CN107446918B - Method for obtaining MYC gene in human tissue by two-step overlapping PCR method - Google Patents
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Abstract
The invention belongs to the technical field of biology, and relates to a method for obtaining MYC genes in human tissues by a two-step overlapping PCR method. The method comprises the following steps: (1) TES water bath dewaxing; (2) extracting DNA of a human tissue sample; (3) searching and selecting 7 MYC gene sequences with the same length; (4) designing 16 MYC gene specific primers; (5) two-step overlapping PCR amplification; (6) purifying the DNA fragment; (7) determining the DNA concentration; (8) and (4) gene detection. The invention has the advantages of high accuracy, high efficiency, better dewaxing effect, less damage to the environment and human body, simple operation and low cost, amplifies the DNA fragments of the MYC target genes with 5279bp different sequences, has higher purity and concentration of the target genes, verifies the fragments of the target genes by a sequencing method, and provides convenience for the research of biomedicine.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for obtaining MYC genes in human tissues by a two-step overlapping PCR method.
Background
Formalin-fixed paraffin embedding is the most widely used technique for clinical pathological diagnosis. In the formalin-fixed paraffin embedding technology, formalin is used as a fixing agent, so that the change of tissue morphology within a certain time can be reduced, and the reliable guarantee is provided for the aspects of subsequent immunohistochemistry, histological analysis, gene diagnosis and the like.
The existing formalin-fixed paraffin embedding technology still has the following defects: firstly, the concentration of DNA extracted from a specimen is low, fragments are short, the quality of the specimen is gradually reduced along with the lapse of time, so that the acquisition of complete genes is quite difficult, the fragmentation of the DNA can cause the deletion integrity of target genes, the detection result is unreliable, the data splicing is more difficult, the haplotype information of the MYC full-length gene cannot be analyzed, and great obstacles are caused to clinical examination and medical science research; secondly, a xylene dewaxing method is usually adopted in the existing paraffin-embedded tissue dewaxing method, but xylene is easy to pollute the environment and is harmful to human bodies, and the extraction process is longer; thirdly, the existing methods for obtaining the target DNA fragment include PCR, enzyme digestion, gene chips, hybridization capture and the like, but the obtained fragment has the problems of easy breakage, low purity and concentration, more impurities, short length and the like, and meanwhile, the second-generation sequencing method which is generally used for formalin-fixed paraffin-embedded human tissue DNA detection also has the problems of short read length, too large sequencing depth, insensitivity to high GC (gas chromatography) regions, repetitive regions and base modification regions and the like, so that the information splicing amount is large, MYC (myo-associated protein) genes cannot be sequenced, and the difficulty in analyzing the long-fragment DNA result is increased. Therefore, there is an urgent need to establish a method for obtaining a long-fragment target gene with high quality, high concentration and strong integrity from formalin-fixed paraffin-embedded human tissues.
Disclosure of Invention
The invention aims to provide a method for obtaining MYC genes in human tissues, which is characterized in that long-fragment DNA (deoxyribonucleic acid), namely MYC genes, is obtained from formalin-fixed paraffin-embedded human tissues by using a TES water bath dewaxing and two-step overlap PCR method, and is verified by using Sanger sequencing.
