CN112760364A - PCR amplification sequencing analysis method by extracting DNA from whole blood - Google Patents
PCR amplification sequencing analysis method by extracting DNA from whole blood Download PDFInfo
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Abstract
The invention discloses a method for extracting DNA from whole blood to carry out PCR amplification sequencing analysis, which comprises the following steps of 1) preparing a required DNA sample, 2) carrying out PCR amplification and analysis, and 3) obtaining folic acid metabolism information; provides effective information, thereby improving the accuracy of folic acid metabolism.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a PCR amplification sequencing analysis method by extracting DNA from whole blood.
Background
Folic acid (pteroylglutamic acid) refers to a class of isovitamin with related biological activity, which is formed by combining pteridine, p-aminobenzoic acid and 1 or more glutamic acids, i.e. a-amino-4-hydroxyperidine is connected with p-aminobenzoic acid, and then is connected with glutamic acid by-NH-CO-bond, and different coenzyme forms can be used as donor or carrier of one-carbon unit to play a role, and mainly participate in the following reactions: firstly, methionine is synthesized; synthesis of purine and pyrimidine; ③ interconversion of serine and glycine; degradation of histidine. Numerous scientific studies have shown that absolute or relative deficiency in folate intake is a direct cause of hyperhomocysteinemia. Hyperhomocysteinemia may lead to neonatal Neural Tube Defects (NTDs), Down Syndrome (DS), Congenital Heart Disease (CHD), cleft lip and palate; pregnancy induced hypertension, premature birth, spontaneous abortion.
Disclosure of Invention
The invention provides a PCR amplification sequencing analysis method by extracting DNA from whole blood.
The scheme of the invention is as follows:
a method for PCR sequencing analysis by whole blood extraction of DNA comprising the steps of:
1) preparing a required DNA sample, extracting the DNA of the sample from whole blood, and storing the obtained DNA of the sample at the temperature of between 20 ℃ below zero and 4 ℃ for later use;
2) PCR amplification and analysis, namely constructing a reaction system in a PCR tube by using the sample DNA obtained in the step 1), then sending the reaction system into a PCR amplification instrument for amplification, setting a PCR circulation program, and obtaining the information of the sample after the amplification is finished;
3) and obtaining folic acid metabolism information, and performing a genotyping PCR experiment through software to obtain an experiment result and obtain folic acid metabolism information.
As a preferred technical scheme, the DNA of the whole blood extracted sample is as follows:
cutting a Tip of a 1-mL Tip, inserting a pipette, extending into a blood collection tube, repeatedly blowing and beating whole blood for 3-4 times, fully suspending precipitated white blood cells, transferring the whole blood to an EP tube, adding MGS lysate, adding proteinase K, uniformly mixing, heating in a water bath kettle at 60 ℃ for 10-15 min, wherein the uniformly mixing time is more than 2 times;
taking out the EP pipe from the water bath kettle, pouring all solution in the EP pipe into a centrifugal adsorption column, putting the adsorption column into a collecting pipe, centrifuging at 8000rpm for 1min, taking out the adsorption column, and pouring out waste liquid in the collecting pipe;
adding cleaning solution into the adsorption column
8000rpm, centrifuging for 30s, taking out the adsorption column, and pouring off the waste liquid in the collection tube;
rinsing twice, taking out the adsorption column after the completion, pouring off the waste liquid in the collection tube, putting the adsorption column back into the collection tube again, centrifuging at 12000rpm for 1min, and removing the residual ethanol;
placing the adsorption column in a new 1.5ml centrifuge tube, and drying at room temperature for 2-3 min; dripping 60 deg.C eluent ET into the adsorption column near the center of the adsorption membrane, centrifuging at 12000rpm for 1-2min, collecting the liquid in the tube as sample DNA solution, covering with a cover, and lightly pulling the liquid in the tube to mix well.
As a preferable technical scheme, the rinsing treatment is to add a rinsing liquid Wash into the adsorption column at 8000rpm, centrifuge for 30s, take out the adsorption column and pour the waste liquid in the collection tube.
As a preferable technical scheme, the reaction system in the step 2) is as follows:
as a preferable technical scheme, the temperature of the change in the circulation in the step 2) is changed to 92 ℃.
