CN112725332B - Mixed fine spot extraction process - Google Patents
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Abstract
The invention relates to a mixed fine spot extraction process. The mixed fine spot extraction process comprises the following steps: performing primary pyrolysis on the detection material, collecting primary precipitate, performing secondary pyrolysis on the primary precipitate, collecting secondary precipitate, performing tertiary pyrolysis on the secondary precipitate, and collecting supernatant to obtain a solution containing male DNA. According to the mixed sperm spot extraction process, the female components are subjected to pyrolysis digestion on the detection material for two times, so that the pyrolysis treatment period can be shortened, the female components can be digested efficiently, and the influence of residual female components on subsequent analysis of male DNA is avoided.
Description
Technical Field
The invention belongs to the technical field of DNA extraction, and relates to a mixed fine spot extraction process.
Background
Female vagina or underpants in rape and residual sperm spots on bed sheets are the most common mixed examination materials, and single male typing can be obtained through digestion of female components. At present, the Chelex-100 method is often used for obtaining sperm DNA, but the operation is complex, the time consumption is long, the efficiency is low, the DNA extraction amount is small, and the rapid detection of cases is not facilitated, so that a great deal of research and attempts are made to improve the existing extraction method.
Zhao Tiejun (Zhao Tiejun. Comparison of three extraction methods for mixed sperm spot DNA [ J ]. J.French medical journal, 2004,020 (004): 237-237, 239.) the Chelex-100 method was modified, and a Chelex-100 secondary treatment method and a Chelex-100+ organic method were proposed, wherein the Chelex-100 secondary treatment method is to add a Chelex-100 digestion process on the basis of the Chelex-100 method; the Chelex-100+ organic method is to extract the supernatant obtained by the Chelex-100 method by using a mixed solution of phenol, chloroform and isoamyl alcohol, and then to treat the supernatant by using an ice ethanol sodium chloride solution. Both the above methods can improve the extraction amount and purity of DNA, but still have the problems of complicated operation, long time consumption and low efficiency.
Li Gongxia (Li Gongxia, chen Xiaohui. 3 extraction methods of materials for spot detection compare [ J ]. J.China journal of forensics, 2007,22 (005): 323-324.). The Chelex-100 method is optimized, and the supernatant obtained by the Chelex-100 method is extracted by using a magnetic bead method, so that the extraction amount of DNA can be remarkably increased and the operation time can be shortened, but the operation is complicated, and the separation effect is affected by the level of experimenters and the environmental change, so that the popularization and the use are not facilitated.
In summary, the mixed refined spot extraction process is simple to operate, short in time consumption and low in cost, and has important significance in the technical field of forensics.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides the mixed fine spot extraction process, which can efficiently separate female components and male components in the detection material to obtain high-purity male DNA, can be automatically carried out in a full-automatic pipetting workstation and can simultaneously process a plurality of detection materials, and has the advantages of simple operation, short time consumption and high efficiency.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a mixed fine spot extraction process, which comprises the following steps of:
performing primary pyrolysis on the detection material, collecting primary precipitate, performing secondary pyrolysis on the primary precipitate, collecting secondary precipitate, performing tertiary pyrolysis on the secondary precipitate, and collecting supernatant to obtain a solution containing male DNA.
In the mixed sperm spot extraction process, the female component is firstly subjected to two-time pyrolysis digestion to obtain the detection material only containing male cells, so that the pyrolysis treatment period can be shortened, the female component can be efficiently digested, and the influence of the residual female component on subsequent analysis of male DNA is avoided.
Preferably, the mixed spot extraction process is performed in a fully automated pipetting station.
According to the invention, the mixed fine spot extraction process is developed on the basis of the full-automatic pipetting workstation, so that the mixed fine spot extraction is automatically carried out in the full-automatic pipetting workstation, a plurality of detection materials can be processed simultaneously, the operation difficulty is reduced, the extraction time is shortened, the processing efficiency is improved, the success rate of detecting pure target men is improved, and the rapid detection of cases is facilitated.
Preferably, the mixed fine spot extraction process further comprises a step of washing the primary precipitate and the secondary precipitate, respectively.
Preferably, the cleaning solution for the cleaning comprises Tris-Cl, sodium chloride and ethylenediamine tetraacetic acid.
Preferably, the concentration of Tris-Cl in the wash solution is 90-110 mM, including but not limited to 91mM, 92mM, 93mM, 94mM, 100mM, 102mM, 104mM, 106mM or 108mM.
Preferably, the concentration of sodium chloride in the wash solution is 18-22 mM, including but not limited to 18.2mM, 18.5mM, 19mM, 19.5mM, 20mM, 21mM, 21.2mM, 21.4mM, 21.6mM, or 21.8mM.
Preferably, the concentration of ethylenediamine tetraacetic acid in the washing solution is 9-11 mM, including but not limited to 9.2mM, 9.4mM, 9.6mM, 10mM, 10.2mM, 10.4mM, 10.6mM, or 10.8mM.
Preferably, the extraction flow of the mixed fine spot extraction process is shown in fig. 1, and comprises the following steps:
(1) Mixing the detection material with a lysate comprising Tris-Cl, sodium chloride and Sodium Dodecyl Sulfate (SDS), centrifuging, and collecting a precipitate;
(2) Mixing the precipitate obtained in the step (1) with the cleaning solution, centrifuging, and collecting the precipitate;
(3) Mixing the precipitate obtained in the step (2) with a lysate comprising Tris-Cl, sodium chloride, ethylenediamine tetraacetic acid (EDTA), sodium dodecyl sulfate, triton-X100, sodium deoxycholate (deoxycholate), magnesium chloride and calcium chloride, centrifuging, and collecting the precipitate;
(4) Mixing the precipitate obtained in the step (3) with the cleaning solution, centrifuging, and collecting the precipitate;
(5) Mixing the precipitate obtained in the step (4) with a lysate comprising Tris-Cl, sodium chloride, sodium dodecyl sulfate, guanidine hydrochloride, dithiothreitol and sodium citrate, centrifuging, and collecting a supernatant to obtain a solution containing male DNA.
