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Method for performing DNA terminal repair/dA addition by adopting one-step method and application

A technique of end repair and footwork, applied in the field of molecular biology, can solve problems such as incompatibility of activity, and achieve the effects of avoiding DNA loss, improving efficiency, and reducing consumption of reagent consumables

Inactive Publication Date: 2016-05-11
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, DNA end repair based on these enzymatic processes is incompatible with DNA 3’-end A addition and 3’-5’ DNA exonuclease activity, resulting in DNA blunt end, phosphorylation, and A addition must be performed separately

Method used

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  • Method for performing DNA terminal repair/dA addition by adopting one-step method and application
  • Method for performing DNA terminal repair/dA addition by adopting one-step method and application
  • Method for performing DNA terminal repair/dA addition by adopting one-step method and application

Examples

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Effect test

Embodiment 1

[0048] A method for repairing DNA ends and adding dA, first preparing a premix, the composition of which is shown in Table 1:

[0049] Table 1

[0050]

[0051] The method comprises the steps of:

[0052] (1) Interrupt the DNA to be repaired, and take 5ng of the interrupted DNA;

[0053] (2) Mix the DNA interrupted in step (1) with the premix solution at a volume ratio of 1:1, react at 20°C for 20 minutes, then raise the temperature to 72°C and react for 15 minutes to complete DNA end repair / addition of dA.

Embodiment 2

[0055] A method for repairing DNA ends and adding dA, first preparing a premix, the composition of which is shown in Table 1:

[0056] Table 1

[0057]

[0058] The method comprises the steps of:

[0059] (1) Interrupt the DNA to be repaired, and take 2ng of the interrupted DNA;

[0060] (2) Mix the DNA interrupted in step (1) with the premix solution at a volume ratio of 1:1, react at 18°C ​​for 23 minutes, then raise the temperature to 68°C for another 20 minutes, and complete DNA end repair / addition of dA.

Embodiment 3

[0062] A method for repairing DNA ends and adding dA, first preparing a premix, the composition of which is shown in Table 1:

[0063] Table 1

[0064]

[0065] The method comprises the steps of:

[0066] (1) Interrupt the DNA to be repaired, and take 10ng of the interrupted DNA;

[0067] (2) Mix the DNA interrupted in step (1) with the premix solution at a volume ratio of 1:1, react at 25°C for 15 minutes, then raise the temperature to 70°C and react for 18 minutes to complete DNA end repair / addition of dA.

[0068] In summary, the method of the present invention adopts the specific ratio of dATP and dCTP+dTTP+dGTP, adjusts the ratio of T4 DNA polymerase, TaqDNA polymerase and T4 polynucleotide kinase by adjusting the buffer, so that T4 DNA polymerase, TaqDNA Polymerase and T4 polynucleotide kinase can meet the reaction conditions at the same time, and by controlling the temperature, the activities of the three enzymes can be adjusted, and the DNA end repair / addition of ...

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Abstract

The invention belongs to the technical field of molecular biology and particularly relates to a method for performing DNA terminal repair / dA addition by adopting a one-step method and application thereof. The method adopts an enzyme system of T4DNA polymerase, Taq DNA polymerase and T4 polynucleotide kinase. The time required by the DNA terminal repair / dA addition in the method is shortened from one and a half hours to 40 minutes, and the reagent consumable consumption is reduced due to the fact that a DNA purification step is omitted, the cost is saved, DNA loss caused by purification is avoided, the preparation efficiency of a DNA library is improved, and the lowest DNA quality required by preparation of the DNA library is reduced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method and application of one-step method for DNA end repair / addition of dA. Background technique [0002] In next-generation sequencing (NGS) based on the Illumina platform, the DNA to be sequenced must first be truncated uniformly to a certain length, and then the adapters necessary for sequencing are added to the two segments to complete the preparation of the DNA library. [0003] Ultrasonic fragmentation or an enzyme mixture capable of randomly cleaving double-stranded DNA is currently the most widely used DNA fragmentation method. DNA fragmented by these methods is heterogeneous in the state of the two segments, with 5'-bulges or 3'-bulges. For ultrasonically fragmented DNA, the phosphate groups or hydroxyl groups at the 5' and 3' ends are not necessarily the same. In order to add adapters necessary for sequencing to both ends of these heterogeneou...

Claims

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Application Information

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IPC IPC(8): C12N15/11C40B40/06
CPCC12N15/11C12N2310/10C40B40/06
Inventor 孙子奎王锋丁方美李嫦娥吴帅来朱月艳王果
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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