CN110551803A - Sequencing method for human HLA gene polymorphism primer insertion or deletion - Google Patents
Sequencing method for human HLA gene polymorphism primer insertion or deletion Download PDFInfo
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- 238000012163 sequencing technique Methods 0.000 title claims abstract description 40
- 238000012217 deletion Methods 0.000 title claims abstract description 19
- 230000037430 deletion Effects 0.000 title claims abstract description 19
- 238000003780 insertion Methods 0.000 title claims abstract description 19
- 230000037431 insertion Effects 0.000 title claims abstract description 19
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- 230000036425 denaturation Effects 0.000 claims abstract description 4
- 230000002093 peripheral effect Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 38
- 108090000623 proteins and genes Proteins 0.000 claims description 27
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 19
- 239000000047 product Substances 0.000 claims description 13
- 238000012408 PCR amplification Methods 0.000 claims description 11
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 9
- 108010006785 Taq Polymerase Proteins 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- 238000004140 cleaning Methods 0.000 claims description 7
- 239000012154 double-distilled water Substances 0.000 claims description 7
- 239000007850 fluorescent dye Substances 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 7
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 6
- 230000000295 complement effect Effects 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000000523 sample Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 5
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 claims description 4
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 claims description 4
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 claims description 4
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims description 4
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 claims description 4
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- 239000000427 antigen Substances 0.000 description 9
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- 102000036639 antigens Human genes 0.000 description 9
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Abstract
the invention discloses a sequencing method for human HLA gene polymorphism primer insertion or deletion, belonging to the technical field of HLA gene sequencing, and the sequencing method for human HLA gene polymorphism primer insertion or deletion comprises the following steps: the DNA is extracted, a fasting peripheral venous blood sample is collected from a target object, a DNA extraction kit is adopted to extract genome DNA from the blood sample, an ultraviolet spectrophotometer is used for measuring the concentration and purity of the DNA, high-temperature denaturation is carried out, and double strands of a DNA template are dissociated into single strands of the DNA.
Description
Technical Field
The invention relates to the technical field of HLA gene sequencing, in particular to a sequencing method for human HLA gene polymorphism primer insertion or deletion.
background
human Leukocyte Antigens (HLA) are a group of glycoprotein molecules present on the cell surface and have been considered as the major antigens responsible for organ transplant rejection. HLA antigens are the expression products of Major Histocompatibility Complex (MHC) in humans, and are mainly responsible for the mutual recognition between cells and the induction of immune responses, the function of regulating immune responses in the immune system.
HLA antigens can be divided into three classes: the I-type molecules are HLA-A, -B and-C series antigens and are widely distributed on the surfaces of nucleated cells of various tissues, including platelets and reticulocytes, and mature red blood cells generally do not contain HLA antigens; the II-class molecules are HLA-D/DR, -DP, DQ series antigens, mainly expressed on B cells and antigen presenting cells, and the two antigens are related to transplantation, wherein the II-class antigens are more important; class III molecules are complement components. The gene coding human HLA is located on chromosome 6, is a gene complex with the highest allelic polymorphism in the known genes so far, and is the most complex genetic polymorphism system of human, and the polymorphism distribution of the HLA genes has obvious group characteristics.
at present, the HLA genes are analyzed at home and abroad, and PCR-SSCP, sequencing method (SBT) and fluorescence PCR are mostly adopted. The sequencing method is most accurate, but the sequencing result needs to be analyzed through professional software, the detection and analysis period is long, the PCR-SSCP method is a classical method, DNA is amplified through a primer and is analyzed through an electrophoresis result, the pollution risk is high, the period is long, the PCR-SSCP method is not suitable for clinical application, the existing fluorescence PCR method adopts a dye to generate signals for the amplified specific fragments, the specificity is weak, and the internal control of the PCR reaction cannot be carried out.
The existing HLA gene sequencing methods (PCR-SSCP, SBT, fluorescence PCR method and the like) have various difficulties and defects, so that the detection result of the HLA gene is not accurate enough.
