Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Monolithic matrix for separating nucleic acids by reverse-phase into-pair high performance liquid chromatography

A reversed-phase ion pair, matrix technology, applied in the field of bio-organic molecule separation, which can solve the problems of limited efficiency, reduced surface area, and high operating pressure

Inactive Publication Date: 2001-10-17
AGILENT TECH INC
View PDF1 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Columns packed with 3-micron particles are used at high operating pressures, while columns packed with 10-micron particles have limited efficiency due to longer diffusion paths and lower binding capacity due to reduced surface area

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Monolithic matrix for separating nucleic acids by reverse-phase into-pair high performance liquid chromatography
  • Monolithic matrix for separating nucleic acids by reverse-phase into-pair high performance liquid chromatography
  • Monolithic matrix for separating nucleic acids by reverse-phase into-pair high performance liquid chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Isolation of single-stranded oligonucleotides

[0062] Prepare a mixture containing 1.0 ml trimethylolpropane trimethacrylate, 0.32 ml hexyl methacrylate, 2.4 ml isooctane and approximately 20 mg azobisisobutyronitrile and inject it into an empty 4.6 mm ID× 5 cm long HPLC column tubing (one end capped with end fitting and stopper). Seal the loaded tube with an end fitting and a stopper at the other end, and then soak overnight in a water bath at 65°C. After the reaction, the stoppers and end fittings were removed, and the end fittings with polyethylene porous sheets were fitted. The entire column was flushed with tetrahydrofuran.

[0063] figure 1 It is a chromatogram of separating single-chain oligothymidylic acid with the C6 monolithic column of the above structure. Specifically, the sample is 10 microliters of oligothymidylates between 12-18 units in length. Oligonucleotides were eluted at a flow rate of 2 ml / min, gradient of 0-12% acetonitrile (prepared with 100 ...

Embodiment 2

[0065] Separation of dsDNA

[0066] Prepare a mixture containing 1.25 ml of trimethylolpropane trimethacrylate, 0.30 ml of lauryl methacrylate, 2.6 ml of a mixture of 95% isooctane and 5% toluene, and 17 mg of azobisisobutyronitrile , and inject it into an empty 4.6 mm ID x 5 cm long HPLC column (capped at one end with an end fitting and stopper). Seal the loaded tube with an end fitting and a stopper at the other end, and then soak overnight in a water bath at 65°C. After the reaction, the stoppers and end fittings were removed, and the end fittings with polyethylene porous sheets were fitted. The entire column was flushed with tetrahydrofuran.

[0067] Figure 2A It is a chromatogram of separating double-stranded DNA fragments with the single-chip C12 column of the above structure. Specifically, the sample was 3 microliters of pUC18 DNA (digested with MSP I restriction enzyme). DNA fragments were eluted at a flow rate of 1.0 ml / min with a 10 minute gradient of 35-60% 20...

Embodiment 3

[0072] Isolation of partially denatured double-stranded polynucleotides

[0073] The invention can also isolate partially denatured double-stranded polynucleotides. Such as Figure 3A with 3B As shown, a porous monolithic column was used to separate a DNA fragment of 304 base pairs in length. exist Figure 3A , the sample contains one type of DNA, Figure 3B The samples in Figure 3A A mixture of the type in and a variant sequence containing a single base substitution. at a certain temperature, Figure 3B The sample shows a second peak, indicating partially denatured DNA with a single base substitution.

[0074] Specifically, in Table 3A, homogeneous double-stranded DNA with a length of 304 base pairs was separated on the monolithic 12 column prepared as above. Table 2 lists the conditions under which this separation was performed, as follows:

[0075] mobile phase:

[0076] observed under similar conditions Figure 3B data, but contains samples other than ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides monolithic polymer matrices for the separation of bio-organic molecules by liquid chromatography. In one embodiment, the matrix is formed from a polymerization mixture including (i) a hydrophobic monomer, (ii) a crosslinking agent, and (iii) a porogenic solvent or mixture of porogenic components. The monolithic matrices of the invention are particularly useful for resolving polynucleotides (e.g., DNA and / or RNA) in samples by way of reversed-phase ion-pairing chromatography.

Description

field of invention [0001] The present invention relates to the separation of bio-organic molecules. Specifically, the present invention provides a monolithic polymer matrix for the separation of polynucleotides by reversed-phase ion-pair chromatography. Background of the invention [0002] The isolation of polynucleotides has grown in importance in recent years. The polynucleotide contained in the isolated sample can be used, for example, for detection and / or quantification of amplification reaction product DNA, and detection of various DNAs (eg, polymorphisms or mutations). However, due mainly to the complex structure and large molecular weight of this molecule, no separation technique has been completely satisfactory to date. [0003] Typically, separation of polynucleotides is performed using electrophoretic techniques, such as slab gel electrophoresis, or more recently capillary electrophoresis. Generally, these methods involve passing an electrical current through a ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): B01J20/285B01D15/08B01D15/32B01D15/36B01J20/26B01J20/28B01J20/281C08F2/44C12N15/09C12N15/10G01N30/52G01N30/88
CPCB01J20/26B01D15/325B01D15/366G01N2030/8827B01J20/3064B01J20/285G01N2030/528B01J2220/54B01J20/267B01J2220/82B01J20/28042C12N15/101
Inventor R·哈奇
Owner AGILENT TECH INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com