Because the technical scheme that the method for carrying out PCR amplification sequencing analysis by extracting DNA from whole blood is adopted, 1) the required DNA sample is prepared, the sample DNA is extracted from the whole blood, and the obtained sample DNA is stored at the temperature of-20-4 ℃ for standby; 2) PCR amplification and analysis, namely constructing a reaction system in a PCR tube by using the sample DNA obtained in the step 1), then sending the reaction system into a PCR amplification instrument for amplification, setting a PCR circulation program, and obtaining the information of the sample after the amplification is finished; 3) and obtaining folic acid metabolism information, and performing a genotyping PCR experiment through software to obtain an experiment result and obtain folic acid metabolism information.
The invention has the advantages that: the operation steps are simple, the detection time is shortened, the detection efficiency is improved, and the detection accuracy is improved;
provides effective information, thereby improving the accuracy of folic acid metabolism.
Drawings
FIG. 1 is a diagram of a Quickstart interface of the StepOnePlus software according to the embodiment of the present invention;
FIG. 2 is an information interface diagram of a genotyping assay according to an embodiment of the present invention;
FIG. 3 is an interface diagram of Run Method according to an embodiment of the present invention;
FIG. 4 is a diagram of an interface after information is filled in according to an embodiment of the present invention;
FIG. 5 is a configuration interface for entering running software according to an embodiment of the present invention;
FIG. 6 is a diagram of sample information setup according to an embodiment of the present invention;
FIG. 7 is a flow chart of the present invention;
FIG. 8 is a graphical parameter diagram of a first result of an embodiment of the present invention;
FIG. 9 is a second resulting convex parameter plot of an embodiment of the present invention.
Detailed Description
In order to remedy the above deficiencies, the present invention provides a method for PCR sequencing analysis by whole blood DNA extraction to solve the above problems in the background art.
A method for PCR sequencing analysis by whole blood extraction of DNA comprising the steps of:
1) preparing a required DNA sample, extracting the DNA of the sample from whole blood, and storing the obtained DNA of the sample at the temperature of between 20 ℃ below zero and 4 ℃ for later use;
2) PCR amplification and analysis, namely constructing a reaction system in a PCR tube by using the sample DNA obtained in the step 1), then sending the reaction system into a PCR amplification instrument for amplification, setting a PCR circulation program, and obtaining the information of the sample after the amplification is finished;
3) and obtaining folic acid metabolism information, and performing a genotyping PCR experiment through software to obtain an experiment result and obtain folic acid metabolism information.
As a preferred technical scheme, the DNA of the whole blood extracted sample is as follows:
cutting a Tip of a 1-mL Tip, inserting a pipette, extending into a blood collection tube, repeatedly blowing and beating whole blood for 3-4 times, fully suspending precipitated white blood cells, transferring the whole blood to an EP tube, adding MGS lysate, adding proteinase K, uniformly mixing, heating in a water bath kettle at 60 ℃ for 10-15 min, wherein the uniformly mixing time is more than 2 times;
taking out the EP pipe from the water bath kettle, pouring all solution in the EP pipe into a centrifugal adsorption column, putting the adsorption column into a collecting pipe, centrifuging at 8000rpm for 1min, taking out the adsorption column, and pouring out waste liquid in the collecting pipe;
adding cleaning solution into the adsorption column
8000rpm, centrifuging for 30s, taking out the adsorption column, and pouring off the waste liquid in the collection tube;
rinsing twice, taking out the adsorption column after the completion, pouring off the waste liquid in the collection tube, putting the adsorption column back into the collection tube again, centrifuging at 12000rpm for 1min, and removing the residual ethanol;
placing the adsorption column in a new 1.5ml centrifuge tube, and drying at room temperature for 2-3 min; dripping 60 deg.C eluent ET into the adsorption column near the center of the adsorption membrane, centrifuging at 12000rpm for 1-2min, collecting the liquid in the tube as sample DNA solution, covering with a cover, and lightly pulling the liquid in the tube to mix well.
As a preferable technical scheme, the rinsing treatment is to add a rinsing liquid Wash into the adsorption column at 8000rpm, centrifuge for 30s, take out the adsorption column and pour the waste liquid in the collection tube.
