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Reversibly modified thermostable enzyme compositions and methods of making and using the same

A thermostable enzyme and thermostable technology, which can be used in biochemical equipment and methods, microbial determination/inspection, enzymes, etc., and can solve the problems of long modification process, cumbersome efficiency, etc.

Active Publication Date: 2009-05-06
SUZHOU NUHIGH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this modification process is long, tedious and inefficient
More importantly, not all proteins are suitable for processing conditions

Method used

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  • Reversibly modified thermostable enzyme compositions and methods of making and using the same
  • Reversibly modified thermostable enzyme compositions and methods of making and using the same
  • Reversibly modified thermostable enzyme compositions and methods of making and using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0186] Preparation of flap endonuclease-1 and Taq DNA polymerase

[0187] The DNA of Archaeoglobus fulgidis was obtained from ATCC (49558D). The gene encoding the flap endonuclease-1 (Afu FEN-1 ) of Archaeoglobulin fulgidis was cloned by PCR as described by Hosfield et al. (Hosfield, 1998, JBC 275(22): 16420-16427). The cloned sequences were verified by direct sequencing. The Afu FEN-1 gene was cloned into pET-28 (Noragen). Overexpression and purification of Afu FEN-1 protein was accomplished according to the method of Hosfield et al. with minor modifications.

[0188] Thermus aquaticus strain YT-1 was obtained from ATCC (25104). The DNA polymerase gene of Thermus aquaticus (Taq) was cloned by PCR using the sequence from GeneBank (Acession No. J04639). Plasmid pET-28 was used to construct the expression vector. Taq DNA polymerization was performed according to the method described by Lawyer et al. (Lawyer et al., 1989, JBC 264(11):6427-37; Lawyer et al.1989, PCR Meth.Ap...

Embodiment 2

[0190] Modification of Afu FEN-1 with citraconic acid

[0191] Modification of Afu FEN-1 with citraconic acid was performed in a buffer containing 20 mM MOPS, pH 8.0 and 100 mM KCl. The concentration of Afu FEN-1 was adjusted to 1 mg / ml.

[0192] Citraconic acid (Aldrich) and N,N'-dicyclohexylcarbodiimide (DCC) (Aldrich) were dissolved in N,N'-dimethyl-formamide (DMF) (Fisher, sequencing grade), The concentration is 1M. 100 μl of 1M citraconic acid and 200 μl of 1M DCC were mixed in a 1.5 ml Eppendorf tube. The mixture was incubated at room temperature for 1 hour. The mixture was then centrifuged at 12,000 rpm for 20 minutes at room temperature. The precipitate was discarded and the supernatant was retained for modification of Afu FEN-1.

[0193]One volume of activated citraconic acid was mixed with 99 volumes of Afu FEN-1. The mixture was then incubated at room temperature for 1 hour to obtain chemically inactivated Afu FEN-1.

Embodiment 3

[0195] Activity assay of modified Afu FEN-1

[0196] The flap endonuclease activity of the modified Afu FEN-1 was determined. Control reaction mixture without enzyme contained 30mM Tris HCl pH8.0, 3mM Mg 2+ , 400 nM 5-ROX (Sigma), 0.01% Tween-20, 100 nM each of the following nucleic acids 18SI, 18SP, 18ST (see sequence information in Table 1). Both 18SI and 18SP consist of sequences complementary to 18ST. 18SI is located upstream of 18SP and overlaps with 18SP by 1 nucleotide. When 18SP is intact, the 6Fam fluorescence of 18SP is quenched. In the presence of Afu FEN-1, 18SP in a complex containing 18SI, 18SP and 18ST is cleaved by Afu FEN-1. This cleavage results in an increase in 6FAM fluorescence. Add 10 ng of chemically modified Afu FEN-1 to a 25 μl reaction. The same amount of unmodified enzyme was used as a control.

[0197] Activity assays were performed on an ABI Prism 7000 to detect changes in fluorescence intensity in real time. The incubation conditions wer...

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Abstract

The present invention provides reversibly modified thermostable enzyme compositions and methods for making the same. The present invention also provides methods of using the reversibly modified thermostable enzyme compositions, as well as kits and systems comprising the reversibly modified thermostable enzymes.

Description

[0001] cross reference [0002] This application claims priority to US Provisional Application 60 / 578,442, filed June 9, 2004, which is incorporated herein by reference. Background technique [0003] Nucleic acid detection techniques such as target amplification and signal amplification are widely used in clinical microbiology, blood screening, food safety, genetic disease diagnosis and prognosis, environmental microbiology, drug target discovery and verification, forensics and other biological Medical Research. As a result, nucleic acid testing is increasingly becoming an essential component of emerging pharmacogenomics, prenatal diagnosis, and molecular-based cancer diagnosis and treatment. Therefore, the stability, specificity, sensitivity, reliability, and availability of accuracy and precision of nucleic acid detection are very important. [0004] Specific amplification of nucleic acid sequences allows sensitive detection of the presence or absence of specific target nu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N9/12
CPCC12N9/1241C12Q1/686C12N9/1252C12N9/99C12N9/22
Inventor 毕万里
Owner SUZHOU NUHIGH BIOTECH
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