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Nucleic acid sample treating method, sequencing method and kit

A sample and polynucleotide kinase technology, which is applied in the field of kits and nucleic acid sample processing, can solve the problems of not disclosing specific formulas, etc., and achieve the effects of saving operating time, simplifying operations, and facilitating industrialization

Active Publication Date: 2021-02-23
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, commercially available kits do not disclose their specific formulations, including the type, characteristics, concentration, and configuration of specific reaction solutions of enzymes, etc.

Method used

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  • Nucleic acid sample treating method, sequencing method and kit
  • Nucleic acid sample treating method, sequencing method and kit
  • Nucleic acid sample treating method, sequencing method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The method for preparing the kit is exemplified below.

[0051] The kit contains tube 1, which is a mixed system with end repair function (also called End Prep Mix). In one case, tube 1 is a solution containing polynucleotide kinases and various DNA polymerases. In another case, tube 1 is a solution containing DNA fragmentation enzymes, polynucleotide kinases and various DNA polymerases.

[0052] The DNA polymerase can fill in the ends of the fragmented nucleic acid fragments. Here, preferably, the selected DNA polymerase has an amplification function, as well as repair and proofreading functions, such as selected from Taq DNA polymerase (referred to as Taq DNA polymerase for short). Enzyme) and one or both of Klenow fragment; polynucleotide kinase can be selected as T4 Polynucleotide Kinase (T4 PolynucleotideKinase).

[0053] In one case, the so-called DNA polymerase is a mixture of Taq enzyme and Klenow fragment. The concentration of Taq enzyme in tube 1 was (1-2) ...

Embodiment 2

[0062] This example provides the application of the kit in Example 1 in library construction. A total of 4 parallel experiments were performed in this example, and the samples used were F4, F5, F6 and F7 genomes. The quality of the genomes needs to be preliminarily evaluated before the genomic DNA is fragmented to ensure that the genomes exist in the absence of metal ion chelators or other salts. solvent, and the genome integrity was checked by gel electrophoresis. The specific steps of library construction are as follows:

[0063] 1. Genomic DNA fragmentation, end repair and dA tailing

[0064] This step fragmented the DNA with blunt end-filling, phosphorylation at the 5' end and dA tailing at the 3' end.

[0065] Configure the reaction system in the PCR tube as shown in Table 1:

[0066] Table 1

[0067] H2O 14ul End Prep Mix 4ul DNA (25ng / ul) 2ul Total 20ul

[0068] Place the PCR tube in the PCR machine and set the reaction conditions a...

Embodiment 3

[0120] This example provides application 2 of the kit in Example 1 in library construction. A total of 12 parallel experiments were performed in this example, and the samples used were N5, N6, N13, N14, N21, N22, N23, N24, O5, O6, O11 and O12 genomes. Do a preliminary assessment to ensure that the genome is present in a solvent free of metal ion chelators or other salts, and to check for genome integrity by gel electrophoresis. The specific steps of library construction are as follows:

[0121] 1. DNA fragmentation, end repair and dA tailing

[0122] This step fragmented the DNA with blunt end-filling, phosphorylation at the 5' end and dA tailing at the 3' end.

[0123] Different samples have the same reaction system, as shown in Table 6, the reaction time at 32°C is different, 20min, 22min, 25min, 30min, 35min, 40min respectively.

[0124] The sample types and corresponding response times are as follows:

[0125] N5: 50ng, 32℃ for 20min; N6: 50ng, 32℃ for 20min;

[0126]...

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Abstract

The invention provides a DNA sample treating method, a sequencing method and a kit. The method for treating a DNA sample comprises the following steps: performing fragmentation, terminal repair and dAtail addition on DNA in a first premixed system to obtain a first product, wherein the first premixed system comprises premixed enzyme, and the premixed enzyme comprises DNA fragmentation enzyme, polynucleotide kinase and DNA polymerase. Based on test experiments and an optimized mixed enzyme system, multiple enzymes are mixed in the same reaction system, DNA breakage, terminal repair and dA tailaddition are realized in one step, the convenience and rapidness are achieved, the operation time of sample treatment before loading is saved, and therefore the industrialization is facilitated.

Description

technical field [0001] The invention belongs to the field of detection and analysis of biological macromolecules, and in particular, relates to a nucleic acid sample processing method, a sequencing method and a kit. Background technique [0002] High-throughput sequencing includes next-generation sequencing and single-molecule sequencing, etc., and involves the processing of nucleic acid samples before testing on the machine. In particular, the nucleic acid sequence determination based on chip detection requires the processing of the nucleic acid to be tested (template) to connect it to the chip to achieve sequence determination. [0003] Currently, commercially available sequencing platforms based on chip detection generally have matching chips for sale. The chips sold in matching chips carry specific sequences (probes), and the templates generally need to undergo various treatments to contain the specific sequence or be related to the specific sequence. At least a portion...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2521/101C12Q2527/101C12Q2521/501C12Q2563/107
Inventor 张萌刘丽春冯燕李改玲林群婷甘广丽
Owner GENEMIND BIOSCIENCES CO LTD
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