As a preferable technical scheme, the reaction system in the step 2) is as follows:
as a preferable technical scheme, the temperature of the change in the circulation in the step 2) is changed to 92 ℃.
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example (b):
extraction of DNA from whole blood
The extraction operation steps are as follows:
1. cutting off a Tip of a 1-mL Tip head, inserting a pipette, extending into a blood collection tube, repeatedly blowing and beating whole blood for 3-4 times to fully suspend precipitated white blood cells, transferring 200 mul of whole blood into a 2mL EP tube, adding 200 mul of MGS lysate and 20 mul of proteinase K, uniformly mixing, heating in a 60 ℃ water bath kettle for 10min (no more than 15min), uniformly mixing for 2-3 times, otherwise, enabling hemoglobin to cohere and agglomerate.
2. Taking out the sample from the water bath, pouring the solution into a centrifugal adsorption column, placing the adsorption column into a collecting tube, centrifuging at 8000rpm for 1min, taking out the adsorption column, and pouring off the waste liquid in the collecting tube.
3. Adding 500 μ l of cleaning solution into the adsorption column
8000rpm, centrifuging for 30s, taking out the adsorption column, and pouring the waste liquid in the collecting pipe.
4. Adding 500 μ l of rinsing liquid Wash to the adsorption column at 8000rpm, centrifuging for 30s, taking out the adsorption column, and pouring off waste liquid in the collection tube.
5. Repeat 4 times.
6. Taking out the adsorption column, pouring off waste liquid in the collection tube, putting the adsorption column back into the collection tube again, and centrifuging at 12000rpm for 1min to remove residual ethanol as much as possible.
7. The adsorption column was placed in a new 1.5ml centrifuge tube and dried for 2-3min at room temperature.
8. And (3) suspending and dropwise adding 50-100 μ l of eluent ET (preheating the eluent ET at 65 ℃ in advance to improve the elution efficiency) into the adsorption column close to the center of the adsorption film. 12000rpm, centrifuging for 1-2min, and collecting liquid in the centrifugal tube to obtain the DNA solution. And lightly stirring the liquid in the centrifugal tube after covering the cover to uniformly mix. The sample DNA is stored at 4 ℃ or-20 ℃ for a long period of time according to the application.
9. Can be directly used for PCR, enzyme digestion, library construction, sequencing reaction and the like.
And (3) folic acid reaction:
II, secondly: PCR amplification and sequencing analysis
(1) PCR System configuration
The operation and each configuration system error are reduced, and the system is simplified as follows:
configuration 20 x MIX (scaled up):
after mixing, 19. mu.L of the mixture was dispensed, and finally 1. mu.L of the DNA sample was added.
2) PCR program set-up using StepOnePlus software
Running the software, and clicking quick start, as shown in FIG. 1;
the cycle number was then changed to 50 in a Run Method, the system defaulted to 20 μ L, and the temperature of the cycle change was changed to 92 ℃. After the program is set up as above, START RUN is clicked. At this point the procedure has begun and the experimental and sample names may be re-labeled later. (FIG. 3)
After running, return to the Plate Setup interface to set up the reaction name and sample information. (FIG. 6)
Setting genotyping experimental information: as shown in fig. 2;
after filling information, the method comprises the following steps: as shown in fig. 4;
sample information was set indicating negative and positive controls. The following were used: as shown in fig. 6;
the corresponding tunnel on the right is selected to specify the reaction name and the sample name.
Finally, the checking experiment setup was error-free and the genotyping PCR experiment setup was completed.
The operation is started.
Result analysis and report generation
And (4) result graphic parameters: fig. 8 and 9.
And reading an experiment result, filling a folic acid metabolism report according to an analysis result of the experiment, and generating a complete report.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. A method for performing PCR amplification sequencing analysis by extracting DNA from whole blood, which is characterized by comprising the following steps:
1) preparing a required DNA sample, extracting the DNA of the sample from whole blood, and storing the obtained DNA of the sample at the temperature of between 20 ℃ below zero and 4 ℃ for later use;
2) PCR amplification and analysis, namely constructing a reaction system in a PCR tube by using the sample DNA obtained in the step 1), then sending the reaction system into a PCR amplification instrument for amplification, setting a PCR circulation program, and obtaining the information of the sample after the amplification is finished;
3) and obtaining folic acid metabolism information, and performing a genotyping PCR experiment through software to obtain an experiment result and obtain folic acid metabolism information.
