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Method and compositions for improved polynucleotide synthesis

A technology of polynucleotides and nucleosides, which is applied in the direction of microorganism-based methods, plant genetic improvement, chemical instruments and methods, etc., and can solve problems such as temperature sensitivity of enzymes

Inactive Publication Date: 2003-04-02
GUANGZHOU FULENGEN
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  • 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

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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

[0099] 3′polynucleotide phosphatase activity detection

[0100] 3'phosphate-modified oligonucleotides were chemically synthesized (Midland Certified Reaget Company, Midland, Tex.). The oligonucleotide sequence is composed as follows: 5'-GCT GCTCTGTGCATCCGAGTGG-p-3' (SEQ ID No: 7)

[0101] T4 polynucleotide kinase without 3' polynucleotide phosphatase activity 32 The P-label is at the 5' end of the oligonucleotide (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' terminal phosphat...

example 2

[0102] Isolation and Purification of 3' Phosphatase from Pfu

[0103] 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 ultrasound and centrifuged at 200 rpm for 10 minutes on a Sorvall GS-3 rotor 8. 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, finally in ...

example 3

[0105] Peptide sequencing of the N-terminus of 3 phosphatases

[0106] 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 (pH8.4), 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. A sequence of 27 amino acids is listed in single letters starting from the N-terminus: FKIDRLRFGTAGIPLSTPKPSTIAGI (SEQ ID NO: 1). Example 4

[0107] Cloning of the gene encoding the 3' phosphatase from the Pfu genome

[0108] Figure 2 is the cloning process of the 3' phosphatase gene, which is briefly described as follows: use the BLAST progr...

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