阅读说明：本技术 一种快速转化胞嘧啶的方法及所用试剂盒 (Method for rapidly transforming cytosine and kit used in same ) 是由 张帆 贾泽川 于 2021-08-25 设计创作，主要内容包括：本发明提供一种快速转化胞嘧啶的方法及所用试剂盒。所述转化试剂盒由DNA修饰液、M-结合液、M-洗涤液、L-脱硫缓冲液、M-洗脱液、I号柱或磁珠、收集管组成。各溶液按比例配制好后,可以更快速的对DNA胞嘧啶进行转化；所述洗涤液有利于清洗修饰过程中引入的其它成分,达到修饰DNA、纯化修饰DNA的目的。(The invention provides a method for quickly converting cytosine and a kit used by the method. The transformation kit consists of DNA modification liquid, M-binding liquid, M-washing liquid, L-desulfurization buffer solution, M-eluent, I-column or magnetic bead and collection tube. After the solutions are prepared in proportion, DNA cytosine can be converted more quickly; the washing solution is beneficial to cleaning other components introduced in the modification process, and the purposes of modifying DNA and purifying the modified DNA are achieved.)

1. A kit for converting cytosine consists of a DNA modification solution, an M-binding solution, an M-washing solution, an L-desulfurization buffer solution, an M-eluent, a No. I column or magnetic bead and a collecting pipe.

2. The kit of claim 1, wherein: the DNA modification solution is used for modifying unmethylated cytosine into uracil, and the M-binding solution, the M-washing solution, the L-desulfurization buffer solution, the M-eluent, the I column or the magnetic bead and the collecting tube are used for purifying the modified DNA.

3. The kit according to claim 1 or 2, characterized in that: the DNA modification solution consists of sodium bisulfite and tris (hydroxymethyl) aminomethane, wherein the sodium bisulfite is 50 g/L to 80 g/L; 2g-10g/L of tris (hydroxymethyl) aminomethane;

the M-binding solution is an aqueous solution of Tween 20, wherein the concentration of Tween 20 is 3-15%;

the M-washing solution is absolute ethyl alcohol with the volume concentration of 80%;

the L-desulfurization buffer solution is a tris (hydroxymethyl) aminomethane group with the concentration of 1-10 mM;

the M-eluent is DNase/RNase-free water;

the aperture of the adsorption membrane of the column I is 20-30 um.

4. A method of using the kit of any one of claims 1-3, comprising the steps of:

1) adding a DNA modification solution into a PCR tube containing a DNA sample, uniformly mixing, and placing on a PCR instrument for reaction;

2) adding M-binding solution to the No. 1 column and placing on a collecting pipe;

3) adding the sample modification solution reacted in the step 1) to the No. 1 column filled with the M-binding solution obtained in the step 2), uniformly mixing, centrifuging, and discarding the liquid in the collecting pipe;

4) adding an M-washing solution to the No. 1 column in the step 3), centrifuging, and discarding the liquid in the collection tube;

5) adding an L-desulfurization buffer solution to the No. 1 column in the step 4), standing for 15-20 minutes at normal temperature, centrifuging, and discarding the liquid in the collecting pipe;

6) adding an M-washing solution to the No. 1 column in the step 5), centrifuging, and discarding the liquid in the collection tube; repeatedly adding M-washing liquid, and centrifuging once;

7) and (3) putting the No. 1 column in the step 6) into a new collecting pipe, adding M-eluent, centrifuging, and collecting the eluent to obtain the purified DNA modification solution.

5. The method of claim 4, wherein: in the step 1), the volume ratio of the DNA modification solution to the DNA sample is 110-: 20 ul;

the reaction was carried out according to the following procedure: reacting at 98 ℃ for 8 minutes and at 54 ℃ for 60 minutes;

in the step 2), the volume ratio of the M-binding solution to the DNA sample is 200-: 20 ul;

in the step 3), the centrifugation conditions are as follows: centrifuging at 1000rpm for 30 s;

in the step 4), the volume ratio of the M-washing solution to the DNA sample is 80-120 ul: 20 ul; the centrifugation conditions were: centrifuging at 1000rpm for 30 s;

in step 5), the volume ratio of the L-desulfurization buffer solution to the DNA sample is 150-: 20 ul;

the centrifugation conditions were: centrifuging at 1000rpm for 30 s;

in step 6), the volume ratio of the M-washing solution to the DNA sample is 150-: 20 ul; the centrifugation conditions were: centrifuging at 1000rpm for 30 s;

in step 7), the volume ratio of the M-eluate to the DNA sample is 5-20 ul: 20 ul;

the centrifugation conditions were: centrifuge at 12000rpm for 30 seconds.

