Unlock instant, AI-driven research and patent intelligence for your innovation.

Methods and compositions for amplification of nucleic acids

a technology of nucleic acids and compositions, applied in the direction of enzyme stabilisation, enzymology, transferases, etc., can solve the problems of patent disclosure of non-detergent surfactants or zwitterionic detergents for stability, and the use of zwitterionic detergents, etc., to achieve the effect of stabilizing polymerase activity

Inactive Publication Date: 2015-06-11
AFFYMETRIX INC
View PDF1 Cites 34 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent discusses the use of certain chemicals, such as zwitterionic derivatives of poly-alkoxylated alkyl derivatives and nonionic poly(propylene oxide)-poly(ethylene oxide) block copolymer surfactants, to stabilize nucleic acid polymerases. These chemicals can help prevent the polymerases from losing their activity when exposed to higher temperatures or other factors that may cause them to denature. Additionally, the patent describes a method for modifying a polymerase with a specific chemical called 2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate (MSEC) which makes it inactive at low temperatures but can be reactivated at higher temperatures. This modification is particularly useful for creating a thermostable polymerase that can withstand higher temperatures.

Problems solved by technology

Although efficient, exponential amplification of target sequences is not an unlimited process.
However, this patent does not disclose the use of non-detergent surfactants or zwitterionic detergents for the stability of thermostable polymerases in PCR reactions.
However, this publication does not disclose the use of zwitterionic detergents alone in improving the amplification of nucleic acids and actually teaches that the zwitterionic detergents used should be selected carefully so as not to inhibit the activity of the DNA polymerase in the reaction.

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
  • Methods and compositions for amplification of nucleic acids
  • Methods and compositions for amplification of nucleic acids
  • Methods and compositions for amplification of nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0050]Amine oxide derivatives of selected surfactants were prepared as follows: amine containing surfactants (10 mmole in amine equivalents) were dissolved in ˜3-4 volumes of ethanol. H2O2 (30%; 2.3 ml; 20 mmole) was added, and the solution stirred at 55° C. for 24-48 hrs. After cooling to room temperature, ˜20 mg of 10% Pt° on C (platinum on activated carbon) was added, and stirring continued for another 4 h to decompose the excess peroxide. The solution was filtered through celite and evaporated under vacuum. The 1H-NMR was recorded in MeOH-d4.

[0051]The procedure above was used to prepare amine N-oxides from the following polyalkoxylated amine surfactants: PEO(15) Laurylamine (DeThoxamine C-15, Deforest); PEO(5) Isodecyloxypropylamine (Tomamine E-14-5, Air Products); PEO(5) Isotridecyloxypropylamine (Tomamine E-17-5, Air Products); PEO(5) Stearylamine; PEO(10) Stearylamine; PEO(15) Stearylamine; PEO(50) Stearylamine; TETRONIC® 304 (MW 1650); TETRONIC® 904; and TETRONIC® 1107. When...

example 2

[0054]In order to test the efficacy of detergents on Taq DNA Polymerase, a stock of Taq DNA Polymerase at a base concentration of 111 units / ul was diluted to 5 units / ul in a storage buffer that lacked detergents. The standard Taq DNA Polymerase storage buffer has 0.5% Tween-20 and 0.5% NP-40. In this example 0.625 units of Taq were added per 25 ul reaction, so that represents a change in final detergent concentration of 0.0025% each detergent to 0.00011% of each. A 455 bp single-copy target from the human numb gene was PCR-amplified from 1 ng human genomic DNA in a 25 ul reaction with 0.625 units of the diluted Taq DNA polymerase. The cycling conditions were 95° C. for 2 minutes; 35 cycles of 95° C. for 10 seconds, 55° C. for 20 seconds, and 72° C. for 30 seconds; 72° C. for 5 minutes; and 10° C. until required. An aliquot of 10 ul was run on a 1.5% agarose / ethidium bromide gel.

[0055]In FIG. 1, the results of the initial screen of several non-ionic and zwitterionic detergents are sh...

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

PropertyMeasurementUnit
cloud pointaaaaaaaaaa
temperaturesaaaaaaaaaa
cloud pointaaaaaaaaaa
Login to View More

Abstract

The present invention provides methods, compositions, and kits for storing and enhancing the activity of polymerases and particularly thermostable polymerases. The methods comprise mixing a thermostable polymerase with at least one zwitterionic or ylide surfactant that has at least one PEO group. In another aspect the polymerase is mixed with a blocker such as PLURONIC® or TETRONIC® or an amine N-oxide derivative thereof. The thermostable polymerase may be reversibly inactivated by treatment with 2-(Methylsulfonyl)ethyl 4-nitrophenyl carbonate. Compositions and kits for performing the process according to the invention are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of U.S. patent application Ser. No. 13 / 791,164 filed Mar. 8, 2013, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61 / 659,542, filed Jun. 14, 2012, the contents of each of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention is generally in the field of nucleic acid amplification.BACKGROUND OF THE INVENTION[0003]Compositions of thermostable nucleic acid polymerases are useful for amplification of nucleic acids by multiple cycles of the polymerase chain reaction. Various compositions for stabilizing polymerases using surfactants have been disclosed. In an early study it was observed that viral DNA polymerase activity was stimulated and stabilized against thermal inactivation by nonionic detergent (see, Wu and Cetta, Biochemistry (1975) 14(4):789-795). U.S. Pat. No. 6,127,155 discloses stabilization of thermostable DNA polymerases in a composit...

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
Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N9/12C12N9/96
CPCC12Q1/686C12N9/1252C12N9/96C12Q1/6818C12N9/1241C12Q2527/125C12P19/34C12Q1/6869
Inventor MCGALL, GLENN H.BARONE, ANTHONY D.KUBU, CHRISTOPHER J.
Owner AFFYMETRIX INC