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Method and compositions for improved polynucleotide synthesis

A polynucleotide and nucleoside technology, applied in microorganism-based methods, plant genetic improvement, chemical instruments and methods, etc., can solve problems such as enzyme temperature sensitivity

Inactive Publication Date: 2010-02-17
GUANGZHOU FULENGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Other proteins reported in this field as markers are green fluorescent protein (reviewed by Misteli and Spector, Nat Biotechnol. 15(10):961-4 (1997); Cormack, Curr Opin Microbiol 1(4): 406-10(1998)). However, these enzymes are temperature sensitive and must be analyzed shortly after sample preparation

Method used

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  • Method and compositions for improved polynucleotide synthesis
  • Method and compositions for improved polynucleotide synthesis
  • Method and compositions for improved polynucleotide synthesis

Examples

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example 1

[0102] 3′polynucleotide phosphatase activity detection

[0103] 3'phosphate-modified oligonucleotides were chemically synthesized (Midland Certified Reaget Company, Midland, Tex.). The oligonucleotide sequence consists of the following:

[0104] 5'-GCT GCTCTGTGCATCCGAGTGG-p-3' (SEQ ID No: 7)

[0105] 32P was labeled at the 5' end of the oligonucleotide with T4 polynucleotide kinase which has no 3' polynucleotide phosphatase activity (Yang, S.W., et al.Proc.Natl.Acad.Sci.U.S.A.93(21) : 11534-9 (1996)). with 32 P-labeled oligonucleotides were mixed with purified 3′ polynucleotide phosphatase in 1×PCR reaction buffer (10Mm Tris-HCl, pH8.3, 50mM MgCl 2) at 72°C for 5 minutes. Add DNA sequencing buffer, heat at 90° C. for 2 minutes, and analyze the sample by 12% polyacrylamide-7M urea gel electrophoresis. Figure 4 is an autoradiogram of samples quantified with PhophorImager (Molecular Dynamics). One unit of 3' phosphatase is defined as the ability to remove 5 μmol of the 3' ...

example 2

[0107] Isolation and Purification of 3' Phosphatase from Pfu

[0108] 100 g wet weight of Pfu cells (purchased from the Marine Biotechnology Center at the University of Maryland, Baltimore, Md) were suspended in lysate, 20 mM Tris-HCl, Ph7.5, 1 mM EDTA, 1 mM DTT, 0.2 M NaCl on ice , 10 mM mercaptoethanol and 2 mM benzylsulfur fluoride. Cells were then lysed by ultrasonication and centrifuged at 8,200 rpm for 10 minutes on a Sorvall GS-3 rotor. Add 0.05 vol of 10% polyethyleneimine solution to the supernatant, mix well and centrifuge, then fractionate with ammonium sulfate (45-80% saturation). Then use phosphocellulose column (P-11; Whatman, Inc.; activity eluted.apprxeq.0.6M NaCl), Source 15S (Pharmacia; activity eluted.apprxeq.0.2M NaCl), double-stranded DNA-cellulose (Sigma; activityeluted.apprxeq.0.15M NaCl), Mono S column (Pharmacia; activityeluted.apprxeq.0.35M NaCl) and heparin agarose, Mono Q column (Pharmacia; activityeluted.apprxeq.0.25M NaCl) chromatography, finall...

example 3

[0111] Peptide sequencing of the N-terminus of 3′ phosphatases

[0112] Part of the purified 3' polynucleotide phosphatase was added to 7% SDS-Tricine PAGE. The protein in the SDS gel was electrotransferred to PVDF membrane (Immobolin-P from Millipore) in transfer buffer (0.5.times.TBE (pH 8.4), 20% methanol, 0.5mMEDTA), 0.5A electrophoresis, transfer 1 Hour. Membranes were stained with Coomassie Brilliant Blue R-250 for 10 minutes and destained twice with 100% methanol. The band corresponding to the 3' polynucleotide phosphatase on the membrane was excised. Its amino acid sequencing was done with Yale University commercial facilities. List the sequence of 26 amino acids in single letters starting from the N-terminus:

[0113] FKIDRLRFGTAGIPLSTPKPSTIAGI (SEQ ID NO: 1).

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Abstract

The sensitivity and specificity of polynucleotide synthesis is increased by protecting the 3'-end of an oligonucleotide used as a primer in the synthesis of the polynucleotide. Protection of the 3'-end of an oligonucleotide prevents non-specific chain elongation. Removal of blocking group at an elevated temperature, using a thermostable enzyme, permits template-specific polynucleotide synthesis. The present invention also provides oligonucleotides with a 3'-end protected by a blocking group and a thermostable enzyme capable of removing the blocking group at an elevated temperature. The compositions and methods of the invention are very useful in a variety of techniques for DNA / RNA amplification and analysis, including medical genetics research and diagnosis, pathogen detection, forensic, and animal and plant genetics applications, among others.

Description

technical field [0001] The invention relates to compositions and methods for improving the sensitivity and specificity of polynucleotide synthesis. The method comprises: when synthesizing polynucleotides, the nucleotides protected by groups at the 3' end are used as primers, which can prevent the extension (reaction) of non-specific chains, and when the temperature rises, the resistance is removed by thermostable enzymes. cleaving groups, allowing template-specific polynucleotide synthesis. The present invention relates to a thermostable 3' polynucleotide phosphatase and its use as a marker protein, as well as to the use of a group to protect the 3' end of an oligonucleotide primer, and to improve the amplification and analysis of DNA from various samples / RNA Sensitivity and Specificity Methods. The invention can be widely used in DNA / RNA amplification and analysis in various samples including medical genetic research, diagnosis, pathogen detection, forensic medicine and an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/62C12N9/16C12P19/34C12Q1/68C12Q1/42C12N15/09C12N1/15C12N1/19C12N1/21C12N5/10C12Q1/44C12R1/01
CPCC07K2319/00C12Q1/6853C12N9/16C12P19/34C12Q2521/525C12Q2525/186C12Q2527/101
Inventor 杨淑伟
Owner GUANGZHOU FULENGEN
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