In the invention, the sample is firstly subjected to two-time pyrolysis treatment to thoroughly digest and separate female components, and then the sample containing male sperm cells is continuously subjected to pyrolysis to obtain the solution containing male DNA, so that the period of digesting and separating female components can be shortened, and the purity of the male DNA solution can be improved.
Preferably, the Tris-Cl concentration of step (1) in the lysate is 90-110 mM, including but not limited to 91mM, 92mM, 93mM, 94mM, 100mM, 102mM, 104mM, 106mM or 108mM.
Preferably, the concentration of sodium chloride in the lysate of step (1) is 9-11 mM, including but not limited to 9.2mM, 9.4mM, 9.6mM, 10mM, 10.2mM, 10.4mM, 10.6mM or 10.8mM.
Preferably, the mass percentage of the sodium dodecyl sulfate in the lysate of the step (1) is 1% -3%, including but not limited to 1.2%, 1.4%, 1.6%, 2%, 2.2%, 2.4%, 2.6% or 2.8%.
Preferably, the temperature of the mixing in step (1) is 50 to 60 ℃, including but not limited to 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃,56 ℃, 58 ℃ or 59 ℃.
Preferably, the mixing time of step (1) is 20-40 min, including but not limited to 21min, 22min, 23min, 25min, 26min, 28min or 29min.
Preferably, the Tris-Cl concentration in step (3) in the lysate is 90-110 mM, including but not limited to 91mM, 92mM, 93mM, 94mM, 100mM, 102mM, 104mM, 106mM or 108mM.
Preferably, the concentration of sodium chloride in the lysate of step (3) is 18-22 mM, including but not limited to 18.2mM, 18.5mM, 19mM, 19.5mM, 20mM, 21mM, 21.2mM, 21.4mM, 21.6mM or 21.8mM.
Preferably, the concentration of ethylenediamine tetraacetic acid in the lysate of step (3) is 9-11 mM, including but not limited to 9.2mM, 9.4mM, 9.6mM, 10mM, 10.2mM, 10.4mM, 10.6mM or 10.8mM.
Preferably, the mass percentage of the sodium dodecyl sulfate in the lysate of the step (3) is 0.5% -1.5%, including but not limited to 0.6%, 0.7%, 0.8%, 1%, 1.2%, 1.3% or 1.4%.
Preferably, the mass percentage of Triton-X100 in the lysate of step (3) is 0.25% -0.75%, including but not limited to 0.28%, 0.3%, 0.4%, 0.5%, 0.55%, 0.6%, 0.7%, 0.72% or 0.74%.
Preferably, the mass percentage of the sodium deoxycholate in the lysate of the step (3) is 0.25% -0.75%, including but not limited to 0.28%, 0.3%, 0.4%, 0.5%, 0.55%, 0.6%, 0.7%, 0.72% or 0.74%.
Preferably, the concentration of magnesium chloride in the lysate of step (3) is 18-22 mM, including but not limited to 18.2mM, 18.5mM, 19mM, 19.5mM, 20mM, 21mM, 21.2mM, 21.4mM, 21.6mM or 21.8mM.
Preferably, the concentration of calcium chloride in the lysate of step (3) is 9-11 mM, including but not limited to 9.2mM, 9.4mM, 9.6mM, 10mM, 10.2mM, 10.4mM, 10.6mM or 10.8mM.
Preferably, the temperature of the mixing in step (3) is 50-60 ℃, including but not limited to 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃,56 ℃, 58 ℃ or 59 ℃.
Preferably, the mixing time of step (3) is 20-40 min, including but not limited to 21min, 22min, 23min, 25min, 26min, 28min or 29min.
Preferably, the Tris-Cl concentration in step (5) in the lysate is 90-110 mM, including but not limited to 91mM, 92mM, 93mM, 94mM, 100mM, 102mM, 104mM, 106mM or 108mM.
Preferably, the concentration of sodium chloride in the lysate of step (5) is 90-110 mM, including but not limited to 91mM, 92mM, 93mM, 94mM, 100mM, 102mM, 104mM, 106mM or 108mM.
Preferably, the mass percentage of the sodium dodecyl sulfate in the lysate of the step (5) is 0.5% -1.5%, including but not limited to 0.6%, 0.7%, 0.8%, 1%, 1.2%, 1.3% or 1.4%.
Preferably, the guanidine hydrochloride of step (5) is present in the lysate at a concentration of 2-6 mM, including but not limited to 2.2mM, 2.4mM, 2.6mM, 2.8mM, 3mM, 3.2mM, 3.4mM, 3.6mM or 3.8mM.
Preferably, the dithiothreitol concentration in the lysate of step (5) is 3-7 mM, including but not limited to 3.1mM, 3.2mM, 3.6mM, 3.8mM, 4mM, 5mM, 6mM, 6.2mM, 6.4mM, 6.6mM, 6.8mM.
Preferably, the sodium citrate of step (5) is present in the lysate at a concentration of 45-55 mM, including but not limited to 46mM, 48mM, 49mM, 50mM, 52mM, 53mM or 54mM.