6. A method of using the kit of any one of claims 1-3, comprising the steps of:

1) adding a DNA modification solution into a PCR tube containing a DNA sample, uniformly mixing, and placing on a PCR instrument for reaction;

2) adding M-binding solution and magnetic beads into a collecting pipe, adding the sample modifying solution reacted in the step 1), uniformly mixing and oscillating;

3) standing for 3-10 min, transferring to a magnetic rack, standing until the magnetic beads are separated from the suspension, and removing the supernatant;

4) taking down the collecting pipe from the magnetic frame, adding M-washing liquid, mixing, oscillating, placing on the magnetic frame until the magnetic beads are separated from the suspension, and removing the supernatant;

5) adding L-desulfurization buffer solution, uniformly mixing and oscillating for 30 minutes, standing for 15-20 minutes, placing on a magnetic frame until the magnetic beads are separated from the suspension, and removing supernatant;

6) adding M-washing solution, mixing uniformly, oscillating, standing for 15-20min, placing on a magnetic rack until the magnetic beads are separated from the suspension, and removing the supernatant;

7) transferring the collecting pipe to a warm bath for heating, adding M-eluent, oscillating and uniformly mixing, standing at 55 ℃ for 4 minutes, placing on a magnetic frame until the magnetic beads are separated from the suspension, and collecting the suspension to obtain the purified DNA modifying solution.

7. The method of claim 6, wherein: in the step 1), the volume ratio of the DNA modification solution to the DNA sample is 110-: 20 ul;

the reaction was carried out according to the following procedure: reacting at 98 ℃ for 8 minutes and at 54 ℃ for 60 minutes;

in the step 2), the volume ratio of the M-binding solution, the magnetic beads and the DNA sample is 350-: 5-20 ul: 20 ul; the oscillation is 12000rpm and 30 seconds;

in the step 3), the standing time is 5 minutes;

in step 4), the volume ratio of the M-washing solution to the DNA sample is 150-: 20 ul; the oscillation is 12000rpm for 30 seconds;

in step 5), the volume ratio of the L-desulfurization buffer solution to the DNA sample is 50-300 ul: 20 ul; the oscillation is 12000rpm and 30 seconds; standing for 15-20 min;

in step 6), the volume ratio of the M-washing solution to the DNA sample is 150-: 20 ul; the oscillation is 12000rpm and 30 seconds; standing for 15-20 min;

in the step 7), the heating of the warm bath is carried out for 20-30 minutes in a heating module at 55 ℃;

the volume ratio of the M-eluent to the DNA sample is 5-50 ul: 20 ul;

the oscillation is 12000rpm and 30 seconds; the placement was at 55 ℃ for 4 minutes.

Technical Field

The invention relates to a method for quickly converting cytosine and a kit used by the same, belonging to the field of epigenetics.

Background

Cytosine methylation is a naturally occurring base modification that occurs in both prokaryotes and eukaryotes, where DNA methylation provides a means of protecting host DNA from digestion by endonucleases whose purpose is to eliminate foreign DNA. DNA methylation in higher eukaryotes plays a role in gene expression regulation. Most DNA methylation in mammals occurs in 5'-CpG-3' dinucleotides, although other patterns also exist. 80% of the 5'-CpG-3' dinucleotides in the mammalian genome are found to be methylated, while the remaining 20% of unmethylated majority are located in the first exon of the promoter or gene. Aberrant DNA methylation has been shown to be a ubiquitous phenomenon in cancer, probably one of the earliest changes in tumorigenesis. DNA methylation has also been shown to play a central role in gene imprinting, embryonic development, X chromosome gene silencing, and cell cycle regulation. The ability to efficiently and accurately detect and quantify DNA methylation has become essential for the study of cancer, gene expression, genetic diseases, and many other important aspects of biology. To date, a number of methods have been developed to detect and quantify DNA methylation, including: capillary electrophoresis (Fraga MF, et al. electrophoresis.2000; 21(14): 2990-. However, the most common techniques at present still rely on bisulfite conversion. Treatment of DNA with bisulfite chemistry modifies unmethylated cytosines to uracil, with methylated cytosines remaining unchanged.

