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Linker for detecting low-frequency variation, linker mixture and corresponding method

A mixture and variation technology, applied in the field of biological information, can solve the problems affecting the application and promotion of double-stranded molecular tag technology, the difficulty of accurate quantification of the final linker concentration, and the low success rate of linker preparation.

Inactive Publication Date: 2019-03-08
XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method involves multi-step enzymatic reactions and multi-step purification steps. The joint preparation process is cumbersome, and the final joint concentration is difficult to quantify accurately. Application and promotion of chain molecule labeling technology

Method used

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  • Linker for detecting low-frequency variation, linker mixture and corresponding method
  • Linker for detecting low-frequency variation, linker mixture and corresponding method
  • Linker for detecting low-frequency variation, linker mixture and corresponding method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 Preparation of adapters containing 7bp+8bp double-stranded molecular tags

[0099] Design the sequences of the linker P5 strand and P7 strand with double-strand molecular tags: the linker mixture is designed to contain 16 kinds of molecular tags, as shown in Table 1 below, 7 pairs of 7 nucleotides in length among the 16 kinds of molecular tags, The remaining 9 pairs are 8 nucleotides in length, and the lengths of the two are staggered by 1 bit. In the first 7 nucleotide sequences of the molecular tag combination, the base ratio of the same vertical position is A:G:T:C=1:1:1:1, and there are at least 3 nucleotide sequences between the molecular tags difference.

[0100] Table 1

[0101]

[0102]

[0103] Centrifuge the synthesized tube at 12,000g for 1 minute to shake the dry powder to the bottom, carefully open the tube cap, dilute the dry powder to 250 μM with LowTE Buffer (10mM Tris-HCl (pH 8.0), 0.1mM EDTA), shake and mix well and place at 4°C Refr...

Embodiment 2

[0114] Example 2 Detection of Low Frequency Somatic Variations in Standards and Tumor Blood Samples

[0115] Preparation of standard DNA: The normal cell line DNA NA18536 was used to dilute the Horizon Discovery standard HD701 and HD753 by different multiples. See Table 4 and Table 5 for the mixture of different dilution multiples and the expected variation frequency. The DNA mixture was sonicated with Covaris S220 until the main peak was around 170bp, which was similar in size to the main peak of cfDNA.

[0116] Table 4

[0117]

[0118]

[0119] table 5

[0120]

[0121] Preparation of cfDNA: Plasma isolated from whole blood of patients was extracted with QIAamp Circulating Nucleic Acid Kit (Qiagen).

[0122] Pre-library preparation: Take KAPA Hyper Prep Kit (Roche) as an example, take 33ng of plasma cfDNA and standard DNA after fragmentation, use KAPA Hyper Prep Kit (Roche) for pre-library preparation, fill in the tail and connect to the example For the linker p...

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PUM

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Abstract

The invention relates to a linker for detecting low-frequency variation. The linker comprises two complementary DNA single chains, wherein one chain P5 comprises a sequence locally coincided with an upstream amplification primer, a sequence combined with an upstream sequencing primer, a molecular label of a specific nucleotide sequence combination and a bulged base T in turn from 5' end to 3' end;and the other chain P7 comprises the following three parts in turn from 5' end to 3' end: a molecular label reversely complementing to the molecular label in the chain P5, a sequence combined with adownstream sequencing primer and a sequence combined with a downstream amplification primer. The invention also relates to a linker mixture for detecting low-frequency variation and a corresponding method. The linker mixture provided by the invention is used for performing library preparation and high-throughput sequencing on the samples containing low-frequency variation and with damaged single chains, and the biological information analysis process and algorithm disclosed by the invention are adopted for effectively increasing the accuracy of variation detection.

Description

technical field [0001] The present invention relates to the field of biological information, in particular to gene detection, specifically to a joint suitable for detecting low-frequency somatic cell variation and single-strand damage variation and an application method thereof. Background technique [0002] Compared with the first-generation sequencing technology, the next-generation sequencing technology has greatly reduced the cost of sequencing by virtue of its excellent performance of simultaneously sequencing millions or even tens of billions of sequences, and rapidly promoted its application in various fields such as scientific research, forensic medicine, and clinical practice. application. For example, the free DNA in the peripheral plasma of pregnant women contains the genetic information of the fetus. Through low-depth whole-genome sequencing of the plasma free DNA (cell-free DNA, cfDNA) of pregnant women, fetal chromosomal abnormalities can be detected, and non-i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6827C12N15/11
CPCC12Q1/6827C12Q2525/191
Inventor 黄炳顶章扬任勇哲王丹丹戴珩史耀舟
Owner XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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