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Methods for production of oligonucleotides

一种寡核苷酸、核苷酸的技术，应用在用于产生寡核苷酸领域，能够解决不提供待扩增DNA序列末端自由设计等问题

Inactive Publication Date: 2008-07-02
ON-SITE RCP CO UNDER THE NAME OF NOVIA CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] This technique requires the additional generation of oligonucleotides for cleaving the rolling circle product and, more importantly, it does not provide free design of the ends of the DNA sequence to be amplified since they are subject to binding / Existence restriction of cleavage site

Method used

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  • Methods for production of oligonucleotides
  • Methods for production of oligonucleotides
  • Methods for production of oligonucleotides

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preparation example Construction

[0142] Preparation of probe (A) (step a)

[0143] The starting nucleic acid sequence (probe (A)) comprising one or more oligonucleotides to be amplified and one or more nicking cassettes can be synthesized by standard chemical methods, such as β-cyanoethylphosphoramidite Chemical. A 5'-phosphate can be added during this synthesis, or alternatively a 5'-phosphate can be enzymatically coupled to the 5'-terminus of the nucleic acid sequence, for example by using T4 polynucleotide kinase.

[0144] Long sequences can be constructed by external templated ligation of multiple oligonucleotides, eg when the oligonucleotides to be amplified are very long, eg 200-1000 nucleotides. In this case, probe (A) can be prepared by external templated ligation of two or more oligonucleotides (Fig. 5). In one embodiment, the invention relates to a method wherein probe (A) is prepared by ligating one or more nucleic acid sequences comprising one or more of said one or more oligonucleotides A plur...

Embodiment 1

[0421] Amplification of the 66 nucleotide oligonucleotide SF86-SC by using the suicide cassette

[0422] By using the present invention, we were able to amplify oligonucleotides approximately 100,000-fold. The present invention uses a suicide cassette as a tool to amplify specific oligonucleotides. The suicide cassette is a hairpin structure containing binding sites for the nicking and restriction enzymes; in this example, the nicking and restriction enzymes are N.Alw I (Nt.Alw I) and Mly I, respectively (Fig. 3, A-B). A linear DNA sequence can be turned into a closed circular sequence by self-templated ligation by placing a suicide cassette at each end of the initially synthesized oligonucleotide. Oligonucleotides can be amplified at either polarity by successive rounds of rolling circle replication, nicking and ligation. In a final step, the suicide cassette can be separated from the oligonucleotide by restriction cleavage with Mly I (Figure 1).

[0423] Oligonucleotide ...

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Abstract

The present invention relates to production of oligonucleotides using rolling circle replication, wherein synthesised multimeric oligonucleotides are reduced to mononucleotides using a nicking cassette. Thus, the invention provides a method for the production of oligonucleotides, enabling efficient amplification of oligonucleotides at lengths up to at least 1000 nucleotides and in high amounts contained within a nicking cassette.

Description

field of invention [0001] The present invention presents improved methods for producing oligonucleotides. The invention also relates to the probes used in the method. Background of the invention [0002] In combination with genome sequencing, automated DNA synthesizers have enabled researchers to obtain virtually any genetic element as an oligonucleotide at considerably low cost, and at least for short oligonucleotides. Two current limitations relate to: I) the mass production of high quality oligonucleotides; and II) the synthesis of very long oligonucleotides (200-1000 nucleotides). Regarding the former question, the quality of oligonucleotides can be affected by factors related to the method of chemical synthesis, including depurination and conversion of dG to dA. The maximum length of synthesized oligonucleotides is typically about 150 nucleotides, with yield and quality decreasing with increasing length. [0003] The main current uses of oligonucleotides are as prime...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12N15/10C12Q1/6844C12P19/34C12Q2531/125C12Q2525/131C12Q2537/149C12Q2525/301C12Q2521/307
Inventor J·克啻M·斯托加德J·S·罗曼
Owner ON-SITE RCP CO UNDER THE NAME OF NOVIA CO
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