阅读说明：本技术 mtDNA的提取和建库试剂 (mtDNA extraction and library construction reagent ) 是由 何怡华 李�杰 孙海瑞 郝晓燕 亦桐 王欣 张思瑶 陈兆艺 杨琪 于 2021-09-30 设计创作，主要内容包括：本发明涉及基因组DNA富集技术领域,尤其涉及mtDNA的提取和建库试剂。本发明提供的mtDNA提取的试剂中裂解液的组成更合理,以该裂解液处理后的人血样品能够富集得到更多的mtDNA。且该mtDNA能够提高测序文库的建库效率,经检测测序效果良好。(The invention relates to the technical field of genomic DNA enrichment, in particular to a mtDNA extraction and library building reagent. The composition of the lysis solution in the mtDNA extraction reagent provided by the invention is more reasonable, and a human blood sample treated by the lysis solution can be enriched to obtain more mtDNA. And the mtDNA can improve the library building efficiency of a sequencing library, and has a good sequencing effect through detection.)

The mtDNA extraction kit comprises lysate and an extraction reagent;

the lysis solution comprises water, sodium salt, magnesium salt, Tris-HCl and surfactant;

the surfactant is IGEPAL-CA 630.

2. The mtDNA extraction kit according to claim 1,

the lysis solution comprises water and 130-150 mM NaCl, 8-12 mM Tris-HCl, 1-10 mM MgCl₂、0.1～1％IGEPAL-CA630。

3. An extraction method of mtDNA, comprising: extracting a sample with the kit of claim 1 or 2.

4. The extraction method according to claim 3, wherein the sample is whole blood.

5. The extraction method according to claim 3 or 4, comprising: mixing whole blood with a lysis solution, performing lysis at 0-4 ℃ for 20-40 min, and centrifuging to obtain a supernatant; and carrying out proteolysis on the supernatant, and purifying to obtain mtDNA.

6. A mtDNA pooling reagent comprising: 2XTD buffer solution, Tn5 transposase, 2% SDS solution, Q5 High-Fidelity DNA Polymerase, Ultra II Q5 Master Mix;

the buffer comprises water and 10mM Tris-Cl, 5mM MgCl₂And 10 wt% N, N-Dimethylformamide.

7. A library construction method of mtDNA, characterized in that mtDNA prepared by the extraction method of any one of claims 3 to 5 is incubated at 85 ℃ and then subjected to library construction by the library construction reagent of claim 6.

8. The library building method of claim 7, wherein the library building specifically comprises:

mixing 1ng of the mtDNA, 10. mu.l of 2XTD buffer, 0.05. mu. of LTn5 transposase, make up 20. mu.L with water, and incubating at 55 ℃ for 10 min;

adding 2 μ l2 wt% SDS, incubating at 70 deg.C for 20 min;

after adding 0.5. mu. l Q5 High-Fidelity DNA Polymerase, 25. mu.l Ultra II Q5 Master Mix, 2. mu.l primer, amplification was performed, and after magnetic bead purification, a sequencing library was obtained.

9. The library construction method of claim 8, wherein the amplification procedure comprises: cycling 1 time at 72 ℃ for 3 minutes; 30 seconds at 98 ℃ and 20 cycles at 75 seconds at 65 ℃.

10. The library construction method of claim 8, wherein the sequence of the primer comprises:

5′-AATGATACGGCGACCACCGA-3′；

5′-CAAGCAGAAGACGGCATACGA-3′。

Technical Field

The invention relates to the technical field of genomic DNA enrichment, in particular to a mtDNA extraction and library building reagent.

Background

Mitochondrial DNA (mtdna) is the genetic material in mitochondria, and the mitochondrial genome is a naked double-stranded molecule of DNA, predominantly circular but also linear. Mitochondrial genomes vary in size from species to species. In general, the mitochondrial genome in animal cells is small, 10-39 kb.

The genetic process of mtDNA strictly follows a parthenogenetic maternity pattern, and compared to the nuclear genome, the mitochondrial genome has the following properties: all genes are located on a single circular DNA molecule. The genetic material is not coated by the nuclear membrane. DNA is not compacted by proteins. The genome does not contain as many non-coding regions (regulatory regions or "introns"). Some codons differ from the universal codon. In contrast, similar to some purple non-sulfur bacteria. Some bases are part of two different genes (overlapping genes): a certain base serves as the end of one gene and at the same time as the start of the next gene. Mitochondrial DNA survives much longer than DNA and is inherited from the mother, and is therefore ideal for confirming familial relationships.

mtDNA has been widely used in association with studies in animal population genetics and evolutionary biology, and has achieved many meaningful results. In the process of researching mtDNA, an effective extraction method is undoubtedly the premise of research development, and library construction in the further sequencing process is also an indispensable step. In order to obtain more accurate research results, the method has important significance on the extraction of mtDNA and the optimization of a database building method.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an extraction and pooling reagent for mtDNA.

The mtDNA extraction kit provided by the invention comprises a lysate and an extraction reagent; the lysis solution comprises water, sodium salt, magnesium salt, Tris-HCl and surfactant.

In the present invention, the surfactant is selected from IGEPAL-CA 630.

In the invention, the sodium salt is NaCl. The magnesium salt is MgCl₂。

In some embodiments, the lysis solution comprises water and 130-150 mM NaCl, 8-12 mM Tris-HCl, 1-10 mM MgCl₂、0.1～1％IGEPAL-CA630。

In some embodiments, the lysis solution consists of water and the following components: 140mM NaCl, 10mM Tris-HCl, 5mM MgCl₂、0.6％IGEPAL-CA630。

In the present invention, the extraction reagent comprises: protease and purification reagents.