Disclosure of Invention
1. Technical problem to be solved
Aiming at the problems in the prior art, the invention aims to provide a sequencing method for human HLA gene polymorphism primer insertion or deletion, which can combine PCR amplification technology and fluorescent marker, takes improvement of DNA purity and concentration as the first premise in the sequencing process, utilizes the advantages of PCR amplification technology and fluorescent marker, reduces the difficulty and defects generated in the sequencing process to the maximum extent, has high sensitivity, strong specificity and simple and convenient operation in the sequencing process, thereby ensuring the sensitivity and accuracy of HLA gene sequencing and accurately obtaining whether the fragment of HLA gene insertion or deletion exists.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
A method for sequencing a human HLA gene polymorphism primer insertion or deletion, comprising the steps of:
The method comprises the following steps: extracting DNA, collecting 5-10mL of a fasting peripheral venous blood sample from a target object, extracting genome DNA from the blood sample by adopting a DNA extraction kit, measuring the concentration and purity of the DNA by using an ultraviolet spectrophotometer, and adjusting the concentration of the DNA to 50-80 ng/mu L and 1.6-2.0;
step two: high temperature denaturation, heating the prepared DNA template to 95-100 ℃ to dissociate the double strands of the DNA template into single DNA strands; then annealing at low temperature, reducing the temperature to 45-50 ℃, adding a PCR primer into the annealing solution, and enabling the PCR primer to be matched and combined with the complementary sequence of the DNA template single strand;
step three: extending the primers, namely adding a PCR buffer solution, dNTP, Taq DNA polymerase, a magnesium chloride solution, double distilled water and a fluorescent probe into the mixture of the PCR primers and the DNA template to obtain a PCR mixture, wherein the step is carried out on a PCR amplification instrument;
step four: purifying a PCR product, namely adding a sufficient amount of magnetic bead suspension into the mixture in the third step, uniformly mixing by vibration, standing for 5-10min, adding an eluent, and separating magnetic beads from the PCR product to obtain a high-purity PCR product;
Step five: and (3) detecting HLA genes, adding formamide solution into the purified product, mixing uniformly, centrifuging, heating to 95-100 ℃ for 3-5 minutes to denature DNA, and then denaturing on a sequencer.
According to the invention, by combining the PCR amplification technology and the fluorescent marker, the advantages of the PCR amplification technology and the fluorescent marker are utilized on the premise of improving the purity and concentration of DNA in the sequencing process, the difficulty and the defect generated in the sequencing process are reduced to the greatest extent, the sequencing process has high sensitivity, strong specificity and is simple and convenient to operate, so that the sensitivity and the accuracy of HLA gene sequencing are ensured, and whether the inserted or deleted fragment exists in the HLA gene is accurately obtained.
further, the weight ratio of the DNA template, the PCR primer, the PCR buffer solution, the dNTP, the Taq DNA polymerase, the magnesium chloride solution, the double distilled water and the fluorescent probe is 1: 2: 10: 0.2: 0.5: 0.2: 100: 2.
Further, the solubility of the magnesium chloride solution is 25 mmol/L.
further, the dNTPs include equal components of dATP, dGTP, dTTP and dCTP.
Further, in the step one the DNA extraction kit includes the urceolus, the inside of urceolus is equipped with netted inner tube, the inner wall fixedly connected with of netted inner tube is a pair of filter plate, and is a pair of the filter plate divide into three-layer about the inside of netted inner tube, a plurality of magnetic balls have all been placed to the interior bottom surface of netted inner tube and the upper end of filter plate, through carrying out the layering with the magnetic ball and placing evenly, can make magnetic ball and DNA combine ground more abundant and quick to improve the concentration and the purity of DNA.
Further, the upper end fixedly connected with apron of netted inner tube, the side of apron rotates and is connected with a pair of L template, the side of urceolus is dug there is the ring channel, the lower extreme of L template is located the inboard of ring channel, moves along the ring channel through the L template and can realize the stable rotation of apron to drive netted inner tube at the urceolus internal rotation.
further, the interior bottom surface fixedly connected with spacing post of urceolus, the lower extreme of netted inner tube is dug a spacing groove, spacing post rotates to be connected in the inside of spacing groove, can further improve netted inner tube pivoted stability through spacing post and spacing groove.
furthermore, the upper end of the cover plate is fixedly connected with a rubber handle, so that the cover plate is convenient to take manually by the aid of the rubber handle.
