Non-diagnostic gene localization method

A gene positioning and purpose technology, applied in the field of detection analysis and biological application, can solve the problems of time-consuming, low accuracy, high cost, etc., and achieve the effect of shortening the detection time and good biocompatibility

Active Publication Date: 2022-01-04
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to overcome the deficiencies of the prior art, the present invention provides a gene localization method for non-diagnostic purposes, which is appl...

Method used

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  • Non-diagnostic gene localization method
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  • Non-diagnostic gene localization method

Examples

Experimental program
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Embodiment 1

[0070] Example 1: Positioning method using only Nb.BssSI digestion and triangular origami probe labeling

[0071] The PCR amplification process of the target sequence in step S1, its concrete operation is:

[0072] PCR reaction of linear DNA: the sequences of the upstream and downstream primers in this process are designed by the software Primer-BLAST. The 50 μL reaction system contains: 10 μL 5×LongAmp Taq reaction solution, 2 μL dNTP mixture (2.5 mM), 1 μL LongAmp Taq DNA polymerase, 4 μL upstream primer (10 μM), 4 μL downstream primer (10 μM), 1.5 μL LambdaDNA, 27.5 μL ultrapure water. The components were mixed quickly on ice and transferred to a PCR machine. Set the PCR reaction program as follows: ① 95 °C, 2 min; ② 98 °C, 10 s; 42 °C, 50 s; 72 °C, 7 min (set 30 cycles); ③ 72 °C, 10 min; slowly cool down to 4 °C for storage. The nucleotide sequence of the Lambda DNA is provided in GenBank Accession No. J02459.1; the full length of the Lambda DNA is 48502 bp, the upstrea...

Embodiment 2

[0081] Example 2: Positioning method using only Nb.BbvCI enzyme digestion and cross-shaped origami probe labeling

[0082] Embodiment 2 except that following steps are different, all the other operations are identical with embodiment 1:

[0083] The enzyme digestion system used in step S2 is: Nb.BbvCI digestion into 30 uL system contains: 1 uL Nb.BbvCI, 3 uLRutSmart™ buffer, 26 uL of the DNA obtained in step S1;

[0084] In step S3, use the triblock primer and T4 DNA Ligase to fill the "gap", the nucleotide sequence of the triblock primer is shown in sequence NO: 8, sequence NO: 8: GCTGAGGTTTTTAAAAAAAAAAA; according to Example 1 Atomic force microscopy characterization in , the results are as follows Figure 4c and 4d as shown, Figure 4c It is a schematic diagram and an atomic force microscope characterization map of gene localization using triangular origami probes after digestion with Nb.BbvC on the short double-stranded template strand after linear double-stranded Lambd...

Embodiment 3

[0086] Example 3: A gene localization method using two nicking endonucleases and corresponding origami probe markers at the same time, the specific operations are as follows:

[0087](1) Restriction endonuclease digestion reaction: This process includes two restriction endonuclease digestion reactions, and the digestion is 30uL. The system contains: 1 uL Nb.BbvCI, 1 uL Nb.BssSI, 3 uL NEBuffer™ r3.1, 25 uL The DNA obtained in step 1 was incubated at 37°C for 6 h, then inactivated at 80°C, and then purified with TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 kit. Since Nb.BbvCI and Nb.BssSI are in NEBuffer™ The activity in r3.1 is 100%, so the above reaction buffer is selected as NEBuffer™r3.1.

[0088] (2) PCR reaction to fill the "gap" DNA: 28 uL reaction system contains: 3 uL T4 DNA LigaseReaction Buffer, 2 uL 1X TA-Mg 2+ Buffer, 23 uL "gapped" DNA. The components were mixed quickly on ice and transferred to a PCR machine. Set the PCR reaction program as follows: ① ...

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Abstract

The invention discloses a non-diagnostic gene localization method. The non-diagnostic gene localization method is suitable for DNA in different forms; the DNA in different forms comprises annular single-stranded DNA and linear double-stranded DNA; and the method comprises the specific operation steps of changing the annular single-stranded DNA into double-stranded DNA and changing the linear double-stranded DNA into double-stranded DNA with proper length through PCR (Polymerase Chain Reaction); and adding restriction endonuclease for digestion to obtain a DNA chain with a gap, then adding a triblock primer and DNA ligase to obtain a required template, finally adding a DNA origami probe, carrying out PCR annealing to enable the template to capture the free DNA origami probe, and carrying out atomic force microscope characterization to achieve visual gene localization. According to the gene positioning method, 6-7 basic groups can be amplified and positioned, higher accuracy is achieved, and meanwhile the method has the advantages of biological sample polymorphism, high resolution, visualization and rapid detection.

Description

technical field [0001] The invention belongs to the field of detection analysis and biological application, and in particular relates to a non-diagnostic gene positioning method. Background technique [0002] In genetics, genetic markers mainly include morphological markers based on the differences in species morphological traits and biochemical markers using isozymes. The essence is to locate genes on chromosomes. Genemapping is genome sequencing, pathogenic bacteria An important means in the field of identification, specifically refers to the determination of the linkage group or chromosome to which the gene belongs and the position of the gene on the chromosome. Gene mapping is a basic work in genetics research, and its purpose is not only to locate gene fragments on the chromosome , but also to determine the order and distance of the linear arrangement of genes on the chromosome. [0003] The results obtained based on the above markers are indirectly reflected in the di...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6816
CPCC12Q1/6806C12Q1/6816C12Q2531/113C12Q2521/301C12Q2521/501C12Q2565/519C12Q2525/30C12Q2565/601
Inventor 晁洁熊金鑫
Owner NANJING UNIV OF POSTS & TELECOMM
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