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Joint composition and application thereof

A composition and linker connection technology, applied in combinatorial chemistry, organic compound libraries, biochemical equipment and methods, etc., can solve problems such as inability to remove false positives, and achieve the goal of improving utilization efficiency, increasing library yield, and improving utilization. Effect

Active Publication Date: 2020-01-31
MGI TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot remove false positives caused by PCR amplification or sequencing

Method used

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  • Joint composition and application thereof
  • Joint composition and application thereof
  • Joint composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 BGI-seq500 platform cfDNA starting single-stranded UID library construction

[0071] Based on BGI-seq500 platform of BGI, this example provides a cfDNA starting single-stranded UID library construction method, in which cfDNA is derived from cfDNA extracted from mixed plasma, and the mixed plasma is derived from BGI Gene Cell Resource Bank, including the following specific steps :

[0072] 1) 3ng cfDNA was dephosphorylated at the 5' end, denatured and melted, and DNA repaired. The specific system preparation process is shown in Table 2 below:

[0073] Table 2

[0074] system volume / μl dna 1-100ng (volume X<=16μl)

Circular Ligase Buffer II 4 MnCl 2

2 Endonuclease VIII (10U / μl) 0.5 nuclease free water 16-X

[0075] Incubate at 37°C for 1 hour; then add 2 μl FastAP (1U / μl), incubate at 37°C for 10 minutes, at 95°C for 2 minutes, and incubate on ice for 2 minutes;

[0076] 2) Connect the first linker to the si...

Embodiment 2

[0103] Gradient optimization of the first joint length in embodiment 2

[0104] Since it was found that the length of the first linker had a greater impact on the ligation efficiency, the choice of linker was due to the fact that the first linker consisted of a 5bp UID sequence + another sequence, which was the reverse sequence truncated from the 3' end of the extension primer. To the complementary sequence, and since the first adapter needs to be blocked by ddc, the end is required to be base C, and the extension primer should be G, that is, this embodiment uses truncated 20bp, 24bp and 34bp adapters for library construction, so that Analyze and study the influence of linkers with different lengths on the connection efficiency and the final library construction effect. The specific steps of the library construction method are the same as in Example 1. The specific sequences and results of the linkers are shown in Table 11 below:

[0105] Table 11

[0106]

[0107]

[0...

Embodiment 3

[0126] Example 3 FFPE severely degraded sample initial single-chain UID library construction

[0127] Since the present application can also build a library for degraded DNA, this embodiment provides a method for building a library of starting single-stranded UID from FFPE severely degraded samples, including the following specific steps:

[0128] 1) Extract FFPE sample DNA;

[0129] 2) Covaris ultrasound interrupts and enriches DNA samples, 12 cycles;

[0130] 3) Purification with 1.5×Agencourt AMPure XP-Medium magnetic beads;

[0131] 4) Wash twice with 80% alcohol, redissolve in 20 μl;

[0132] 5) Qubit quantification;

[0133] 6) Take 30ng of interrupted FFPE genomic DNA, and build a library according to the method of building a library in Implementation 1;

[0134] 7) Qubit quantification and Agilent 2100 detection, the results are as follows Figure 6 (A)- Figure 6 (C) and Figure 7 shown.

[0135] From Figure 6 (A)- Figure 6 (C) It can be seen that the dist...

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Abstract

The invention provides a joint composition and application thereof. The joint composition comprises a first joint and a second joint, wherein the first joint is a single-chain joint and contains a UID(unique identifier) sequence and a segment of single-chain nucleotide; the 3'-end of the first joint has sealed modification; the second joint is a double-chain joint formed by a long chain of nucleic acid and a short chain of nucleic acid in complementary pairing; the 5'-end of the long chain of the nucleic acid of the second joint has phosphoric acid modification; and the 5'-end of the short chain of the nucleic acid of the second joint does not undergo phosphorylation modification. Since a synthesized joint with the UID sequence is directly introduced, and the UID sequence is introduced into a double-chain library through extension, a tedious joint preparation process in a conventional UID joint database construction technique is avoided, the experiment efficiency can be improved, theexperiment difficulties can be reduced, and the experiment cost can be reduced.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a linker composition and its application, especially to a linker composition, a method for constructing a sequencing library using the linker composition, the constructed sequencing library and its application. Background technique [0002] Peripheral blood free DNA (cell-free DNA, cfDNA) refers to the circulating DNA in peripheral blood. The content in the peripheral blood of healthy people is low, and the content in the peripheral blood of cancer patients is increased. These cfDNA mainly come from the tumor source. Foci, metastases, circulating tumor cells, and normal tissues. At present, scholars generally believe that nucleic acid is released into the blood at the early stage of tumor development, and the tumor cell DNA circulating in the blood is called ctDNA (circulating tumor DNA). ctDNA is considered to carry tumor histogenetics and epigenetics Variation information ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12Q1/6806C40B50/06C40B40/06
CPCC12Q1/6876C12Q1/6806C40B50/06C40B40/06C12Q2525/191C12Q2521/501
Inventor 刘军郑志雄吴逵彭昌盛刘静齐晓娟
Owner MGI TECH CO LTD
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