Preferably, the temperature of the mixing in step (5) is 50-60 ℃, including but not limited to 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃,56 ℃, 58 ℃ or 59 ℃.
Preferably, the mixing time of step (5) is 20-40 min, including but not limited to 21min, 22min, 23min, 25min, 30min, 36min, 38min or 39min.
Preferably, the mixed spot extraction process further comprises a step of purifying DNA.
Preferably, the purification comprises adsorbing DNA using magnetic beads, and washing and eluting to obtain purified DNA.
Preferably, the adsorbed binding solution comprises isopropyl alcohol.
Preferably, the volume fraction of isopropyl alcohol is 80% to 90%, including but not limited to 81%, 82%, 83%, 85%, 86%, 88% or 89%.
Preferably, the eluted eluent comprises Tris-Cl, sodium chloride and ethylenediamine tetraacetic acid.
As an optimal technical scheme, the mixed fine spot extraction process comprises the following steps of:
(1) In a full-automatic pipetting workstation, adding a lysate comprising 90-110 mM Tris-Cl, 9-11 mM sodium chloride and 1-3% sodium dodecyl sulfate into a sample to be mixed, incubating for 20-40 min at 50-60 ℃, transferring the mixed solution to a centrifuge by a mechanical gripper to centrifuge, and collecting precipitate;
(2) Mixing the precipitate obtained in the step (1) with a cleaning solution comprising 90-110 mM Tris-Cl, 18-22 mM sodium chloride and 9-11 mM ethylenediamine tetraacetic acid by a transfer pump, transferring the mixed solution to a centrifugal machine by a mechanical clamping hand for centrifugation, and collecting the precipitate;
(3) Mixing the precipitate obtained in the step (2) with a lysate comprising 90-110 mM Tris-Cl, 18-22 mM sodium chloride, 9-11 mM ethylenediamine tetraacetic acid, 0.5-1.5% sodium dodecyl sulfate, 1000.25-0.75% Triton-X, 0.25-0.75% sodium deoxycholate, 18-22 mM magnesium chloride and 9-11 mM calcium chloride by a transfer pump, incubating for 20-40 min at 50-60 ℃, transferring the mixed solution to a centrifugal machine by a mechanical clamping hand, centrifuging, and collecting the precipitate;
(4) Mixing the precipitate obtained in the step (3) with a cleaning solution comprising 90-110 mM Tris-Cl, 18-22 mM sodium chloride and 9-11 mM ethylenediamine tetraacetic acid by a transfer pump, transferring the mixed solution to a centrifugal machine by a mechanical clamping hand for centrifugation, and collecting the precipitate;
(5) Mixing the precipitate obtained in the step (4) with a lysate comprising 90-110 mM Tris-Cl, 90-110 mM sodium chloride, 0.5-1.5% sodium dodecyl sulfate, 2-6 mM guanidine hydrochloride, 3-7 mM dithiothreitol and 45-55 mM sodium citrate by a transfer pump, incubating for 20-40 min at 50-60 ℃, transferring the mixed solution to a centrifugal machine by a mechanical gripper for centrifugation, and collecting supernatant to obtain a solution containing male DNA;
(6) Transferring the solution containing the male DNA obtained in the step (5) and the isopropanol solution with the volume fraction of 80% -90% to a combination plate by a liquid transfer pump, transferring the combination plate to a magnetic suction plate by a mechanical clamp to carry out magnetic bead adsorption, and then sucking out the solution in the combination plate by the liquid transfer pump and discarding the solution;
(7) Adding a cleaning solution comprising Tris-Cl, naCl, guanidine hydrochloride, isopropanol and ethanol into the binding plate after the adsorption in the step (6) by a pipetting pump for cleaning, and then carrying out magnetic bead adsorption and discarding the cleaning solution;
(8) Adding ethanol into the binding plate adsorbed in the step (7) by a transfer pump for cleaning, then carrying out magnetic bead adsorption and discarding the cleaning liquid;
(9) And (3) adding an eluent comprising Tris-Cl, sodium chloride and ethylenediamine tetraacetic acid into the binding plate after the adsorption in the step (8) by a pipetting pump for eluting, and collecting the eluent to obtain purified male DNA.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the mixed sperm spot extraction process, the female components are cracked and digested twice on the detection material, so that the cracking treatment period can be shortened, the female components can be digested efficiently, and the influence of residual female components on subsequent analysis of male DNA is avoided;
(2) The mixed fine spot extraction process can realize automatic mixed fine spot extraction in a full-automatic pipetting workstation, can simultaneously process a plurality of detection materials (1-24), reduces operation difficulty, shortens extraction time (shortened to within 3 h), improves treatment efficiency, and is beneficial to quickly detecting cases.