The purification reagent comprises silica gel column or magnetic beads.

The invention also provides an extraction method of mtDNA, which comprises the following steps: the kit is used for extracting samples.

In the present invention, the sample in the extraction method is whole blood. Human whole blood is preferred.

The extraction method specifically comprises the following steps: mixing whole blood with a lysis solution, performing lysis at 0-4 ℃ for 20-40 min, and centrifuging to obtain a supernatant; and carrying out proteolysis on the supernatant, and purifying by using a column to obtain mtDNA. In some embodiments, the time for lysis is 30 min. The volume ratio of the whole blood to the lysate is 1: 2.

The invention also provides a library establishing reagent of mtDNA, which comprises: 2XTD buffer solution, transposase, 2% SDS solution, Q5 High-Fidelity DNApolymerase, Ultra II Q5 Master Mix;

the buffer comprises water and 10mM Tris-Cl, 5mM MgCl₂And 10 wt% N, N-Dimethylformamide.

The invention also provides a library construction method of mtDNA, which comprises the step of carrying out library construction by using the library construction reagent after the mtDNA prepared by the extraction method is incubated at 85 ℃. In the invention, the incubation temperature is 70-85 ℃, but the warehouse building efficiency can be obviously improved when the incubation temperature is 85 ℃. The incubation time is 1-10 min, preferably 5 min. After incubation, the cells were stored at 55 ℃.

In the invention, the database building specifically comprises: mixing 1ng of the mtDNA, 10. mu.l of 2XTD buffer, 0.05. mu. of LTn5 transposase, make up 20. mu.L with water, and incubating at 55 ℃ for 10 min;

adding 2 μ l2wt ‰ SDS, and incubating at 70 deg.C for 20 min;

after adding 0.5. mu. l Q5 High-Fidelity DNA Polymerase, 25. mu.l ultra II Q5 Master Mix, 2. mu.l primer, amplification was performed, and after magnetic bead purification, a sequencing library was obtained.

In the library construction method of the present invention, the amplification procedure comprises: cycling 1 time at 72 ℃ for 3 minutes; 30 seconds at 98 ℃ and 20 cycles at 75 seconds at 65 ℃. The sequence of the primer is

5'-AATGATACGGCGACCACCGA-3' and

5′-CAAGCAGAAGACGGCATACGA-3。

the library established by the library construction method provided by the invention can be directly subjected to computer sequencing.

The invention provides a reagent for extracting mtDNA, wherein the composition of a lysate is more reasonable, and a human blood sample treated by the lysate can be enriched to obtain more mtDNA. And the mtDNA can improve the library building efficiency of a sequencing library, and has a good sequencing effect through detection.

Drawings

FIG. 1 shows the enrichment and banking effect of mtDNA obtained by lysate extraction;

FIG. 2 shows the effect of incubation temperature on the effect of mtDNA enrichment pooling.

Detailed Description

The invention provides a reagent for extracting and establishing a library of mtDNA, and a person skilled in the art can use the content for reference and appropriately improve the process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market. Wherein the mtDNA extraction kit is from magenta with the product number of D3018-03.

The invention is further illustrated by the following examples:

example 1

To 100. mu.l of whole blood was added 200. mu.l of lysate [140mM NaCl (Invitrogen, Cat. No. AM9759),10mM Tris-HCl (amresco, Cat. No. E199-500 mL),5mM MgCl (Invitrogen, Cat. No. AM9530G), 0.6% IGEPAL-CA630(MP, Cat. No.19856) ], and the mixture was gently blown with a gun or inverted 5 times, and then allowed to stand at 4 ℃ or on ice for 30 minutes. Centrifuge at 3000g for 10min and gun carefully remove 150ul of supernatant (avoid disturbing the bottom nuclear pellet). The DNA in the supernatant was extracted with a commercial kit (magenta, Cat. No. D3018-03) (mtDNA enrichment effect is shown in FIG. 1, the effect of the lysate is better than the mtDNA enrichment effect after lysis under other conditions).

Before library construction, the incubation of DNA at 85 ℃ for 5 minutes is helpful for improving the library construction efficiency of mtDNA. The pool building effect of mtDNA samples obtained at different incubation temperatures was shown in FIG. 2, stored at 55 ℃ until the next step.

1ng of the above DNA was collected and subjected to library construction. The reaction system included 10ul of 2xTD buffer [10mM Tris-Cl (pH 7.6),5mM MgCl₂,10％N,N-Dimethylformamide]0.05. mu.L of Tn5 transposase (Vazyme, Cat. No. TD501-01), supplemented with water to 20 ul. After incubation at 55 ℃ for 10min, 2ul of 2% SDS was added and incubation at 70 ℃ for 20 min. Amplification was performed by the following procedure with the addition of 0.5ul Q5 High-Fidelity DNApolymerase (NEB, Cat. No. M0491L) and 25ul ultra II Q5 Master Mix (NEB, Cat. No. M0544L) and 2ul of the corresponding primers (single primer concentration 5 uM): cycling 1 time at 72 ℃ for 3 minutes; 30 seconds at 98 ℃ and 20 cycles at 75 seconds at 65 ℃. And sequencing by magnetic bead purification.

The primer sequence is as follows:

5′-aatgatacggcgaccaccga-3′；5′-caagcagaagacggcatacga-3′。

sequencing results show that the mtDNA content (more than 30%) is obviously higher than the theoretical content (1 per thousand-1%) of the non-enriched mtDNA, and the average mitochondrial genome coverage of 1000x can be obtained only by 50Mbp sequencing.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.