The technical scheme for solving the technical problems comprises the following steps:
a method for obtaining MYC genes in human tissues by using a two-step overlapping PCR method comprises the following steps:
(1) taking a formalin-fixed paraffin-embedded human tissue sample, slicing the sample according to the thickness of 8-12 microns, and then carrying out TES water bath dewaxing operation, wherein the TES water bath dewaxing operation comprises the following steps: adding 800-1200 mu L of TES (trihydroxymethyl methylaminoethanesulfonic acid) solution, incubating for 25-35 min at 50-60 ℃ after full oscillation, fully oscillating again and mixing uniformly, centrifuging for 10-15 min at 4 ℃ and a centrifuge with the rotation speed of 8000-14000 rpm, discarding supernatant, and repeating the TES water bath dewaxing operation for 2-3 times;
(2) extracting DNA of the dewaxed human tissue sample;
(3) searching and selecting 7 MYC gene sequences with the same length;
(4) designing 16 MYC gene specific primers according to the MYC gene sequence found in the step (3), wherein the sequence numbers of the primers are as follows:
item 1: F1-AGAAGGGCAGGGCTTCTCA, R1-ATCTAACTCGCTGTAGTA;
item 2: F2-AAAGAACGGAGGGAGGGAT, R2-TATGGGCAAAGTTTCGTGG;
item 3: F3-CGAAACTTTGCCCATAGC, R3-TGGACTTCGGTGCTTACCTG;
item 4: F4-GGTAAGCACCGAAGTCCAC, R4-AAAGCAGGAATGTCCGAC;
item 5: F5-CGGACATTCCTGCTTTATTG, R5-AGGAGAATCGGACACATCC;
item 6: F6-TGTCCGATTCTCCTGGA, R6-GCACTCAATACGGAGATGC;
item 7: F7-TCTCCGTATTGAGTGCGA, R7-TTAGTGAACCAGCGGCTTG;
item 8: F8-AGCCGCTGGTTCACTAA, R8-GTCGCAGTAGAAATACGGC;
item 9: F9-GTATTTCTACTGCGACGAGG, R9-CCAAGGTTTCAGAGGTGATG;
item 10: F10-AACCTTGGGCTTTAGCG, R10-TTCATACACTCCCTGCCAC;
item 11: F11-CACTTCTTCTTACCTCCCGT, R11-TTCCCAAGCACCTCCTAT;
item 12: F12-AGGTGCTTGGGAATGTG, R12-CACCTGTAATCCCAGCACT;
item 13: F13-CCAAAGTGCTGGGATTACA; R13-TGCGTAGTTGTGCTGATG
Item 14: F14-CACATCAGCACAACTACGC, R14-TGGACGGACAGGATGTATGC;
item 15: F15-CATACATCCTGTCCGTCCA, R15-AGACTCAGCCAAGGTTGTGA;
item 16: F16-CCTTGGCTGAGTCTTGAGA, R16-AATAAAATAACTGGCA;
(5) overlapping PCR amplification: wherein, the overlapped PCR amplification is divided into two steps, specifically:
the first step of PCR amplification: carrying out PCR (polymerase chain reaction) amplification experiments on the DNA extracted in the step (2) by using the specific primers of the 16 MYC genes in the step (4), so as to amplify DNA fragments of the MYC genes and obtain 16 amplification products;
the conditions for PCR amplification were: pre-denaturation: 93-95 ℃ for 4-6 min; denaturation: 93-95 ℃ for 30-45 s; annealing: 50-60 ℃ for 30 s-1 min; extension: at 72 ℃ for 40 s-1 min; final extension: 72 ℃ for 7-10 min; and (3) storage: 4 ℃; wherein, the process of denaturation, annealing and extension is circulated for 30-40 times;
the second step of PCR amplification: taking 2 mu L of each 16 amplification products amplified by the PCR in the first step, and fully mixing the 16 amplification products with 19 mu L of forward primers and 19 mu L of reverse primers to carry out PCR amplification reaction, thereby amplifying DNA fragments with the full length of 5279 bp; wherein the forward primer is F1-AGAAGGGCAGGGCTTCTCA in the step (4), and the reverse primer is R16-AATAAAATAACTGGCA in the step (4);
the conditions of the PCR amplification reaction are as follows: pre-denaturation: 95 deg.C for 4 min; denaturation: 95 ℃ for 15 s; annealing: 56 ℃ for 30 s; extension: 72 ℃ for 6 min; final extension: 72 ℃ for 10 min; and (3) storage: 4 ℃; wherein, the process of denaturation, annealing and extension is circulated for 35 times;
(6) purification of DNA fragment: adding magnetic beads, wherein the volume of the magnetic beads is 0.45-0.60 times of that of the DNA fragments, carrying out vortex for 10min in a vortex instrument with the rotating speed of 1800-2000 rpm, and then removing liquid in the vortex instrument; adding 400-600 mu L of ethanol with the mass concentration of 70%, uniformly mixing and removing internal liquid, adding 400-600 mu L of ethanol with the mass concentration of 70% again, uniformly mixing and removing internal liquid; adding 50-100 mul of elution buffer solution, and whirling in a vortex instrument with the rotating speed of 2000rpm for 1min, and keeping the concentration of the DNA fragment at 100 ng/mul; repeatedly purifying the DNA fragment for 1 time to obtain a MYC target gene;
(7) determination of DNA concentration: quantifying MYC target gene, determining its DNA concentration, keeping the DNA concentration of target gene at 100ng/μ L, and temporarily storing at 4 deg.C or-20 deg.C for 1 month;
(8) gene detection: and carrying out detection comparison and data analysis on MYC target genes.