Further, the method for extracting DNA in the first step comprises the following steps:
S1, adding the blood specimen into the outer cylinder, adding a sufficient amount of lysate into the outer cylinder, shaking up, destroying the structure of cells, and separating DNA from impurities such as protein and polysaccharide;
S2, inserting the meshed inner cylinder with the magnetic balls into the outer cylinder, so that the magnetic balls are completely immersed in the mixed solution of the outer cylinder, and the DNA and the magnetic balls are combined together through special groups on the surfaces of the magnetic balls;
When the magnetic ball is inserted into the limiting groove, the limiting column is inserted into the limiting groove, the L-shaped plate can be downwards rotated, the end part of the L-shaped plate is inserted into the annular groove, the rubber handle is manually rotated to drive the reticular inner cylinder to rotate in the outer cylinder, and the magnetic ball is more fully combined with the DNA;
s3, taking the mesh inner cylinder out of the outer cylinder, putting the mesh inner cylinder into another outer cylinder filled with washing liquid, washing the magnetic ball, and washing away impurities remained on the magnetic ball; and taking out the meshed inner cylinder after cleaning, and then putting the meshed inner cylinder into the other outer cylinder filled with eluent to separate the magnetic ball from the DNA so as to obtain the required DNA template.
Furthermore, the aperture on the reticular inner cylinder is the same as the aperture on the filter plate, the aperture of the reticular inner cylinder is smaller than the diameter of the magnetic ball, DNA can pass through the through holes on the reticular inner cylinder and the filter plate without obstacles, and the magnetic ball can not provide the reticular inner cylinder and the filter plate, so that the magnetic ball is convenient to collect, and the DNA extraction process is quicker and simpler.
3. Advantageous effects
compared with the prior art, the invention has the advantages that:
(1) according to the scheme, the PCR amplification technology and the fluorescent marker are combined, the purity and the concentration of DNA are improved in the sequencing process as the first premise, the advantages of the PCR amplification technology and the fluorescent marker are utilized, the difficulty and the defects generated in the sequencing process are reduced to the greatest extent, the sequencing process is high in sensitivity, strong in specificity and simple and convenient to operate, the sensitivity and the accuracy of HLA gene sequencing are guaranteed, and whether the inserted or deleted fragment exists in the HLA gene or not is accurately obtained.
(2) The weight ratio of the DNA template, the PCR primer, the PCR buffer solution, the dNTP, the TaqDNA polymerase, the magnesium chloride solution, the double distilled water and the fluorescent probe is 1: 2: 10: 0.2: 0.5: 0.2: 100: 2.
(3) The solubility of the magnesium chloride solution was 25mmol/L, and the dNTPs included equal components of dATP, dGTP, dTTP and dCTP.
(4) The DNA extraction kit in the first step comprises an outer barrel, a mesh-shaped inner barrel is arranged in the outer barrel, a pair of filter plates is fixedly connected to the inner wall of the mesh-shaped inner barrel, the inside of the mesh-shaped inner barrel is divided into an upper layer and a lower layer by the pair of filter plates, a plurality of magnetic balls are placed on the inner bottom surface of the mesh-shaped inner barrel and the upper ends of the filter plates, and the magnetic balls are placed uniformly in layers, so that the magnetic balls and DNA can be combined more sufficiently and rapidly, and the concentration and purity of the DNA are improved.
(5) The upper end fixedly connected with apron of netted inner tube, the side of apron rotates and is connected with a pair of L template, and the side of urceolus is dug there is the annular groove, and the lower extreme of L template is located the inboard of annular groove, moves the stable rotation that can realize the apron through the L template along the annular groove to drive netted inner tube at the urceolus internal rotation.
(6) the interior bottom surface fixedly connected with of urceolus has spacing post, and the lower extreme of netted inner tube is dug a spacing groove, and spacing post rotates to be connected in the inside of spacing groove, can further improve netted inner tube pivoted stability through spacing post and spacing groove.
(7) the upper end fixedly connected with rubber handle of apron improves the convenient manual apron of taking of rubber handle.
(8) The aperture on the reticular inner cylinder is the same as that on the filter plate, the aperture of the reticular inner cylinder is smaller than the diameter of the magnetic ball, DNA can pass through the through holes on the reticular inner cylinder and the filter plate without obstacle, and the magnetic ball can not provide the reticular inner cylinder and the filter plate, thereby realizing convenient collection of the magnetic ball and enabling the DNA extraction process to be faster and simpler.
(9) Through the cooperation use of urceolus, netted inner tube, filter plate, magnetic ball, apron, L template and ring channel, can furthest improve the solubility and the purity of the DNA of extracting, still have the effect of simplifying the operation mode simultaneously, improvement extraction rate.
Drawings
FIG. 1 is a perspective view of a DNA extraction kit of the present invention;
FIG. 2 is an internal perspective view of the DNA extraction kit of the present invention;
FIG. 3 is a schematic front view of the DNA extraction kit of the present invention;
FIG. 4 is a schematic view of the structure of the DNA extraction kit of the present invention in use;
FIG. 5 is a flow chart of the present invention.
