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Nucleic acid separation and detection by electrophoresis with a counter-migrating high-affinity intercalating dye

An electrophoretic separation and affinity technology, applied in the field of electrophoretic separation and detection of nucleic acids, can solve the problems of high cost, inability to provide resolution, low affinity and low sensitivity of DNA detection, and achieve the effect of convenient sensitivity and resolution

Inactive Publication Date: 2005-07-27
BECKMAN COULTER INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These dyes usually involve modification of the quinolinium portion of the dye and are quite expensive
[0009] In conclusion, some conventional intercalating dyes such as EB provide high DNA resolution, but their affinity and DNA detection sensitivity are low
On the other hand, other conventional intercalating dyes such as TO provide high detection sensitivity but not ideal resolution

Method used

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  • Nucleic acid separation and detection by electrophoresis with a counter-migrating high-affinity intercalating dye
  • Nucleic acid separation and detection by electrophoresis with a counter-migrating high-affinity intercalating dye
  • Nucleic acid separation and detection by electrophoresis with a counter-migrating high-affinity intercalating dye

Examples

Experimental program
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Effect test

Embodiment 1

[0092] CE settings

[0093] A capillary (Polymicro Technology, Tucson, Arizona) with an inner diameter of 100 μm and a length of 27 cm was used in the following study. The capillary is coated with polyvinyl alcohol (PVA) before use. EB, SYBR Green I and TO were purchased from Molecular Probes (Eugene, Oregon).

[0094] The gel matrix was prepared with polyethylene oxide (PEO) purchased from Aldrich Co. (Milwaukee, Wisconsin). In short, the preparation of PEO gel is as follows. At room temperature, 2.5 g PEO (average molecular weight 4,000,000) and 2 g PEO (average molecular weight 900,000) were slowly (overnight) added to 445 ml of water under stirring. Then, 50 ml of 250 mM MOPS-Tris pH 7.55 and 325 μl of 10% NaN3 were added to the above solution. The resulting gel is stored at room temperature.

[0095] PEO gel and EB (final concentration of 30μM) were mixed to prepare washing buffer. The PEO gel was mixed with SYBR Green I (1:500 dilution) to prepare the outlet buffer. In anoth...

Embodiment 2

[0099] DNA analysis by CE-LIF using two intercalating dyes

[0100] The capillary is filled with gel buffer containing PEO gel and EB. The outlet electrode vial (anode) is filled with outlet buffer containing PEO gel and SYBR Green I or TO. The DNA sample is injected into the capillary from the cathode. After a voltage is applied between the cathode and the anode, a DNA-EB complex is formed and moves toward the anode. At the same time, SYBR Green I or TO molecules migrate from the anode to the cathode in the opposite direction due to their positive charge.

[0101] Once the anti-migrating SYBR Green I or TO molecules come into contact with the isolated DNA-EB complex, compared with EB, because the anti-migrating molecules have higher DNA affinity, they replace the EB in the DNA complex. The result is the formation of a new DNA-SYBR Green I or DNA-TO complex. Since the absolute value of the DNA charge is greater than SYBR Green I and TO, the total charge of the new complex is negat...

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Abstract

The invention provides novel electrophoretic methods for high-resolution separation and high-sensitivity detection of nucleic acids. The methods involve the use of a high-resolution buffer and a counter-migrating high-affinity intercalating dye in an electrophoresis system to achieve superior separation and detection of nucleic acids. A kit for separation and detection of nucleic acids in a sample by electrophoresis is also provided. The kit comprises a gel buffer containing a high-resolution intercalating dye with a resolution of at least 2.0 between the 271-bp and 282-bp phiX 171 HAIII nucleic acid fragments nucleic acid fragments; and a high-affinity intercalating dye having a positive charge and DNA affinity constant of at least 10<6>M<-1>.

Description

Invention field [0001] The present invention generally relates to the separation and detection of nucleic acids by electrophoresis. Specifically, the present invention relates to electrophoresis using anti-migrating intercalating dyes. Background of the invention [0002] Various methods for separating and detecting deoxyribonucleic acid (DNA) from liquid biological samples are well known in the art. One such technique is electrophoresis, which involves the migration of charged substances when they are dissolved or suspended in an electrolyte through which an electric current flows. Various conventional forms of electrophoresis are known, including free zone electrophoresis, gel electrophoresis, isoelectric focusing, isotachophoresis, and capillary electrophoresis. [0003] Conventionally, the separation and identification of nucleic acids has been accomplished by gel electrophoresis on polymer gels such as agarose gels or polyacrylamide gels. The preparation types and sizes of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/483C12N15/09C12Q1/68G01N21/78G01N27/447G01N33/50
CPCC12Q1/68C12Q2565/125C12Q2563/173
Inventor 刘明孙陈夫泰
Owner BECKMAN COULTER INC
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