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Preparation method and application of DNA capturing probe

A technology of capturing probes and magnetic beads, applied in the field of DNA capture probe preparation, can solve the problems of rising probe synthesis cost and high cost of artificial base synthesis method, and achieve the effect of reducing preparation cost and avoiding cost increase.

Active Publication Date: 2015-01-14
FOURTH MILITARY MEDICAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0006] The biggest drawback of the "tile coverage design, artificially synthesized base" method is that the cost is too high, and with the increase of the target area, the cost of probe synthesis rises sharply

Method used

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  • Preparation method and application of DNA capturing probe
  • Preparation method and application of DNA capturing probe
  • Preparation method and application of DNA capturing probe

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Embodiment Construction

[0028] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

[0029] (1) The preparation method of human mitochondrial DNA capture probe comprises the following steps:

[0030] 1. Amplify the full-length mitochondrial DNA sequence with three pairs of primers (1F and 1R, 2F and 2R, 3F and 3R);

[0031] The conditions for the amplification are:

[0032] (1) PCR reaction system:

[0033] The DNA polymerase used is Toyobo KOD FX, product number KFX-101.

[0034] reaction system:

[0035]

[0036] (2) The PCR reaction program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 8 min; 40 cycles; extension at 72°C for 15 min.

[0037] The sequences of the primers are shown in Table 1:

[0038] Table 1

[0039]

[0040] 2. Mix the amplification products of the three pairs of primers equimolarly, and break them into fragments of about 200bp...

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Abstract

The invention provides a preparation method and application of a DNA capturing probe. The preparation method of the probe comprises the steps of amplifying a target gene; randomly breaking; and labeling with biotin. The traditional tile coverage is replaced with the design thought of random coverage; compared with the traditional probe preparation method in which a great number of DNA coverage target regions are needed to be synthesized, the preparation method provided by the invention has the advantages that only amplification primers of the target regions are needed to be synthesized, and the problem of cost increase caused by direct synthesis of the probe is avoided, so that the preparation cost is greatly reduced and the capturing sensitivity and specificity are same as those of a probe prepared by using the traditional probe preparation method.

Description

technical field [0001] The invention belongs to the field of nucleic acid probe preparation, and in particular relates to a preparation method and application of a DNA capture probe. Background technique [0002] Traditional Sanger sequencing utilizes a DNA polymerase to extend a primer bound to a template to be sequenced until a chain-terminating nucleotide is incorporated. Each reaction contains all four deoxynucleotide triphosphates (dNTPs) mixed with limited amounts of fluorescently labeled dideoxynucleoside triphosphates (ddNTPs) of different colors. Since ddNTPs lack the 3-OH group required for elongation, elongated oligonucleotides are selectively terminated at G, A, T, or C. The termination point is determined by the corresponding dideoxygen in the reaction. They have a common starting point, but end at different nucleotides. Fragments of different sizes can be separated by capillary electrophoresis, and the sequence can be deduced by observing fluorescent labels o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B30/04
Inventor 邢金良李德洋
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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