Disclosure of Invention

The invention aims to solve the problems of insufficient conversion and long conversion time of a conversion kit in the use process in the prior art, and provides a conversion kit based on sodium bisulfite.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a transformation kit.

The transformation kit provided by the invention consists of DNA modification liquid, M-binding liquid, M-washing liquid, L-desulfurization buffer solution, M-eluent, No. I column or magnetic bead and a collecting pipe.

The DNA modification solution is used for modifying unmethylated cytosine into uracil, and the M-binding solution, the M-washing solution, the L-desulfurization buffer solution, the M-eluent, the I column or the magnetic bead and the collecting tube are used for purifying the modified DNA.

The DNA modification solution consists of sodium bisulfite and tris (hydroxymethyl) aminomethane, and specifically comprises the following components: 50g-80g/L of sodium bisulfite, specifically 60 g/L; the tris (hydroxymethyl) aminomethane is 2g-10g/L, specifically 5.5g/L or 8 g/L.

The M-binding solution is a Tween 20 aqueous solution, wherein the concentration of Tween 20 can be 3-15% (namely, 100ml of water contains 3-15ml of Tween 20);

the M-wash was 80% absolute ethanol by volume (i.e., 80ml absolute ethanol, 20ml water in 100ml M-wash).

The L-desulfurization buffer solution is 1-10mM of tris (hydroxymethyl) aminomethane.

The M-eluent is DNase/RNase-free water;

the aperture of the adsorption membrane of the column I is 20-30 um.

In another aspect, the present invention provides a method of using the transformation kit as described above.

The use method of the transformation kit containing the No. 1 column provided by the invention comprises the following steps:

1) adding a DNA modification solution into a PCR tube containing a DNA sample, uniformly mixing, and placing on a PCR instrument for reaction;

2) adding M-binding solution to the No. 1 column and placing on a collecting pipe;

3) adding the sample modification solution reacted in the step 1) to the No. 1 column filled with the M-binding solution obtained in the step 2), uniformly mixing, centrifuging, and discarding the liquid in the collecting pipe;

4) adding an M-washing solution to the No. 1 column in the step 3), centrifuging, and discarding the liquid in the collection tube;

5) adding an L-desulfurization buffer solution to the No. 1 column in the step 4), standing for 15-20 minutes at normal temperature, centrifuging, and discarding the liquid in the collecting pipe;

6) adding an M-washing solution to the No. 1 column in the step 5), centrifuging, and discarding the liquid in the collection tube; repeating the operation once;

7) and (3) placing the No. 1 column in the step 6) on a new collecting pipe, adding M-eluent, centrifuging, and collecting the eluent to obtain the purified DNA modification solution.

In step 1) of the method, the volume ratio of the DNA modification solution to the DNA sample may be 110-: 20ul, specifically 130 ul: 20 ul;

the reaction was carried out according to the following procedure: reacting at 98 ℃ for 8 minutes and at 54 ℃ for 60 minutes;

in step 2), the volume ratio of the M-binding solution to the DNA sample may be 200-: 20ul, specifically 600 ul:

20ul；

in step 3), the centrifugation conditions may be: centrifuging at 1000rpm for 30 s;

in step 4), the volume ratio of the M-washing solution to the DNA sample can be 80-120 ul: 20ul, specifically 100 ul: 20 ul; the conditions of the centrifugation may be: centrifuging at 1000rpm for 30 s;

in step 5), the volume ratio of the L-desulfurization buffer solution to the DNA sample can be 150-: 20ul, specifically 200 ul: 20 ul;

the conditions of the centrifugation may be: centrifuging at 1000rpm for 30 s;

in step 6), the volume ratio of the M-washing solution to the DNA sample can be 150-: 20ul, specifically 200 ul: 20 ul; the conditions of the centrifugation may be: centrifuging at 1000rpm for 30 s;

in step 7), the volume ratio of the M-eluate to the DNA sample may be 5-20 ul: 20ul, specifically 10 ul: 20 ul;

the conditions of the centrifugation may be: centrifuge at 1200rpm for 30 seconds.