The method for obtaining MYC genes in human tissues by using the two-step overlapped PCR method has the advantages that the dewaxing operation is performed by using TES to replace dimethylbenzene, the dewaxing effect is better, the damage to the environment and the human body is less, the operation is simple, and the cost is low; the extracted DNA fragment has shorter length, and the DNA fragments of the MYC target genes with different sequences of 5279bp are amplified by using a two-step overlapping PCR method, so that the purity and the concentration of the target genes are greatly improved; the Sanger sequencing method is applied to verify the target gene fragment, so that convenience is provided for further scientific research work in the aspect of biomedicine; the method can splice fragment DNA into long fragment target genes, has high accuracy and high efficiency, and the obtained DNA fragments of the target genes have high purity and content.
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FIG. 1 is a two-step overlap PCR method for amplifying 16 MYC target gene fragments;
FIG. 2 is a two-step overlap PCR method for amplifying 5279bp MYC target gene fragment;
FIG. 3 shows the comparison result of MYC target gene fragment amplified by primers F2 and R2 and Blast determined by Sanger sequencing method;
FIG. 4 shows the comparison result of MYC target gene fragment amplified by primers F15 and R15 and Blast determined by Sanger sequencing method.
Detailed Description
A method for obtaining MYC genes in human tissues by using a two-step overlapping PCR method comprises the following steps:
(1) taking a formalin-fixed paraffin-embedded human tissue sample, slicing the sample according to the thickness of 8-12 microns, loading the sample into a 1.5mL EP tube, and performing TES water bath dewaxing operation; wherein, the TES water bath dewaxing operation process is as follows: adding 800-1200 mu L TES solution, incubating for 25-35 min at 50-60 ℃ after full oscillation, fully oscillating again and mixing uniformly, centrifuging for 10-15 min at 4 ℃ and a centrifugal machine with the rotation speed of 8000-14000 rpm, discarding supernatant, and repeating TES water bath dewaxing operation for 2-3 times;
(2) extracting DNA of the dewaxed human tissue sample, wherein the extraction method refers to QIAGEN Kit specification, in particular to GeneRead DNA FFPE Kit No. 180134;
(3) searching and selecting 7 MYC gene sequences with the same length from Pubmed, wherein the specific sequence names are respectively as follows:
1.>ref|NC_000008.11|:127736069-127741434Homo sapiens chromosome 8,GRCh38.p7；
2.>ref|NG_007161.1|:5000-10365Homo sapiens MYC proto-oncogene,bHLH transcription factor(MYC),RefSeqGene on chromosome 8；
3.>emb|CU310075.1|:55001-60366Human DNA sequence from clone XX-NCIH2171_7C07,complete sequence；
4.>emb|CU310073.1|:65021-70386Human DNA sequence from clone XX-NCIH2171_15F08,complete sequence；
5.>gb|AC103819.3|:87653-93018Homo sapiens chromosome 8,clone CTD-3056O22,complete sequence；
6.>gb|AY214166.1|:1872-7237Homo sapiens v-myc myelocytomatosis viral oncogene homolog(avian)(MYC)gene,complete cds；
7.>gb|AF315312.2|:61930-67295Homo sapiens chromosome 8clone RP1-80K22map 8q24.3,complete sequence；
(4) designing 16 MYC gene specific primers according to the MYC gene sequence found in the step (3), wherein the sequence numbers of the primers are as follows:
item 1: F1-AGAAGGGCAGGGCTTCTCA, R1-ATCTAACTCGCTGTAGTA;
item 2: F2-AAAGAACGGAGGGAGGGAT, R2-TATGGGCAAAGTTTCGTGG;
item 3: F3-CGAAACTTTGCCCATAGC, R3-TGGACTTCGGTGCTTACCTG;
item 4: F4-GGTAAGCACCGAAGTCCAC, R4-AAAGCAGGAATGTCCGAC;
item 5: F5-CGGACATTCCTGCTTTATTG, R5-AGGAGAATCGGACACATCC;
item 6: F6-TGTCCGATTCTCCTGGA, R6-GCACTCAATACGGAGATGC;
