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A molecule adaptor and applications thereof

A molecular linker and sequence technology, applied in the field of sequencing, can solve the problems of high sequencing background noise, complicated experimental process, and low effective data rate, and achieve the effects of increased detection sensitivity, accurate detection results, and simple preparation

Active Publication Date: 2017-06-30
SUZHOU PREMED MEDICAL LAB CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the extremely low content of ctDNA in plasma, the experimental process is complicated, the amount of samples and the number of experiments are limited, and there are losses in sample preparation and sequencing pre-library construction and hybridization capture, so the high-throughput sequencing (next-generation sequencing) technology was used to obtain The effective data rate is low; in addition, ctDNA samples in plasma are easily contaminated by genomic DNA, resulting in high sequencing background noise; in addition, during the sequencing process, there are varying degrees of oxidative damage in library enrichment, subsequent hybridization capture, and sequencing, resulting in false positives Mutations, will mask rare mutations in samples, especially limited ctDNA in plasma, limiting detection sensitivity
Therefore, the traditional adapters connected to the tested samples can only distinguish different samples through molecular labels. However, due to the low amount of sample DNA, high background signal, false positive mutations, etc., it is difficult to eliminate interference during data analysis and cannot truly reflect the samples. Tumor information carried by DNA, especially the detection of ctDNA

Method used

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  • A molecule adaptor and applications thereof
  • A molecule adaptor and applications thereof
  • A molecule adaptor and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Joint annealing and extension steps

[0049] (1) The sequence of the key-shaped molecular linker is SEQ ID No.1 ( figure 1 ):

[0050] PHO-5’-TTCTACAGTACNNNNNNNN AGATCGGAAGAG CACACGTCTGAACTCCAGTCACdUACACTCTTTCCCTACACGACG CTCTTCCGATC*T -3...

[0051]Note: PHO stands for phosphorylation at the 5' end, where N stands for any base in A / T / G / C, dU stands for deoxyuracil, the left and right underlines of dU are complementary regions, * stands for sulfur modification, dotted line "… ..." represents the extension region, and the italic part is the restriction endonuclease recognition region.

[0052] (2) Reagents required for one-step annealing extension of key-shaped joints:

[0053] Linker sequence (synthesized by Jinweizhi Biotechnology Co., Ltd.), KAPA HiFi Hotstat ReadyMix (KAPA company kk2602), sterilized ultrapure water

[0054] (3) The key-shaped joint adopts one-step annealing and extension steps:

[0055] The synthesized dry powder linker sequence wa...

Embodiment 2

[0069] Example 2 DNA library construction of plasma and tissue samples

[0070] The samples in this example come from the General Hospital of Shenyang Military Region, 5 patients with clinically diagnosed stage III adenocarcinoma of lung cancer. The plasma (2ml) and tissue samples were taken before the preoperative medication, and the free DNA (cfDNA) and tissue DNA were extracted. The tissue DNA was ultrasonically analyzed. Fragmented into a size of 150-250bp, cfDNA and tissue fragmented DNA were qualified by the Agilent 2100 bioanalyzer, and the libraries were constructed according to the following steps.

[0071] (1) Sample DNA end repair

[0072] Configure the mixed reaction according to Table 4, use the KAPA LTP Library Preparation Kit (KK8233) EndRepair, put all the plasma cfDNA, and input 100ng of the fragmented DNA sample.

[0073] Table 4. Sample DNA end repair system

[0074] Fragmented DNA sample (150bp) 50ul KAPA End Repair Buffer(10X) 7ul K...

Embodiment 3

[0097] Practical Example 3 Cellular DNA Sensitivity Experiment of Known Mutation Sites

[0098] The cell samples used in this example come from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, including H1975 cell lines (known EGFR L858 and T790M mutations), H1650 cell lines (known EGFR exon 19 deletion), negative MRC cell lines (no EGFR mutation). DNA was extracted from H1975 cells and H1650 cells, and mixed according to the mass ratio of 1:1 after being interrupted by ultrasound, and then blended with negative cell line MRC fragmented DNA samples according to 1%, 0.1%, 0.05%, and 0%, for library construction, and then Two rounds of specific hybridization capture were carried out, and the library after capture was detected by fluorescent quantitative PCR method for corresponding mutation sites, and finally double-end sequencing was performed to judge the detection sensitivity of molecular joints.

[0099] The specific library constru...

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Abstract

A molecule adaptor having characteristics of high stability, a high sample DNA connection efficiency and correction functions is designed based on illumina sequencing adaptor optimization. The molecule adaptor can detect mutation sites the mutation frequency of which is as low as 0.05%. The molecule adaptor is used for identifying real mutations in a sample sequencing library constructing process and false-positive mutations introduced by an operating process. In addition, a method of constructing a sequencing library for a sample to be detected is provided.

Description

technical field [0001] The invention relates to the field of sequencing technology, and is used for molecular joints and applications in building a database of tested samples; at the same time, it is applied to molecular joints and applications for ultra-low frequency gene mutation detection; especially the preparation of molecular joints with identification functions and the construction of test samples for sequencing library method. Background technique [0002] Tumors are a mixture of heterogeneous cells. Sequencing can detect rare mutations in them. Next-generation sequencing has the advantages of multiple samples and multiple genes. It can also discover unknown mutation sites, so next-generation sequencing can be used in the early stages of tumors. Screening and diagnosis, recurrence monitoring, efficacy evaluation, etc. [0003] ctDNA is free DNA (circulating tumor DNA, ctDNA) in the body fluids of tumor patients, released from tumor cell necrosis or apoptosis, and ex...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68C40B50/06C40B80/00
CPCC12N15/1093C12N15/11C12Q1/6806C12Q1/6869C40B50/06C40B80/00C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 王弢王景李宗飞代玉环周美玲杜帅
Owner SUZHOU PREMED MEDICAL LAB CO LTD
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