The reference numbers in the figures illustrate:
The device comprises an outer cylinder 1, a reticular inner cylinder 2, a filter plate 3, a magnetic ball 4, a limiting column 5, a limiting groove 6, a cover plate 7, an 8L-shaped plate, an annular groove 9 and a rubber handle 10.
Detailed Description
The drawings in the embodiments of the invention will be combined; the technical scheme in the embodiment of the invention is clearly and completely described; obviously; the described embodiments are only some of the embodiments of the invention; but not all embodiments, are based on the embodiments of the invention; all other embodiments obtained by a person skilled in the art without making any inventive step; all fall within the scope of protection of the present invention.
in the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "top/bottom", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplification of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
in the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "sleeved/connected," "connected," and the like are to be construed broadly, e.g., "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Example (b):
Referring to fig. 5, a method for sequencing human HLA gene polymorphism primer insertions or deletions, comprising the steps of:
the method comprises the following steps: extracting DNA, collecting 5mL of a fasting peripheral venous blood sample from a target object, extracting genome DNA from the blood sample by adopting a DNA extraction kit, measuring the concentration and purity of the DNA by using an ultraviolet spectrophotometer, and adjusting the concentration of the DNA to 50 ng/mu L and the concentration of the DNA to 1.6;
step two: high-temperature denaturation, namely heating the prepared DNA template to 95 ℃ to dissociate double strands of the DNA template into single strands of DNA; then annealing at low temperature, reducing the temperature to 45 ℃, and adding a PCR primer into the annealing so that the PCR primer is matched and combined with the complementary sequence of the DNA template single strand;
step three: extending the primers, namely adding a PCR buffer solution, dNTP, Taq DNA polymerase, a magnesium chloride solution, double distilled water and a fluorescent probe into the mixture of the PCR primers and the DNA template to obtain a PCR mixture, wherein the step is carried out on a PCR amplification instrument;
Under the action of Taq DNA polymerase, a DNA template and primer combination takes dNTP as a reaction raw material and a target sequence as a template, a new semi-reserved replication chain complementary with a DNA template chain is synthesized according to the base complementary pairing and semi-reserved replication principles, more semi-reserved replication chains can be obtained by repeating the second step and the third step of the cycle, the new chain can also become a template of the next cycle, 2-4 minutes are needed for completing one cycle, and millions of times of amplification can be carried out on a target gene to be amplified within 2-3 hours.
step four: purifying a PCR product, namely adding a sufficient amount of magnetic bead suspension into the mixture in the third step, uniformly mixing by vibration, standing for 5min, then adding an eluent, and separating the magnetic beads from the PCR product to obtain a high-purity PCR product;
and DNA in the PCR product is extracted by combining the DNA and the magnetic beads, so that other impurities in the PCR product are removed, and the purity of the DNA in the PCR product is improved.
step five: and (3) detecting HLA genes, adding formamide solution into the purified product, mixing uniformly, centrifuging, heating to 95 ℃ for 3 minutes to denature DNA, and then denaturing on a sequencer.
According to the invention, by combining the PCR amplification technology and the fluorescent marker, the advantages of the PCR amplification technology and the fluorescent marker are utilized on the premise of improving the purity and concentration of DNA in the sequencing process, the difficulty and the defect generated in the sequencing process are reduced to the greatest extent, the sequencing process has high sensitivity, strong specificity and is simple and convenient to operate, so that the sensitivity and the accuracy of HLA gene sequencing are ensured, and whether the inserted or deleted fragment exists in the HLA gene is accurately obtained.
The weight ratio of the DNA template, the PCR primer, the PCR buffer solution, the dNTP, the TaqDNA polymerase, the magnesium chloride solution, the double distilled water and the fluorescent probe is 1: 2: 10: 0.2: 0.5: 0.2: 100: 2.
the solubility of the magnesium chloride solution was 25 mmol/L.
dNTPs include equal components of dATP, dGTP, dTTP and dCTP.
Referring to fig. 1, in the first step, the DNA extraction kit includes an outer cylinder 1, referring to fig. 2, a mesh inner cylinder 2 is disposed inside the outer cylinder 1, referring to fig. 3, a pair of filter plates 3 is fixedly connected to an inner wall of the mesh inner cylinder 2, the mesh inner cylinder 2 is divided into an upper layer and a lower layer by the pair of filter plates 3, a plurality of magnetic balls 4 are disposed on an inner bottom surface of the mesh inner cylinder 2 and an upper end of each filter plate 3, and the magnetic balls 4 are uniformly layered, so that the magnetic balls 4 can be more sufficiently and rapidly combined with DNA to improve the concentration and purity of DNA.