item 7: F7-TCTCCGTATTGAGTGCGA, R7-TTAGTGAACCAGCGGCTTG;
item 8: F8-AGCCGCTGGTTCACTAA, R8-GTCGCAGTAGAAATACGGC;
item 9: F9-GTATTTCTACTGCGACGAGG, R9-CCAAGGTTTCAGAGGTGATG;
item 10: F10-AACCTTGGGCTTTAGCG, R10-TTCATACACTCCCTGCCAC;
item 11: F11-CACTTCTTCTTACCTCCCGT, R11-TTCCCAAGCACCTCCTAT;
item 12: F12-AGGTGCTTGGGAATGTG, R12-CACCTGTAATCCCAGCACT;
item 13: F13-CCAAAGTGCTGGGATTACA; R13-TGCGTAGTTGTGCTGATG
Item 14: F14-CACATCAGCACAACTACGC, R14-TGGACGGACAGGATGTATGC;
item 15: F15-CATACATCCTGTCCGTCCA, R15-AGACTCAGCCAAGGTTGTGA;
item 16: F16-CCTTGGCTGAGTCTTGAGA, R16-AATAAAATAACTGGCA;
(5) overlapping PCR amplification: wherein, the overlapped PCR amplification is divided into two steps, specifically:
the first step of PCR amplification: carrying out PCR amplification experiments on the DNA extracted in the step (2) by using the specific primers of the 16 MYC genes in the step (4) so as to amplify DNA fragments of the MYC genes and obtain 16 amplification products, wherein the PCR amplification process refers to KAPA LongRange HotStart PCR Kit (KK 3501-07959141001);
the conditions for PCR amplification were: pre-denaturation: 93-95 ℃ for 4-6 min; denaturation: 93-95 ℃ for 30-45 s; annealing: 50-60 ℃ for 30 s-1 min; extension: at 72 ℃ for 40 s-1 min; final extension: 72 ℃ for 7-10 min; and (3) storage: 4 ℃; wherein, the process of denaturation, annealing and extension is circulated for 30-40 times;
the specific conditions for PCR amplification of the specific primers of the 16 MYC genes are as follows:
the second step of PCR amplification: taking 2 mu L of each 16 amplification products amplified by the PCR in the first step, fully mixing the 16 amplification products with 19 mu L of forward primers and 19 mu L of reverse primers, preparing a mixed solution by referring to KAPA LongRange HotStart PCR Kit (KK3501-07959141001) according to a system expansion 3 times, and carrying out PCR amplification reaction so as to amplify a DNA fragment with the total length of 5279 bp; wherein, the forward primer is: F1-AGAAGGGCAGGGCTTCTCA, wherein the reverse primer is as follows: R16-AATAAAATAACTGGCA; the conditions of the amplification reaction are as follows: pre-denaturation: 95 deg.C for 4 min; denaturation: 95 ℃ for 15 s; annealing: 56 ℃ for 30 s; extension: 72 ℃ for 6 min; final extension: 72 ℃ for 10 min; and (3) storage: 4 ℃; wherein, the process of denaturation, annealing and extension is circulated for 35 times;
(6) purification of DNA fragment: adding an AMPure PB magnetic bead, wherein the volume of the AMPure PB magnetic bead is 0.45-0.60 times of that of the DNA fragment, swirling for 10min in a vortex instrument with the rotation speed of 1800-2000 rpm, and removing liquid in the vortex instrument; adding 400-600 mu L of ethanol with the mass concentration of 70%, flicking for 30-60 s, uniformly mixing and removing internal liquid, adding 400-600 mu L of ethanol with the mass concentration of 70% again, flicking for 30-60 s, uniformly mixing and removing internal liquid; adding 50-100 mul of elution buffer solution, and whirling in a vortex instrument with the rotating speed of 2000rpm for 1min, and keeping the concentration of the DNA fragment at 100 ng/mul; repeating the purified DNA fragment for 1 time to obtain MYC target gene;
(7) determination of DNA concentration: quantifying by using Nanodrop or Qubit, determining the DNA concentration of MYC target gene, keeping the DNA concentration of the target gene at 100 ng/muL, and storing at 4 ℃ for 2 days or at-20 ℃ for 1 month;
(8) gene detection: and (3) detecting and verifying MYC target genes by using a Sanger sequencing method, and comparing the obtained sequence with sequences in a database and analyzing data.