Drawings
FIG. 1 is a flow chart of a mixed fine spot extraction process;
fig. 2 is an internal layout diagram of a full-automatic pipetting workstation, wherein 1 is a first standby plate position, 2 is an oscillating incubator, 3 is a pipetting pump, 4 is a first liquid tank position, 5 is a second liquid tank position, 6 is a first suction head position, 7 is a second suction head position, 8 is a second standby plate position, 9 is a magnetic suction plate position, 10 is a waste blanking area, 11 is a centrifuge, 12 is a first constant temperature incubator, 13 is a second constant temperature incubator, 14 is a mechanical clamping hand, and 15 is a sample plate separation area;
FIG. 3 is a schematic view of consumable placement, wherein 16 is a first liquid tank, 17 is a second liquid tank, 18 is a 1000 μL gun head box, 19 is a 50 μL gun head box, 20 is a first lower plate, 21 is a second lower plate, 22 is a product plate, 23 is a sample plate, 24 is a mating plate, and 25 is a bonding plate;
FIG. 4 is a graph showing STR typing results of male DNA extracted in example 1;
FIG. 5 is a graph showing STR typing results of male DNA extracted in example 2;
FIG. 6 is a graph showing STR typing results of male DNA extracted in example 3;
FIG. 7 is a graph showing STR typing results of male DNA extracted in example 4;
FIG. 8 is a graph showing STR typing results of male DNA extracted in example 5;
FIG. 9 is a graph showing STR typing results of male DNA extracted in comparative example 1;
FIG. 10 is a graph showing STR typing results of male DNA extracted in comparative example 2.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
In an embodiment of the invention, the first wash solution contains 100mM Tris-Cl (pH 7.5), 20mM NaCl and 10mM EDTA; lysate C contains 100mM Tris-Cl (pH 7.5), 100mM NaCl, 1.0% SDS, 4M guanidine hydrochloride, 5mM DTT and 50mM sodium citrate (pH 6.5); the binding liquid is isopropanol with the volume fraction of 80%; the second wash contained 100mM Tris-Cl (pH 7.5), 100mM NaCl, 5mM EDTA, 1M guanidine hydrochloride, 10% isopropanol, and 15% ethanol; the third cleaning liquid is ethanol with the volume fraction of 75%; the eluate contained 10mM Tris-Cl (pH 7.5), 10mM NaCl and 1mM EDTA.
Example 1
The embodiment provides a mixed fine spot extraction process, wherein the internal layout of a full-automatic pipetting workstation (X-Pure 96Plus silica beads/magnetic beads integrated DNA extraction quantitative workstation) in the extraction process is shown in FIG. 2, a consumable placement schematic diagram is shown in FIG. 3, and a lysate A contains 100mM Tris-Cl (pH 7.5), 10mM NaCl and 2.0% SDS; lysate B contains 100mM Tris-Cl (pH 8.0), 20mM NaCl, 10mM EDTA, 1.0% SDS, 0.5% Triton-X100, 0.5% deoxyplate, 20mM MgCl 2 And 10mM CaCl 2 The extraction process comprises the following steps:
(1) Placing corresponding reagents and consumables according to FIG. 3;
(2) Placing a test material (female cotton) in an upper plate of the sample plate 23, and placing the sample plate 23 in a full-automatic pipetting workstation;
(3) The transfer pump 3 sucks the lysate A from the first liquid tank 16 into the upper plate of the sample plate 23, and incubate at a constant temperature of 56 ℃ for 30min;
(4) After incubation, the mechanical clamping hand 14 simultaneously transfers the sample plate 23 and the trimming plate 24 to the centrifugal machine 11 for solid-liquid separation;
(5) The mechanical gripper 14 grips the first lower plate 20 to the first constant temperature incubator 12;
(6) The mechanical gripper 14 takes out the sample plate 23 from the centrifuge 11 to the sample plate separating region 15 for separating the upper and lower plates, and the mechanical gripper 14 transfers the upper plate to the first lower plate 20;
(7) The pipetting pump 3 draws the first washing liquid from the first liquid bath 16 into the upper plate of the sample plate 23;
(8) The mechanical clamping hand 14 transfers the sample plate 23 into the oscillation incubator 2 for oscillation cleaning;
(9) After the cleaning is finished, the mechanical clamping hand 14 transfers the sample plate 23 from the oscillating incubator 2 to the centrifugal machine 11 for solid-liquid separation;
(10) Centrifuging while the mechanical gripper 14 transfers the original lower plate of the sample plate 23 from the sample plate separation zone 15 to the first constant temperature incubator 12, and the liquid transfer pump 3 transfers the liquid (female supernatant) therein to the binding plate 25 (A1-H2 position) for later use;
(11) After centrifugation, the mechanical clamping hand 14 takes out the sample plate 23 to the sample plate separation area 15 to separate the upper layer plate from the lower layer plate, and the mechanical clamping hand 14 transfers the upper layer plate to the original lower layer plate (the position of the first constant temperature incubator 12) of the sample plate 23;
(12) The pipetting pump 3 sucks the lysate B from the first liquid tank 16 to the upper plate of the sample plate 23 and incubate at a constant temperature of 56℃for 30min;
(13) The liquid pump 3 sucks the waste liquid of the cleaning liquid of the first lower layer plate 20 to the waste blanking area 10 for discarding during incubation;
(14) After incubation, the mechanical clamping hand 14 transfers the sample plate 23 from the first constant temperature incubator 12 to the centrifuge 11 for solid-liquid separation;
(15) Centrifuging while the mechanical gripper 14 transfers the first lower plate 20 from the sample plate separation zone 15 to the first incubator 12;
(16) After centrifugation, the mechanical clamping hand 14 transfers the sample plate 23 from the centrifuge 11 to the sample plate separation area 15 for upper and lower plate separation, and the mechanical clamping hand 14 transfers the upper plate to the first lower plate 20;
