By using primers to connect regions of the polymerase and assay

Through high-throughput polymerase PCR analysis and microfluidic systems, specific primers are designed to add nucleotides to the ends of the DNA chains, solving the problems of low DNA connection efficiency and poor accuracy in existing technologies, and achieving efficient and accurate haplotype typing and sequencing.

CN113924370BActive Publication Date: 2025-09-23THE CHINESE UNIVERSITY OF HONG KONG
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Patent Information

Application Number
CN202080040284.0
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Priority Date
2019-05-10
Filing Date
2020-05-11
Publication Date
2025-09-23
Estimated Expiration
2040-05-11

AI Technical Summary

Technical Problem

Existing methods for determining individual haplotypes are costly, inaccurate, and low-throughput, and are unable to effectively connect and sequence distant DNA regions. Traditional PCR methods are also inefficient and inaccurate in connecting DNA molecules.

Method used

High-throughput polymerase chain reaction (PCR) analysis is used to design specific forward and reverse primers. Additional nucleotides are added to the 3' end of the DNA chain to improve the ligation efficiency. A microfluidic system is then used to generate tiny droplets for PCR amplification, ensuring accurate ligation and sequencing of DNA molecules.

Benefits of technology

It achieves accurate determination of individual haplotypes at low cost and high throughput, improves the efficiency of DNA molecule connection and sequencing accuracy, and is suitable for haplotype typing in multiple distant regions.

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Abstract

Specific forward primers and reverse primers can be used to connect remote regions of DNA molecules of the same size into smaller DNA molecules. Reverse primer R1 can have a first portion complementary to the terminator sequence of region A and can include a second portion with an overlapping sequence. Forward primer F2 can have a first portion complementary to the starting sequence of region B, wherein the forward primer includes a complementary overlapping sequence (e.g., identical first or second portion) that is complementary to the overlapping sequence. The first portion of F2 can be an entire primer. Smaller DNA molecules can be used to determine the haplotype of a region. A test kit comprising specific forward primers and reverse primers has also been described.
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