The obtained purified DNA modification solution can be subjected to subsequent methylation detection or stored in a refrigerator at-80 ℃.

The use method of the transformation kit containing the magnetic beads, provided by the invention, comprises the following steps:

1) adding a DNA modification solution into a PCR tube containing a DNA sample, uniformly mixing, and placing on a PCR instrument for reaction;

2) adding M-binding solution and magnetic beads into a collecting pipe, adding the sample modifying solution reacted in the step 1), uniformly mixing and oscillating;

3) standing for 3-10 min (specifically 5 min), transferring to a magnetic rack, standing until the magnetic beads are separated from the suspension, and removing the supernatant;

4) taking down the collecting pipe from the magnetic frame, adding M-washing liquid, mixing, oscillating, placing on the magnetic frame until the magnetic beads are separated from the suspension, and removing the supernatant;

5) adding L-desulfurization buffer solution, uniformly mixing and oscillating for 30 minutes, standing for 15-20 minutes, placing on a magnetic frame until the magnetic beads are separated from the suspension, and removing supernatant;

6) adding M-washing solution, mixing uniformly, oscillating, standing for 15-20min, placing on a magnetic rack until the magnetic beads are separated from the suspension, and removing the supernatant;

7) transferring the collecting pipe to a warm bath for heating, adding M-eluent, oscillating and uniformly mixing, standing at 55 ℃ for 4 minutes, placing on a magnetic frame until the magnetic beads are separated from the suspension, and collecting the suspension to obtain the purified DNA modifying solution.

In step 1) of the method, the volume ratio of the DNA modification solution to the DNA sample may be 110-: 20ul, specifically 130 ul: 20 ul;

the reaction was carried out according to the following procedure: reacting at 98 ℃ for 8 minutes and at 54 ℃ for 60 minutes;

in the step 2), the volume ratio of the M-binding solution, the magnetic beads and the DNA sample can be 350-: 5-20 ul: 20ul, specifically 600 ul: 10 ul: 20 ul; the oscillation is 12000rpm and 30 seconds;

in the step 3), the standing time can be 5 minutes;

in step 4), the volume ratio of the M-washing solution to the DNA sample may be 150-: 20ul, specifically 400 ul: 20 ul; the oscillation can be 12000rpm oscillation for 30 seconds;

in step 5), the volume ratio of the L-desulfurization buffer solution to the DNA sample can be 50-300 ul: 20ul, specifically 200 ul: 20 ul; the oscillation may be 12000rpm oscillation for 30 seconds; the standing time can be 15-20 min;

in step 6), the volume ratio of the M-washing solution to the DNA sample can be 150-: 20ul, specifically 400 ul: 20 ul; the oscillation may be 12000rpm oscillation for 30 seconds; the standing time can be 15-20 min;

in the step 7), the heating in the warm bath can be carried out in a heating module at 55 ℃ for 20-30 minutes;

the volume ratio of the M-eluate to the DNA sample may be 5-50 ul: 20ul, specifically 25 ul: 20 ul;

the oscillation may be 12000rpm oscillation for 30 seconds; the placement is carried out for 4 minutes at 55 ℃;

the obtained purified DNA modification solution can be subjected to subsequent methylation detection or stored in a refrigerator at-80 ℃.

Compared with the prior art, the beneficial effect of this discovery is:

the transformation kit comprises DNA modification liquid, M-binding liquid, M-washing liquid, L-desulfurization buffer solution, M-eluent, a No. I column or magnetic beads and a collecting pipe. After the solution is prepared in proportion, DNA cytosine can be converted more quickly; the washing solution is beneficial to cleaning other components introduced in the modification process, and the purposes of modifying DNA and purifying the modified DNA are achieved.

Drawings

FIG. 1 is a schematic diagram of DNA cytosine conversion.

FIG. 2 shows the genomic prosequence of the amplified region.

FIG. 3 shows the conversion of methylated cytosine.