The aperture on netted inner tube 2 is the same with the aperture on the filter plate 3, and the aperture of netted inner tube 2 is less than the diameter of magnetic ball 4, and DNA can pass in the through-hole on netted inner tube 2 and filter plate 3 without hindrance, and magnetic ball 4 can't provide netted inner tube 2 and filter plate 3 to the realization conveniently collects magnetic ball 4, makes the DNA extraction process quick simple and convenient more.
Referring to fig. 3, a cover plate 7 is fixedly connected to the upper end of the mesh inner cylinder 2, a pair of L-shaped plates 8 is rotatably connected to the side ends of the cover plate 7, referring to fig. 1, an annular groove 9 is cut on the side end of the outer cylinder 1, the lower ends of the L-shaped plates 8 are located inside the annular groove 9, and the cover plate 7 can stably rotate by moving the L-shaped plates 8 along the annular groove 9, so that the mesh inner cylinder 2 is driven to rotate in the outer cylinder 1.
Referring to fig. 3, the inner bottom surface of the outer cylinder 1 is fixedly connected with a limiting post 5, the lower end of the mesh-shaped inner cylinder 2 is provided with a limiting groove 6, the limiting post 5 is rotatably connected inside the limiting groove 6, and the rotation stability of the mesh-shaped inner cylinder 2 can be further improved through the limiting post 5 and the limiting groove 6.
Referring to fig. 3, a rubber handle 10 is fixedly connected to the upper end of the cover plate 7, and the rubber handle 10 is raised to facilitate manual access to the cover plate 7.
The method for extracting DNA in the first step comprises the following steps:
S1, adding the blood specimen into the outer cylinder 1, adding a sufficient amount of lysate into the outer cylinder 1, shaking up, destroying the structure of cells, and separating DNA from impurities such as protein, polysaccharide and the like;
S2, inserting the mesh inner cylinder 2 with the magnetic balls 4 into the outer cylinder 1, so that the magnetic balls 4 are completely immersed in the mixed solution of the outer cylinder 1, and combining the DNA with the magnetic balls 4 through special groups on the surfaces of the magnetic balls 4;
When the magnetic ball 4 is inserted into the limiting groove 6, the limiting column 5 is inserted into the limiting groove 6, the L-shaped plate 8 can be downwards rotated, the end part of the L-shaped plate 8 is inserted into the annular groove 9, the rubber handle 10 is manually rotated, the reticular inner cylinder 2 is driven to rotate in the outer cylinder 1, and the magnetic ball 4 is more fully combined with DNA;
S3, taking the mesh inner barrel 2 out of the outer barrel 1, putting the mesh inner barrel into the other outer barrel 1 filled with the cleaning solution, cleaning the magnetic ball 4, and washing away impurities remained on the magnetic ball 4; and taking out the reticular inner cylinder 2 after cleaning, and then putting the reticular inner cylinder into the other outer cylinder 1 filled with eluent to separate the magnetic ball 4 from the DNA so as to obtain the required DNA template.
through the cooperation use of urceolus 1, netted inner tube 2, filter plate 3, magnetic ball 4, apron 7, L template 8 and ring channel 9, can furthest improve the solubility and the purity of the DNA of extracting, still have the simplified operation mode simultaneously, improve the effect of extraction rate.
The above; but are merely preferred embodiments of the invention; the scope of the invention is not limited thereto; any person skilled in the art is within the technical scope of the present disclosure; the technical scheme and the improved concept of the invention are equally replaced or changed; are intended to be covered by the scope of the present invention.
Claims (10)
1. a method for sequencing a human HLA gene polymorphism primer insertion or deletion, comprising the steps of:
The method comprises the following steps: extracting DNA, collecting 5-10mL of a fasting peripheral venous blood sample from a target object, extracting genome DNA from the blood sample by adopting a DNA extraction kit, measuring the concentration and purity of the DNA by using an ultraviolet spectrophotometer, and adjusting the concentration of the DNA to 50-80 ng/mu L and 1.6-2.0;
step two: high temperature denaturation, heating the prepared DNA template to 95-100 ℃ to dissociate the double strands of the DNA template into single DNA strands; then annealing at low temperature, reducing the temperature to 45-50 ℃, adding a PCR primer into the annealing solution, and enabling the PCR primer to be matched and combined with the complementary sequence of the DNA template single strand;
step three: extending the primers, namely adding a PCR buffer solution, dNTP, Taq DNA polymerase, a magnesium chloride solution, double distilled water and a fluorescent probe into the mixture of the PCR primers and the DNA template to obtain a PCR mixture, wherein the step is carried out on a PCR amplification instrument;
Step four: purifying a PCR product, namely adding a sufficient amount of magnetic bead suspension into the mixture in the third step, uniformly mixing by vibration, standing for 5-10min, adding an eluent, and separating magnetic beads from the PCR product to obtain a high-purity PCR product;
Step five: and (3) detecting HLA genes, adding formamide solution into the purified product, mixing uniformly, centrifuging, heating to 95-100 ℃ for 3-5 minutes to denature DNA, and then denaturing on a sequencer.
