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FFPE DNA library building kit, use thereof and library building method

A kit and buffer technology, applied in the field of FFPEDNA library building kits, can solve the problems of low ligation efficiency, low pre-amplification efficiency, and poor capture efficiency, etc.

Inactive Publication Date: 2018-04-20
深圳裕策生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason for the above situation is that the connection efficiency is low in the process of building the library, and the efficiency of pre-amplification (pre-PCR) is low, resulting in a small amount of captured templates, resulting in poor capture efficiency and high repetition.

Method used

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  • FFPE DNA library building kit, use thereof and library building method
  • FFPE DNA library building kit, use thereof and library building method
  • FFPE DNA library building kit, use thereof and library building method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] In this embodiment, the first reagent is used to combine different adapters for library construction, and the specific steps are as follows:

[0122] 1. End repair plus A reaction, as shown in Table 6:

[0123] Table 6

[0124] components

Dosage

interrupted dsDNA fragment

50μl (fill up with ultrapure water without nucleic acid contamination)

End repair of the first reagent plus A buffer

7μl

End Repair Plus A Enzyme Mix for First Reagent

3μl

total capacity

60μl

[0125] Run the PCR instrument: heat lid 85°C; 20°C, 30min; 65°C, 30min; 4°C∞.

[0126] 2. Connection

[0127] 2.1 The first reagent + the third adapter, as shown in Table 7:

[0128] Table 7

[0129] components

Dosage

DNA obtained in step 1

60μl

Third Linker (15μM)

5μl

Ultrapure water without nucleic acid contamination

5μl

Adapter ligation buffer for the first reagent

30μl

DNA ligase ...

Embodiment 2

[0167] In this embodiment, the second reagent is used in combination with different adapters for library construction. The specific steps are as follows:

[0168] 1. End repair plus A reaction, as shown in Table 15:

[0169] Table 15

[0170] components

[0171] PCR instrument: hot lid 90°C; 22°C, 20min; 72°C, 20min; 4°C∞.

[0172] 2. Joint connection

[0173] 2.1 The second reagent + the third adapter, as shown in Table 16:

[0174] Table 16

[0175] components

[0176] Incubate at 20°C for 30 min; use 1.2 times XP magnetic beads to purify and measure the concentration with Qubit, and calculate the total amount.

[0177] 2.2 The second reagent + the first adapter, as shown in Table 17:

[0178] Table 17

[0179] components

[0180] Incubate at 20°C for 30 min; use 1.2 times XP magnetic beads to purify and measure the concentration with Qubit, and calculate the total amount.

[0181] 2.3 The second reagent + the fourth adapter, as shown in ...

Embodiment 3

[0211] In this embodiment, the third reagent is used in combination with different adapters to perform library building operations, and the specific steps are as follows:

[0212] 1. End repair plus A reaction, as shown in Table 24:

[0213] Table 24

[0214] components

Dosage

interrupted dsDNA fragment

40μl (make up with ultrapure water without nucleic acid contamination)

End repair of the third reagent plus A buffer

6.5μl

End Repair Plus A Enzyme Mix for Third Reagent

3.5μl

total capacity

50μl

[0215] PCR instrument: hot lid 90°C; 20°C, 30min; 65°C, 30min; 4°C∞.

[0216] 2. Joint connection

[0217] 2.1 The third reagent + the third adapter, as shown in Table 25:

[0218] Table 25

[0219] components

Dosage

DNA obtained in step 1

50μl

Ligation buffer for the third reagent

6μl

Ligation Enhancer for Third Reagent

38μl

T4 DNA ligase

5μl

Third Linker...

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Abstract

The invention relates to an FFPE DNA library building kit, use thereof and a library building method. The kit includes combination of specific joint connection buffer solution, a DNA ligase solution and joint sequences. The kit provided by the invention can maximumly enhance the connection efficiency and PCR amplification efficiency in FFPE DNA library building, thus lowering the initial amount ofDNA library building, reducing the amplification cycle number, and meeting the demand of probe captured pre-amplification product quantity, lowering repetition generated in the sequencing process, and improving data utilization.

Description

technical field [0001] The invention relates to the technical field of nucleic acid library construction, in particular to a FFPE DNA library construction kit, its use and a library construction method. Background technique [0002] Next-generation sequencing (NGS) is a commonly used high-throughput sequencing method for effectively obtaining genomic information of human cancers. It can obtain various types of genetic information such as point mutations, gene copy number variations, gene expression, and gene fusion, and can outline tumors. A panorama of associated genetic variation. At present, NGS has gradually penetrated into the field of molecular diagnosis of tumor pathology, and the type of patient samples is no longer limited to blood, and pathological tissue can also be used as the initial sample for detection, which greatly enriches the content of tumor genetic diagnosis. [0003] The most commonly used standard pathological diagnostic sample for tumor genetic testi...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6862
CPCC12Q1/6862C40B50/06C12Q2525/191
Inventor 谢洪涛高志博李淼刘佳慧周专
Owner 深圳裕策生物科技有限公司
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