FIG. 4 shows the conversion of unmethylated cytosine.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

There is provided a kit for rapid transformation of cytosine, the kit comprising:

test examples

The test example uses the kit for rapid transformation of cytosine provided in the example, and the transformation steps are as follows:

1. 130ul of the modified DNA solution was added to a PCR tube containing 20ul of a DNA sample (methylated or unmethylated DNA, zymousearch, Inc.: D5014, and the genomic nucleotide sequence of the amplified region is shown in FIG. 2), and the mixture was thoroughly mixed. The reaction was carried out on a PCR instrument according to the following procedure: the reaction was carried out at 98 ℃ for 8 minutes and at 54 ℃ for 60 minutes. Subsequent experiments were then performed or stored at 4 ℃ for no more than 20 hours.

2. The reacted sample modifier was added to column 1 containing M-conjugate (600ul), mixed well and centrifuged at 1000rpm for 30 seconds.

3. 100ul of M-wash was added and centrifuged at 1000rpm for 30 seconds.

4. 200ul of L-desulfurization buffer was added thereto, and the mixture was left at room temperature (20-30 ℃ C.) for 15-20 minutes. Centrifuge at 1000rpm for 30 seconds.

5. 200ul of M-wash was added and centrifuged at 1000rpm for 30 seconds; the operation was repeated once.

6. Column # 1 was placed in a fresh collection tube, 10ul of M-eluent was added, and the column was allowed to dry for 30 seconds at full speed.

7. The obtained DNA was amplified using a primer (methylated or unmethylated DNA, product number: D5014) and the amplified product was sequenced. Sequencing results showed that neither methylated cytosine was transformed (FIG. 3), while neither unmethylated cytosine was transformed (FIG. 4).

Example 2

This example provides a kit for rapid transformation of cytosine, the kit comprising:

test examples

The test example uses the kit for rapid transformation of cytosine provided in the example, and the transformation steps are as follows:

1. 130ul of the modified DNA solution was added to a PCR tube containing 20ul of a DNA sample (methylated or unmethylated DNA, product number D5014 from zymoresearch), and the mixture was thoroughly mixed. The reaction was carried out on a PCR instrument according to the following procedure: the reaction was carried out at 98 ℃ for 8 minutes and at 54 ℃ for 60 minutes. Subsequent experiments were then performed or stored at 4 ℃ for no more than 20 hours.

2. 600ul of M-binding solution and 10ul of magnetic beads were added to the collection tube; the 1 treated sample was added, mixed well and shaken at 12000rpm for 30 seconds.

3. After standing for 5 minutes, the suspension was transferred to a magnetic rack and then allowed to stand for 5 minutes until the magnetic beads were separated from the suspension, and the supernatant was removed.

4. The magnetic frame in the collection tube was removed, 400ul of M-wash was added, mixed well and shaken at 12000rpm for 30 seconds, placed on the magnetic frame for 3 minutes until the beads separated from the suspension, and the supernatant removed.

5. 200ul of L-desulfurization buffer was added thereto, mixed well and shaken at 12000rpm for 30 seconds, and left at room temperature (20 ℃ C. -30 ℃ C.) for 15-20 minutes. The suspension was placed on a magnetic stand for 3 minutes until the beads were separated from the suspension, and the supernatant was removed.

6. 400ul of M-washing solution was added thereto, mixed well and shaken at 12000rpm for 30 seconds, and left at room temperature (20 ℃ C. -30 ℃ C.) for 15-20 minutes. The suspension was placed on a magnetic stand for 3 minutes until the beads were separated from the suspension, and the supernatant was removed.

7. Transferring the collecting tube to a heating module at 55 deg.C, warm-bathing for 20-30 min, adding 25ul M-eluent, shaking at 12000rpm for 30 s, mixing, and standing at 55 deg.C for 4 min. Placing on a magnetic frame for 1 minute until the magnetic beads are separated from the suspension, and collecting the suspension.

8. The obtained DNA was amplified using a primer (methylated or unmethylated DNA, product number: D5014) and the amplified product was sequenced. Sequencing results showed that neither methylated cytosine was transformed (FIG. 3), while neither unmethylated cytosine was transformed (FIG. 4).

The DNA transformation process of this example only took 60 minutes, and the results of the transformation (FIGS. 3 and 4) showed that unmethylated cytosines were sufficiently transformed.