2. the method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 1, wherein: the weight ratio of the DNA template, the PCR primer, the PCR buffer solution, the dNTP, the TaqDNA polymerase, the magnesium chloride solution, the double distilled water and the fluorescent probe is 1: 2: 10: 0.2: 0.5: 0.2: 100: 2.
3. the method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 1, wherein: the solubility of the magnesium chloride solution is 25 mmol/L.
4. The method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 1, wherein: the dNTPs include equal components of dATP, dGTP, dTTP and dCTP.
5. the method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 1, wherein: in the first step, the DNA extraction kit comprises an outer barrel (1), a mesh-shaped inner barrel (2) is arranged inside the outer barrel (1), a pair of filter plates (3) is fixedly connected to the inner wall of the mesh-shaped inner barrel (2), the mesh-shaped inner barrel (2) is divided into an upper layer and a lower layer by the pair of filter plates (3), and a plurality of magnetic balls (4) are placed on the inner bottom surface of the mesh-shaped inner barrel (2) and the upper ends of the filter plates (3).
6. The method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 5, wherein: the upper end fixedly connected with apron (7) of netted inner tube (2), the side of apron (7) is rotated and is connected with a pair of L template (8), the side of urceolus (1) is dug and is had annular groove (9), the lower extreme of L template (8) is located the inboard of annular groove (9).
7. The method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 5, wherein: the interior bottom surface fixedly connected with of urceolus (1) spacing post (5), spacing groove (6) are dug to the lower extreme of netted inner tube (2), spacing post (5) rotate to be connected in the inside of spacing groove (6).
8. The method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 6, wherein: the upper end of the cover plate (7) is fixedly connected with a rubber handle (10).
9. The method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 1, wherein: the method for extracting DNA in the first step comprises the following steps:
S1, adding the blood specimen into the outer cylinder (1), adding a sufficient amount of lysate into the outer cylinder (1), shaking up, destroying the structure of cells, and separating DNA from impurities such as protein, polysaccharide and the like;
S2, inserting the mesh inner cylinder (2) with the magnetic balls (4) into the outer cylinder (1), completely immersing the magnetic balls (4) in the mixed solution of the outer cylinder (1), and combining the DNA and the magnetic balls (4) through special groups on the surfaces of the magnetic balls (4);
S3, taking the mesh inner cylinder (2) out of the outer cylinder (1), putting the mesh inner cylinder into the other outer cylinder (1) filled with the cleaning solution, cleaning the magnetic ball (4), and washing away the residual impurities on the magnetic ball (4); and taking out the reticular inner cylinder (2) after cleaning, and then putting the reticular inner cylinder into the other outer cylinder (1) filled with eluent to separate the magnetic ball (4) from the DNA so as to obtain the required DNA template.
10. the method for sequencing human HLA gene polymorphism primer insertions or deletions according to claim 5, wherein: the aperture on the reticular inner cylinder (2) is the same as the aperture on the filter plate (3), and the aperture of the reticular inner cylinder (2) is smaller than the diameter of the magnetic ball (4).
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| WO2022116456A1 (en) * | 2020-12-04 | 2022-06-09 | 深圳荻硕贝肯精准医学有限公司 | Analysis method and analysis and processing device for loss of heterozygosity in human hla chromosomal region |
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| US20030165884A1 (en) * | 1999-12-20 | 2003-09-04 | Stemcyte, Inc. | High throughput methods of HLA typing |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030165884A1 (en) * | 1999-12-20 | 2003-09-04 | Stemcyte, Inc. | High throughput methods of HLA typing |
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| WO2022116456A1 (en) * | 2020-12-04 | 2022-06-09 | 深圳荻硕贝肯精准医学有限公司 | Analysis method and analysis and processing device for loss of heterozygosity in human hla chromosomal region |
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