigate whether hybridization in a buffer containing a strong chaotropic agent such as guanidine hydrochloride (GnHCl) can exhibit sufficiently high stringency that single bases can be isolated, the following experiments were performed.

[0124] name

EQ number

SEQ ID

NO

sequence

feature

C8

EQ-3133

4

5’-AQ-tac atg tta tgc ttt

GAC met C met GT GTg-3'

5' anthraquinone modified

LNA

T8

EQ-3134

9

5’-AQ-tac atg tta tgc ttt

GAC met T GT GTg-3'

5' anthraquinone modified

LNA

Wild type

target molecule

EQ-3185

7

5’ttg aat tcc aag agc aca

cgg tct tca gtg aag ctg cag

ggc act tcc aa3'

wild type, intended

Meaning g / c pos.

9756 (50-mer)

mutant

target molecule

EQ-3187

10

5’ttg aat tcc a...

Embodiment 3

[0137] Embodiment 3GnSCN makes the hybridization in sodium citrate and phosphate buffer saline solution possible and promotes it to carry out

[0138] Standard lysis buffers for RNA preparation are usually based on sodium citrate buffer, eg Glisin (1974) Biochemistry 13, 2633 and Chirwin (1979) Biochemistry 18, 5294. The following experiments were used to compare the performance of hybridization in the presence of guanidinium thiocyanate (GnSCN) containing sodium citrate or phosphate based buffers.

[0139] name

EQ number

SEQ ID

NO

sequence

feature

C8

EQ-3133

4

5’-AQ-tac atg tta tgc ttt

GAC met C met GT GTg-3'

5' anthraquinone modified

LNA

T8

EQ-3134

9

5’-AQ-tac atg tta tgc ttt

GAC met T GT GTg-3'

5' anthraquinone modified

LNA

Wild type

target molecule

EQ-3185

7

5’ttg aat tcc aag agc aca cgg

tct tca gtg aag ctg...

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PUM

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Abstract

The present invention relates to a method for detecting said nucleic acid while releasing the nucleic acid from a complex biological sample. The present invention relates to the use of cell lysis buffers containing strong chaotropic agents such as guanidinium thiocyanate to promote cell lysis and release of cellular nucleic acids. The invention also relates to the use of new forms of bicyclic nucleotide analogs to lock nucleic acids (LNA) Detection of specific nucleic acids released during cell lysis by nucleic acid hybridization. In particular, the covalent attachment method for capturing LNA-oligomers is described. New methods for sample preparation, such as polyadenylated mRNA, have also been proposed. The present invention further provides reagents for the method and applications of the method.

Description

Background of the invention [0001] A brief description of related technologies [0002] Organic solvents such as phenol and chloroform have traditionally been used in techniques for isolating nucleic acids from prokaryotic and eukaryotic cells or complex biological samples. Typical nucleic acid isolation begins with enzymatic digestion with proteases, followed by cell lysis with ionic detergents, and extraction with phenol or a phenol / chloroform mixture. The organic and aqueous phases are then separated, while the nucleic acids partition into the aqueous phase and can be recovered by alcohol precipitation. However, phenol or phenol / chloroform mixtures are corrosive to human skin and are considered hazardous waste that must be handled with care and discarded properly. Moreover, the extraction method is time-consuming and labor-intensive. Marmur, J.Mol.Biol., 3:208-218 (1961) describes a standard preparation method for the extraction and purification of high molecular weight ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C07K14/00C12Q1/68C12Q1/6806C12Q1/6816C12Q1/6837
CPCC12Q1/6816C07K14/003C12Q1/6806C12Q1/6837C12Q1/68C12Q2525/307C12Q2527/125C12Q2565/101C12Q2537/125C12Q2537/101C12Q2527/101
Inventor J·斯科夫
Owner EXIQON AS
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