2. The method of claim 1, wherein the whole blood extracted sample DNA is as follows:
cutting a Tip of a 1-mL Tip, inserting a pipette, extending into a blood collection tube, repeatedly blowing and beating whole blood for 3-4 times, fully suspending precipitated white blood cells, transferring the whole blood to an EP tube, adding MGS lysate, adding proteinase K, uniformly mixing, heating in a water bath kettle at 60 ℃ for 10-15 min, wherein the uniformly mixing time is more than 2 times;
taking out the EP pipe from the water bath kettle, pouring all solution in the EP pipe into a centrifugal adsorption column, putting the adsorption column into a collecting pipe, centrifuging at 8000rpm for 1min, taking out the adsorption column, and pouring out waste liquid in the collecting pipe;
adding cleaning solution into the adsorption column
8000rpm, centrifuging for 30s, taking out the adsorption column, and pouring off the waste liquid in the collection tube;
rinsing twice, taking out the adsorption column after the completion, pouring off the waste liquid in the collection tube, putting the adsorption column back into the collection tube again, centrifuging at 12000rpm for 1min, and removing the residual ethanol;
placing the adsorption column in a new 1.5ml centrifuge tube, and drying at room temperature for 2-3 min; dripping 60 deg.C eluent ET into the adsorption column near the center of the adsorption membrane, centrifuging at 12000rpm for 1-2min, collecting the liquid in the tube as sample DNA solution, covering with a cover, and lightly pulling the liquid in the tube to mix well.
3. The method of claim 1 for PCR sequencing analysis by whole blood DNA extraction, wherein: and the rinsing treatment comprises the steps of adding a rinsing liquid Wash into the adsorption column at 8000rpm, centrifuging for 30s, taking out the adsorption column, and pouring out waste liquid in the collecting pipe.
4. The method for PCR amplification sequencing analysis by whole blood DNA extraction according to claim 1, wherein the reaction system in step 2) is as follows:
5. the method of claim 1 for PCR sequencing analysis by whole blood DNA extraction, wherein: the temperature change in the cycle in step 2) was changed to 92 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013104106A1 (en) * | 2012-01-10 | 2013-07-18 | 北京贝瑞和康生物技术有限公司 | Method for construction of plasma dna sequencing library and kit thereof |
| CN107828870A (en) * | 2017-11-16 | 2018-03-23 | 沈阳迪安医学检验所有限公司 | One kind detects MTHFR and MTRR gene pleiomorphisms kit and method simultaneously using molecular beacon probe melting curve method |
| CN110257507A (en) * | 2019-07-11 | 2019-09-20 | 上海联吉医学检验所有限公司 | A kind of pregnancy period folic acid metabolism, calcium metabolism and H-type hypertension associated detecting method |
| CN111019940A (en) * | 2019-12-25 | 2020-04-17 | 上海派森诺生物科技股份有限公司 | Extracting solution for directly extracting whole blood genome DNA and extracting method thereof |
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- 2020-12-29 CN CN202011596524.3A patent/CN112760364A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013104106A1 (en) * | 2012-01-10 | 2013-07-18 | 北京贝瑞和康生物技术有限公司 | Method for construction of plasma dna sequencing library and kit thereof |
| CN107828870A (en) * | 2017-11-16 | 2018-03-23 | 沈阳迪安医学检验所有限公司 | One kind detects MTHFR and MTRR gene pleiomorphisms kit and method simultaneously using molecular beacon probe melting curve method |
| CN110257507A (en) * | 2019-07-11 | 2019-09-20 | 上海联吉医学检验所有限公司 | A kind of pregnancy period folic acid metabolism, calcium metabolism and H-type hypertension associated detecting method |
| CN111019940A (en) * | 2019-12-25 | 2020-04-17 | 上海派森诺生物科技股份有限公司 | Extracting solution for directly extracting whole blood genome DNA and extracting method thereof |
Non-Patent Citations (1)
| Title |
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| 刘云龙等: "全血直接扩增结合焦磷酸测序法测定亚甲基四氢叶酸还原酶基因多态性", 分析化学, vol. 40, no. 07, pages 1037 - 1042 * |
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