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Method for rapidly detecting mismatching quantity of bases through capillary electrophoresis

A technology of capillary electrophoresis and capillary, applied in the field of biological analysis, to achieve the effect of high repeatability, simple operation, and expanded application

Active Publication Date: 2015-12-23
CHANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The invention overcomes the deficiency of detecting the number of mismatched bases in complementary DNA in the prior art, provides a method for quickly detecting the number of base mismatches by capillary electrophoresis, and broadens its application in the field of biological analysis

Method used

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  • Method for rapidly detecting mismatching quantity of bases through capillary electrophoresis
  • Method for rapidly detecting mismatching quantity of bases through capillary electrophoresis

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Experimental program
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Effect test

Embodiment 1

[0019] 1. The capillary adopts a quartz capillary with an inner diameter of 75 μm, a total length of 60 cm, and an effective length of 35 cm. The sample to be tested and the buffer solution flow in the capillary through an 18kV high-voltage power supply.

[0020] 2. (1) Design DNA1-FAM fluorescent probe, which includes two parts of fluorescent dye 6-FAM and DNA1. DNA1 is 5'-GGTTGGTGTGGTTGG-3', and the 3' end is coupled with a fluorescent dye; (2) Design five complementary DNAs, which are 5'-CCAACCACACCAACC-3' (zero mismatched bases), 5'-CCAACCACACCAAC G -3' (one mismatch base), 5'-CCAACCACACCAA GG -3' (two mismatched bases), 5'-CCAACCACACC GGGG -3' (four mismatched bases), 5'-CCAACCACA GGGGGG -3' (six mismatched bases); (3) DNA1-FAM and complementary DNA containing different numbers (0, 1, 2, 4, 6) of mismatched bases at the same concentration ratio 2:2, with an interval of 20s in the capillary, inject samples for 20s respectively, and detect by fluorescence capillary el...

Embodiment 2

[0023] 1-2 steps are the same as in Example 1.

[0024] 3. DNA3 to be detected, the sequence is 5'-CCAA GG ACACCAACC-3', carry out capillary electrophoresis, get S 1 / S Total is 0.2, compared with the standard curve, the number of mismatches detected is 2.

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Abstract

The invention relates to the technical field of biological analysis, and discloses a method for rapidly detecting the mismatching quantity of bases through capillary electrophoresis. The method comprises the following steps: 1, designing DNA of a 3' end coupling fluorescence dye to form a fluorescence probe; 2, designing several complementary DNAs having mismatching bases with the quantity different from that of the DNA fragment in step 1; and 3, respectively hybridizing the several complementary DNAs with the fluorescence probe in a capillary tube, carrying out fluorescence detection, calculating the peak area ratio (S1 / STotal) to obtain a standard curve of the peak area ratio and the quantity of the mismatching bases, hybridizing a sample to be detected with the fluorescence probe in the capillary tube, calculating the electrophoresis peak area, and comparing with the standard curve to determine the quantity of the mismatching bases in the sample to be detected. The method for rapidly detecting the quantity of mismatching bases in the capillary tube has the advantages of simple operation, high repeatability, convenient and fast detection of the quantity of the mismatching bases in the sample, and further development of the application of the DNA fluorescence probe in the field of biological analysis.

Description

technical field [0001] The invention relates to the technical field of biological analysis, in particular to a method for quickly detecting the number of base mismatches by capillary electrophoresis. Background technique [0002] Methods for detecting base sequences include Sanger method, pending sequencing method, single-molecule sequencing, etc., but detection generally requires relatively large detection instruments, and the detection cost is also high. [0003] Fluorescent dye is a kind of bright color that can absorb ultraviolet or visible light and convert it from short wavelength to longer wavelength visible light and reflect it, which can be seen by naked eyes. Due to its high sensitivity and convenient operation, fluorescent dyes have gradually replaced radioactive isotopes as detection labels. They are widely used in fluorescent probes, cell staining, and specific DNA staining. [0004] On the other hand, capillary electrophoresis, as a fast, effective, highly sen...

Claims

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Application Information

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IPC IPC(8): G01N27/447
Inventor 王建浩秦玉琴滕一万陈瑶李进晨蒋鹏举邱琳王车礼杨丽张晨澄樊杰柳丽
Owner CHANGZHOU UNIV
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