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A method for preparing long dna probes containing multiple repeating units

A technology of repeating units and long probes, applied in the field of oligonucleotide probe preparation, can solve the problems of limiting the magnification of bDNA structure and lower connection efficiency, and achieve the effect of increasing the magnification and increasing the sensitivity

Active Publication Date: 2022-04-05
沈阳中科赛尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insufficient probe length will affect the number of repeat units in the preamplifier and amplifier, severely limiting the magnification of bDNA constructs
In order to increase the number of repeating units in the preamplifier and amplifier, some literatures spliced ​​together three probes with lengths of 86nt, 79nt and 73nt by bridging to obtain a 239nt multi-repeating unit probe 【4】 , but as the number of spliced ​​fragments increases, the connection efficiency will be greatly reduced, and it is still limited by the length

Method used

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  • A method for preparing long dna probes containing multiple repeating units
  • A method for preparing long dna probes containing multiple repeating units
  • A method for preparing long dna probes containing multiple repeating units

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of 800nt, 20 repeat unit DNA long probes

[0031] (1) Preparation of a plasmid containing 20 repeating unit sequences, the specific steps are as follows:

[0032] 1. Design 20 repeating unit sequences (sequence R, see Sequence Listing 1), with restriction sites EcoRV and Nb.BbvCI at both ends.

[0033] Synthesized by Sangon Bioengineering Co., Ltd. and linked into pUC57 to obtain a new plasmid pR20 containing the sequence R (attached figure 1 ) and stored in Escherichia coli.

[0034]2. Inoculate Escherichia coli containing pR20 in 3ml Amp LB medium, and culture at 37°C 200r / min for 16h.

[0035] 3. Use Axygen's plasmid mini-extraction kit (AP-MN-P-50) for plasmid extraction. (The steps are consistent with those provided in the kit)

[0036] 4. EcoRV (NEB R0195S), Nb.BbvCI (NEB R0631S) double-digest pR20, the specific system is:

[0037]

[0038] The reaction conditions are: 37°C, 2h; 80°C, 10min.

[0039] 5. Use Axygen's PCR Cleanup kit (AP-PCR-50G...

Embodiment 2

[0075] Preparation of 400nt, 10 repeat unit DNA long probes

[0076] (1) Preparation of a plasmid containing 10 repeating unit sequences, the specific steps are as follows:

[0077] 1. Design 10 repeating unit sequences (sequence T, see Sequence Table 2), the restriction sites at both ends are SmaI and Nt.BbvCI respectively, synthesized by Sangon Bioengineering Co., Ltd. and ligated into pUC57. Obtain new plasmid pT10 containing sequence T (attached image 3 ) and stored in Escherichia coli.

[0078] 2. Inoculate Escherichia coli containing pT10 in 3ml Amp LB medium and culture at 37°C 200r / min for 16h.

[0079] 3. Use Axygen's plasmid mini-extraction kit (AP-MN-P-50) for plasmid extraction. (The steps are consistent with those provided in the kit)

[0080] 4. SmaI (NEB R0141V), Nt.BbvCI (NEB R0632S) double digestion of pT10, the specific system is:

[0081]

[0082] The reaction conditions are: 30°C, 2h; 80°C, 10min.

[0083] 5. Use Axygen's PCR Cleanup kit (AP-PCR-5...

Embodiment 3

[0105] Preparation of 200nt, 5 repeat unit DNA long probes

[0106] (1) Preparation of a plasmid containing 5 repeating unit sequences, the specific steps are as follows:

[0107]1. Design 5 repeating unit sequences (sequence F, see Sequence Table 3), the restriction sites at both ends are EcoRV and Nt.BbvCI respectively, synthesized by Sangon Bioengineering Co., Ltd. and ligated into pUC57. A new plasmid pF5 containing sequence F was obtained (attached Figure 4 ) and stored in Escherichia coli.

[0108] 2. Inoculate Escherichia coli containing pT10 in 3ml Amp LB medium and culture at 37°C 200r / min for 16h.

[0109] 3. Use Axygen's plasmid mini-extraction kit (AP-MN-P-50) for plasmid extraction. (The steps are consistent with those provided in the kit)

[0110] 4. EcoRV (NEB R0195S), Nt.BbvCI (NEB R0632S) double enzyme digestion pF5, the specific system is:

[0111]

[0112] The reaction conditions are: 37°C, 2h; 80°C, 10min.

[0113] 5. Use Axygen's PCR Cleanup kit ...

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Abstract

The invention relates to the preparation of oligonucleotide probes, in particular to a method for preparing long DNA probes containing multiple repeating units. Using blunt-end endonuclease and nicking endonuclease to simultaneously cut the plasmid containing multiple repeat unit DNA double strands, and then use the principle of lambda exonuclease to specifically cut, after cutting, separate by electrophoresis to obtain multiple repeat units Single-stranded DNA of interest for the unit. The method of the present invention is suitable for preparing DNA probes of all sequence components, especially for sequences that are difficult to perform PCR amplification, such as those containing multiple repeating units, and the length of the prepared probe can reach 800nt, solving the problem of multiple repeating unit DNA Technical issues in the preparation of long probes.

Description

technical field [0001] The invention relates to the preparation of oligonucleotide probes, in particular to a method for preparing long DNA probes containing multiple repeating units. Background technique [0002] Lambda exonuclease is a highly persistent exonuclease that selectively cleaves phosphorylated single strands from the 5'-3' end of double-stranded DNA. The most suitable substrate for Lambda exonuclease is 5' phosphorylated blunt-ended double-stranded DNA. It uses the 3'-end nucleic acid strand as a template to cut sequentially from the 5' end of the DNA, and forms at the center of both ends. single chain 【1】 . Another characteristic of Lambda exonuclease is that it cannot cut from the 5' end of the nick (nicking) site and the gap site 【2】 . [0003] Branched-DNA signal amplification technology (branched-DNA, bDNA) is a nucleic acid hybridization signal amplification technology introduced by Chiron Company. This detection method has the characteristics of high ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12Q1/6806C12Q2521/301C12Q2521/319C12Q2565/125
Inventor 郭海燕黄妙玲王伟伟
Owner 沈阳中科赛尔生物科技有限公司
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