(17) The pipetting pump 3 draws the first washing liquid from the first liquid bath 16 into the upper plate of the sample plate 23;
(18) The mechanical clamping hand 14 transfers the sample plate 23 into the oscillation incubator 2 for oscillation cleaning;
(19) After the cleaning is finished, the mechanical clamping hand 14 transfers the sample plate 23 from the oscillating incubator 2 to the centrifugal machine 11 for solid-liquid separation;
(20) Centrifuging, and sucking the waste liquid of the cleaning liquid in the original lower layer plate of the sample plate 23 to a waste blanking area 10 by a mechanical clamping hand 14 for discarding;
(21) The mechanical gripper 14 transfers the original lower plate of the sample plate 23 from the sample plate separation zone 15 to the first spare plate location 1 and the second lower plate 21 from the second spare plate location 8 to the first incubator 12;
(22) After centrifugation, the manipulator 14 removes the sample plate 23 from the centrifuge 11 to the sample plate separation zone 15, while transferring the upper plate to the second lower plate 21;
(23) The mechanical gripper 14 transfers the first lower plate 20 from the sample plate separation zone 15 to the second spare plate location 8;
(24) The pipetting pump 3 sucks the lysate C from the second liquid tank 17 into the upper plate of the sample plate 23, and incubates at a constant temperature of 56 ℃ for 30min;
(25) Incubating while transferring the original lower plate of the sample plate 23 to the second spare plate position 8;
(26) After incubation, the mechanical clamping hand 14 transfers the sample plate 23 from the first constant temperature incubator 12 to the centrifuge 11 for solid-liquid separation;
(27) Centrifuging while the liquid pump 3 sucks the combined liquid from the second liquid tank 17 into the combining plates 25 (A1 to H4);
(28) After centrifugation, the mechanical clamping hand 14 transfers the sample plate 23 from the centrifuge 11 to the sample plate separation area 15 for upper and lower plate separation, and the mechanical clamping hand 14 transfers the upper plate to the first lower plate 20 (the second standby plate position 8);
(29) The transfer pump 3 transfers the lower plate 2 liquid (male pellet) to the combining plate 25 (A3-H4);
(30) The mechanical clamping hand 14 transfers the combination plate 25 to the oscillation incubator 2 to carry out normal-temperature oscillation combination for 10min;
(31) After the combination is finished, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(32) After the adsorption is finished, the liquid transferring pump 3 sucks the waste liquid in the combining plate 25 and discharges the waste liquid from the waste blanking port;
(33) The transfer pump 3 sucks the second cleaning liquid from the second liquid tank 17 to the combining plates 25 (A1 to H4);
(34) The mechanical clamping hand 14 transfers the combination plate 25 to the oscillation incubator 2 for oscillation cleaning;
(35) After the cleaning is finished, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(36) After the adsorption is finished, the liquid transferring pump 3 sucks the waste liquid in the combining plate 25 and discharges the waste liquid from the waste blanking port;
(37) The liquid transfer pump 3 sucks the third cleaning liquid from the second liquid tank 17 to the combining plates 25 (A1 to H4);
(38) The mechanical clamping hand 14 transfers the combination plate 25 to the oscillation incubator 2 for oscillation cleaning;
(39) After the cleaning is finished, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(40) After the adsorption is finished, the liquid transferring pump 3 sucks the waste liquid in the combining plate 25 and discharges the waste liquid from the waste blanking port;
(41) The mechanical clamping hand 14 transfers the combination plate 25 from the magnetic suction plate position 9 to the oscillation incubator 2 for oscillation heating and drying;
(42) After drying, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9;
(43) The pipetting pump 3 draws eluent from the second liquid tank 17 into the combining plates 25 (A1-H4);
(44) The mechanical clamping hand 14 transfers the combination plate 25 from the magnetic suction plate position 9 to the oscillation incubator 2 for oscillation heating elution;
(45) After the elution is finished, the mechanical clamping hand 14 transfers the binding plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(46) After the completion of the adsorption, the pipetting pump 3 transfers the liquid aspirates from the binding plate 25 to the product plate 22 in order to obtain purified male DNA.
Example 2
The only difference compared to example 1 is that the lysate A contains 90mM Tris-Cl (pH 7.5), 11mM NaCl and 1% SDS; the lysate B contains 110mM Tris-Cl (pH 8.0), 18mM NaCl, 9mM EDTA, 0.5% SDS, 0.75% Triton-X100, 0.25% deoxyplate, 22mM MgCl 2 And 11mM CaCl 2 The other steps are the same as in example 1.
Example 3
The only difference compared to example 1 is that the lysate A contains 110mM Tris-Cl (pH 7.5), 9mM NaCl and 3% SDS; the lysate B contains 90mM Tris-Cl (pH 8.0), 22mM NaCl, 11mM EDTA, 1.5% SDS, 0.25% Triton-X100, 0.75% deoxyplate, 18mM MgCl 2 And 9mM CaCl 2 The other steps are the same as in example 1.
Example 4
To demonstrate the universality of the mixed spot extraction process of the present invention, the difference compared to example 1 is that the test material is derived from different victims, otherwise the same as in example 1
Example 5
To demonstrate the universality of the mixed spot extraction process of the present invention, the difference compared to example 1 is only that the source of the test material is different from the victim (the source of the test material is also different from that of example 4), and the other is the same as that of example 1.
