DNA fragment production method

Single-primer PCR with tailored primers addresses the inefficiencies of E. coli-based plasmid production and incomplete DNA amplification, enabling cost-effective AAV vector production by directly amplifying DNA fragments with transgenes between ITR sequences.

WO2026127114A1 Publication Date: 2026-06-18YAMAGUCHI UNIV
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Patent Information

Authority / Receiving Office
WO · WO
Patent Type
Applications
Current Assignee / Owner
YAMAGUCHI UNIV
Filing Date
2025-12-12
Publication Date
2026-06-18

AI Technical Summary

⚠Technical Problem

Existing methods for producing AAV vectors are time-consuming and costly due to the need for plasmid vector production in E. coli, and current PCR-based detection methods fail to amplify DNA fragments containing transgenes sandwiched between ITR sequences, leading to incomplete virus construction.

⚗Method used

A method using single-primer PCR with specifically designed primers that anneal to the ITR sequences, allowing for the production of DNA fragments containing a transgene sandwiched between two ITR sequences, followed by transfection into packaging cells to produce AAV vectors.

🎯Benefits of technology

Enables efficient and cost-effective production of AAV vectors by directly amplifying DNA fragments with transgenes between ITR sequences, overcoming the limitations of traditional methods and facilitating virus construction.

✦ Generated by Eureka AI based on patent content.

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Abstract

The present invention addresses the problem of providing a method for producing, by single primer PCR, a DNA fragment having ITR sequences on both ends. In the present invention, a DNA fragment including a transgene is produced by single primer PCR with a DNA having ITR sequences serving as a template DNA, using: (A-i) a polynucleotide A comprising an artificially designed polynucleotide a1 comprising at least 5 bases which are any of the bases selected from adenine, guanine, cytosine, and thymine and a polynucleotide a2 linked to the 3' end of the polynucleotide a1, the polynucleotide a2 comprising the full length sequence from 1st to 54th of the base sequence represented by SEQ ID NO: 1 or comprising a consecutive partial sequence of at least 16 consecutive bases in the base sequence from the 12th to 39th of the base sequence represented by SEQ ID NO: 1; (A-ii) the polynucleotide A of (A-i) with one or more bases deleted, substituted, added or inserted; or the like.
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