Comparative example 1
This comparative example provides a mixed spot extraction process differing from example 4 only in that the lysate A contains 50mM Tris-Cl (pH 7.5), 10mM NaCl and 1% SDS, and omits the step of lysis using lysate B, the extraction process comprising the steps of:
(1) Placing corresponding reagents and consumables according to FIG. 3;
(2) Placing the test material in the upper plate of the sample plate 23, and placing the sample plate 23 in a fully automatic pipetting station;
(3) The transfer pump 3 sucks the lysate A from the first liquid tank 16 into the upper plate of the sample plate 23, and incubate at a constant temperature of 56 ℃ for 30min;
(4) After incubation, the mechanical clamping hand 14 simultaneously transfers the sample plate 23 and the trimming plate 24 to the centrifugal machine 11 for solid-liquid separation;
(5) The mechanical gripper 14 grips the first lower plate 20 to the first constant temperature incubator 12;
(6) The mechanical gripper 14 takes out the sample plate 23 from the centrifuge 11 to the sample plate separating region 15 for separating the upper and lower plates, and the mechanical gripper 14 transfers the upper plate to the first lower plate 20;
(7) The pipetting pump 3 draws the first washing liquid from the first liquid bath 16 into the upper plate of the sample plate 23;
(8) The mechanical clamping hand 14 transfers the sample plate 23 into the oscillation incubator 2 for oscillation cleaning;
(9) After the cleaning is finished, the mechanical clamping hand 14 transfers the sample plate 23 from the oscillating incubator 2 to the centrifugal machine 11 for solid-liquid separation;
(10) Centrifuging while the mechanical gripper 14 transfers the original lower plate of the sample plate 23 from the sample plate separation zone 15 to the first constant temperature incubator 12, and the liquid transfer pump 3 transfers the liquid (female supernatant) therein to the binding plate 25 (A1-H2 position) for later use;
(11) After centrifugation, the mechanical clamping hand 14 takes out the sample plate 23 to the sample plate separation area 15 to separate the upper layer plate from the lower layer plate, and the mechanical clamping hand 14 transfers the upper layer plate to the original lower layer plate (the position of the first constant temperature incubator 12) of the sample plate 23;
(12) The pipetting pump 3 sucks the lysate C from the second liquid tank 17 into the upper plate of the sample plate 23, and incubates at a constant temperature of 56 ℃ for 30min;
(13) Incubating while transferring the original lower plate of the sample plate 23 to the second spare plate position 8;
(14) After incubation, the mechanical clamping hand 14 transfers the sample plate 23 from the first constant temperature incubator 12 to the centrifuge 11 for solid-liquid separation;
(15) Centrifuging while the liquid pump 3 sucks the combined liquid from the second liquid tank 17 into the combining plates 25 (A1 to H4);
(16) After centrifugation, the mechanical clamping hand 14 transfers the sample plate 23 from the centrifuge 11 to the sample plate separation area 15 for upper and lower plate separation, and the mechanical clamping hand 14 transfers the upper plate to the first lower plate 20 (the second standby plate position 8);
(17) The transfer pump 3 transfers the second lower plate 21 liquid (male pellet) into the combining plate 25 (A3-H4);
(18) The mechanical clamping hand 14 transfers the combination plate 25 to the oscillation incubator 2 to carry out normal-temperature oscillation combination for 10min;
(19) After the combination is finished, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(20) After the adsorption is finished, the liquid transferring pump 3 sucks the waste liquid in the combining plate 25 and discharges the waste liquid from the waste blanking port;
(21) The transfer pump 3 sucks the second cleaning liquid from the second liquid tank 17 to the combining plates 25 (A1 to H4);
(22) The mechanical clamping hand 14 transfers the combination plate 25 to the oscillation incubator 2 for oscillation cleaning;
(23) After the cleaning is finished, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(24) After the adsorption is finished, the liquid transferring pump 3 sucks the waste liquid in the combining plate 25 and discharges the waste liquid from the waste blanking port;
(25) The liquid transfer pump 3 sucks the third cleaning liquid from the second liquid tank 17 to the combining plates 25 (A1 to H4);
(26) The mechanical clamping hand 14 transfers the combination plate 25 to the oscillation incubator 2 for oscillation cleaning;
(27) After the cleaning is finished, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(28) After the adsorption is finished, the liquid transferring pump 3 sucks the waste liquid in the combining plate 25 and discharges the waste liquid from the waste blanking port;
(29) The mechanical clamping hand 14 transfers the combination plate 25 from the magnetic suction plate position 9 to the oscillation incubator 2 for oscillation heating and drying;
(30) After drying, the mechanical clamping hand 14 transfers the combination plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9;
(30) The pipetting pump 3 draws eluent from the second liquid tank 17 into the combining plates 25 (A1-H4);
(32) The mechanical clamping hand 14 transfers the combination plate 25 from the magnetic suction plate position 9 to the oscillation incubator 2 for oscillation heating elution;
(33) After the elution is finished, the mechanical clamping hand 14 transfers the binding plate 25 from the oscillation incubator 2 to the magnetic suction plate position 9 for magnetic bead adsorption;
(34) After the completion of the adsorption, the pipetting pump 3 transfers the liquid aspirates from the binding plate 25 to the product plate 22 in order to obtain purified male DNA.
Comparative example 2
The comparative example provides a process for extracting a common fine spot in the prior art, which comprises the following steps:
(1) Clipping the test material (same as the test material source in the example 5) to a 1.5mL centrifuge tube;
(2) 900. Mu.L of lysate A and 100. Mu.L of proteinase K (10 mg/mL) were added to the centrifuge tube and incubated at 56℃for 4h;
(3) Centrifuging at 10000rpm for 2min, and carefully removing the solid sample by using a pipette;
(4) Adding 500 mu L of a first cleaning solution into the solid sample, and lightly blowing for a plurality of times;
(5) Centrifuge at 10000rpm for 2min, carefully remove supernatant with a pipette, taking 50 μl of liquid;
(6) Repeating the steps 4-5;
(7) Adding 200-400 mu L of lysate A, adding dithiothreitol solution with the final concentration of 10mM, and incubating for 30min at 56 ℃;
(8) Adding a binding solution with the same volume as the lysate A and 20 mu L of magnetic beads, and carrying out shaking incubation for 10min;
(9) Placing the centrifuge tube on a magnetic rack for magnetic attraction, and carefully removing the supernatant by using a pipette;
(10) Adding 500 mu L of a second cleaning solution, and carrying out vortex oscillation cleaning for 1min;
(11) Placing the centrifuge tube on a magnetic rack for magnetic attraction, and carefully removing the supernatant by using a pipette;
(12) Adding 500 mu L of third cleaning solution, and performing vortex oscillation cleaning for 1min;
(13) Placing the centrifuge tube on a magnetic rack for magnetic attraction, and carefully removing the supernatant by using a pipette;
(14) Naturally drying;
(15) Adding 50-100 mu L of eluent, and incubating for 10min at 65 ℃ in an oscillating way;
(16) And (3) placing the centrifuge tube in a magnetic rack for magnetic attraction, and collecting supernatant by using a pipettor to obtain purified sperm spot DNA.
Test example quantitative detection
This test example amplified the male DNA obtained in examples 1-5 and comparative examples 1 and 2 and tested for STR typing, short stretch of repetitive sequences (short tandem repeat, STR) being widely present in the human and mammalian genomes. The number of repetitions of the repeated sequence at a particular position on the chromosome is fixed for a particular individual, whereas the number of repetitions at the same position is different for different individuals, constituting polymorphisms of these repeated sequences in a population, and different individuals can be distinguished clearly by detecting such polymorphisms. The amplification typing results are shown in FIGS. 4 to 10.
As can be seen from fig. 4 to 10, the male DNA obtained in examples 1 to 5 can be amplified to obtain a complete STR typing without influence of female typing background, which indicates that the mixed sperm spot extraction process of the present invention can extract male DNA with high efficiency; in comparison with example 4, the step of secondary cleavage was omitted in comparative example 1, and the genotyping results of the obtained genes showed interference in female genotyping (D6S 1043 (12), D13S317 (16), D16S539 (6), CSF1PO (15) and vWA (19)), and male genotyping partial sites were lost; compared to example 5, comparative example 2 is a conventional manual extraction process of the prior art, which is time consuming, with partial site loss of male typing (D6S 1043 (20), penta E (13), CSF1PO (10), vWA (17), D12S391 (18) and FGA (19)), and lower peak value of the resulting typing (RFU: relative fluorescence intensity).
In summary, the mixed fine spot extraction process disclosed by the invention is used for cracking and digesting female components twice on the detection material, so that the cracking treatment period can be shortened, the female components can be efficiently digested, the influence of residual female components on subsequent analysis of male DNA (deoxyribonucleic acid) is avoided, meanwhile, the mixed fine spot extraction process is developed on the basis of a full-automatic pipetting workstation, a plurality of detection materials can be processed simultaneously, the operation difficulty is reduced, the extraction time is shortened, the treatment efficiency is improved, the success rate of detecting a pure target male is improved, and the rapid detection of cases is facilitated.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (9)
1. The mixed fine spot extraction process is characterized by comprising the following steps of:
performing primary pyrolysis on the detection material, collecting primary precipitation, performing secondary pyrolysis on the primary precipitation, collecting secondary precipitation, performing tertiary pyrolysis on the secondary precipitation, and collecting supernatant to obtain a solution containing male DNA;
the mixed fine spot extraction process comprises the following steps:
(1) Mixing the detection material with a lysate comprising Tris-Cl, sodium chloride and sodium dodecyl sulfate at 50-60 ℃ for 20-40 min, centrifuging, and collecting a precipitate;
(2) Mixing the precipitate obtained in the step (1) with a cleaning solution, centrifuging, and collecting the precipitate;
(3) Mixing the precipitate obtained in the step (2) with a lysate comprising Tris-Cl, sodium chloride, ethylenediamine tetraacetic acid, sodium dodecyl sulfate, triton-X100, sodium deoxycholate, magnesium chloride and calcium chloride at 50-60 ℃ for 20-40 min, centrifuging, and collecting the precipitate;
(4) Mixing the precipitate obtained in the step (3) with a cleaning solution, centrifuging, and collecting the precipitate;
(5) Mixing the precipitate obtained in the step (4) with a lysate comprising Tris-Cl, sodium chloride, sodium dodecyl sulfate, guanidine hydrochloride, dithiothreitol and sodium citrate, centrifuging, and collecting a supernatant to obtain a solution containing male DNA;
the concentration of Tris-Cl in the lysate is 90-110 mM, the concentration of sodium chloride in the lysate is 9-11 mM, and the mass percentage of sodium dodecyl sulfate in the lysate is 1% -3%;
the concentration of Tris-Cl in the lysate is 90-110 mM, the concentration of sodium chloride in the lysate is 18-22 mM, the concentration of ethylenediamine tetraacetic acid in the lysate is 9-11 mM, the mass percentage of sodium dodecyl sulfate in the lysate is 0.5-1.5%, the mass percentage of Triton-X100 in the lysate is 0.25-0.75%, the mass percentage of sodium deoxycholate in the lysate is 0.25-0.75%, the concentration of magnesium chloride in the lysate is 18-22 mM, and the concentration of calcium chloride in the lysate is 9-11 mM;
the concentration of Tris-Cl in the lysate is 90-110 mM, the concentration of sodium chloride in the lysate is 90-110 mM, the mass percentage of sodium dodecyl sulfate in the lysate is 0.5% -1.5%, the concentration of guanidine hydrochloride in the lysate is 2-6 mM, the concentration of dithiothreitol in the lysate is 3-7 mM, and the concentration of sodium citrate in the lysate is 45-55 mM;
the cleaning solution for cleaning comprises Tris-Cl, sodium chloride and ethylenediamine tetraacetic acid, wherein the concentration of the Tris-Cl in the cleaning solution is 90-110 mM, the concentration of the sodium chloride in the cleaning solution is 18-22 mM, and the concentration of the ethylenediamine tetraacetic acid in the cleaning solution is 9-11 mM.
2. The hybrid fine spot extraction process of claim 1, wherein the hybrid fine spot extraction process is performed in a fully automated pipetting workstation.
3. The process for extracting mixed fine spots according to claim 1, wherein the temperature of the mixing in the step (5) is 50-60 ℃, and the mixing time is 20-40 min.
4. The mixed spot extraction process of claim 1, further comprising the step of purifying DNA.
5. The process of claim 4, wherein purifying comprises adsorbing DNA with magnetic beads, and washing and eluting to obtain purified DNA.
6. The hybrid fine spot extraction process of claim 5, wherein the adsorbed binding fluid comprises isopropyl alcohol.
7. The process of claim 6, wherein the isopropyl alcohol is 80-90% by volume.
8. The mixed spot extraction process of claim 5, wherein the eluted eluate comprises Tris-Cl, sodium chloride, and ethylenediamine tetraacetic acid.
9. The process for extracting mixed fine spots according to any one of claims 1 to 8, comprising the steps of:
(1) In a full-automatic pipetting workstation, adding a lysate comprising 90-110 mM Tris-Cl, 9-11 mM sodium chloride and 1-3% sodium dodecyl sulfate into a sample to be mixed, incubating for 20-40 min at 50-60 ℃, transferring the mixed solution to a centrifuge by a mechanical gripper to centrifuge, and collecting precipitate;
(2) Mixing the precipitate obtained in the step (1) with a cleaning solution comprising 90-110 mM Tris-Cl, 18-22 mM sodium chloride and 9-11 mM ethylenediamine tetraacetic acid by a transfer pump, transferring the mixed solution to a centrifugal machine by a mechanical clamping hand for centrifugation, and collecting the precipitate;
(3) Mixing the precipitate obtained in the step (2) with a lysate comprising 90-110 mM Tris-Cl, 18-22 mM sodium chloride, 9-11 mM ethylenediamine tetraacetic acid, 0.5-1.5% sodium dodecyl sulfate, 1000.25-0.75% Triton-X, 0.25-0.75% sodium deoxycholate, 18-22 mM magnesium chloride and 9-11 mM calcium chloride by a transfer pump, incubating for 20-40 min at 50-60 ℃, transferring the mixed solution to a centrifugal machine by a mechanical clamping hand, centrifuging, and collecting the precipitate;
(4) Mixing the precipitate obtained in the step (3) with a cleaning solution comprising 90-110 mM Tris-Cl, 18-22 mM sodium chloride and 9-11 mM ethylenediamine tetraacetic acid by a transfer pump, transferring the mixed solution to a centrifugal machine by a mechanical clamping hand for centrifugation, and collecting the precipitate;
(5) Mixing the precipitate obtained in the step (4) with a lysate comprising 90-110 mM Tris-Cl, 90-110 mM sodium chloride, 0.5-1.5% sodium dodecyl sulfate, 2-6 mM guanidine hydrochloride, 3-7 mM dithiothreitol and 45-55 mM sodium citrate by a transfer pump, incubating for 20-40 min at 50-60 ℃, transferring the mixed solution to a centrifugal machine by a mechanical gripper for centrifugation, and collecting supernatant to obtain a solution containing male DNA;
(6) Transferring the solution containing the male DNA obtained in the step (5) and the isopropanol solution with the volume fraction of 80% -90% to a combination plate by a liquid transfer pump, transferring the combination plate to a magnetic suction plate by a mechanical clamp to carry out magnetic bead adsorption, and then sucking out the solution in the combination plate by the liquid transfer pump and discarding the solution;
(7) Adding a cleaning solution comprising Tris-Cl, naCl, guanidine hydrochloride, isopropanol and ethanol into the binding plate after the adsorption in the step (6) by a pipetting pump for cleaning, and then carrying out magnetic bead adsorption and discarding the cleaning solution;
(8) Adding ethanol into the binding plate adsorbed in the step (7) by a transfer pump for cleaning, then carrying out magnetic bead adsorption and discarding the cleaning liquid;
(9) And (3) adding an eluent comprising Tris-Cl, sodium chloride and ethylenediamine tetraacetic acid into the binding plate after the adsorption in the step (8) by a pipetting pump for eluting, and collecting the eluent to obtain purified male DNA.
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Inventor after: Luo Shenheng Inventor after: Peng Shan Inventor after: Zhu Dongming Inventor after: Du Zhou Inventor after: Wang Chuanhai Inventor after: Li Xiangqin Inventor after: Yu Ruiguo Inventor after: Yin Lu Inventor after: Qu Dongyang Inventor after: Wang Weini Inventor after: Zhang Bo Inventor before: Luo Shenheng Inventor before: Peng Shan Inventor before: Zhu Dongming Inventor before: Du Zhou Inventor before: Wang Chuanhai Inventor before: Li Xiangqin Inventor before: Yu Ruiguo Inventor before: Yin Lu Inventor before: Qu Dongyang Inventor before: Wang Weini Inventor before: Zhang Bo |