As shown in FIG. 1, MYC gene fragments of 87bp, 263bp, 216bp, 309bp, 408bp, 230bp, 513bp, 317bp, 844bp, 202bp, 489bp, 456bp, 357bp, 309bp, 226bp and 356bp in length are amplified by a two-step overlap PCR method.
As shown in FIG. 2, a MYC gene fragment 5279bp in length was amplified by a two-step overlap PCR method.
As shown in FIG. 3, primers F2 and R2 were selected to amplify a MYC target gene fragment, and the homology similarity between the detected sequence and two MYC sequences in a database is 100%, and the coverage rate is 100%, which indicates that the sequence detected in the application is identical to the MYC target gene.
As shown in FIG. 4, primers F15 and R15 were selected to amplify a MYC target gene fragment, and the homology similarity between the detected sequence and two MYC sequences in a database is 100%, and the coverage rate is 100%, which indicates that the sequence detected in the application is identical to the MYC target gene.
MYC target gene fragments amplified by 16 specific primers are respectively determined by a Sanger sequencing method, and are compared with two sequences of MYC in a Blast database, so that the homology similarity between the determined sequences and the two sequences of MYC in the database is 100%, the coverage rate is 100%, and the determined sequences are consistent with the MYC target gene.
In conclusion, the method for obtaining the MYC genes in the human tissues by formalin-fixed paraffin embedding and two-step overlapped PCR amplification has the advantages of high accuracy, high efficiency and high sample purity and content, and can splice fragment DNA into long fragment target genes.
Claims (9)
1. A method for obtaining MYC genes in human tissues by using a two-step overlapping PCR method is characterized by comprising the following steps:
(1) cutting a formalin-fixed paraffin-embedded human tissue sample into slices, and then carrying out TES water bath dewaxing operation; the TES water bath dewaxing operation process comprises the following steps: adding 800-1200 mu L of TES solution, fully shaking, incubating at 50-60 ℃ for 25-35 min, fully shaking again and mixing uniformly, centrifuging at 4 ℃ for 10-15 min, discarding supernatant, and repeating TES water bath dewaxing operation for 2-3 times;
(2) extracting DNA of a human tissue sample;
(3) searching and selecting 7 MYC gene sequences with the same length;
(4) designing 16 MYC gene specific primers, wherein the sequence numbers of the primers are respectively as follows:
item 1: F1-AGAAGGGCAGGGCTTCTCA, R1-ATCTAACTCGCTGTAGTA;
item 2: F2-AAAGAACGGAGGGAGGGAT, R2-TATGGGCAAAGTTTCGTGG;
item 3: F3-CGAAACTTTGCCCATAGC, R3-TGGACTTCGGTGCTTACCTG;
item 4: F4-GGTAAGCACCGAAGTCCAC, R4-AAAGCAGGAATGTCCGAC;
item 5: F5-CGGACATTCCTGCTTTATTG, R5-AGGAGAATCGGACACATCC;
item 6: F6-TGTCCGATTCTCCTGGA, R6-GCACTCAATACGGAGATGC;
item 7: F7-TCTCCGTATTGAGTGCGA, R7-TTAGTGAACCAGCGGCTTG;
item 8: F8-AGCCGCTGGTTCACTAA, R8-GTCGCAGTAGAAATACGGC;
item 9: F9-GTATTTCTACTGCGACGAGG, R9-CCAAGGTTTCAGAGGTGATG;
item 10: F10-AACCTTGGGCTTTAGCG, R10-TTCATACACTCCCTGCCAC;
item 11: F11-CACTTCTTCTTACCTCCCGT, R11-TTCCCAAGCACCTCCTAT;
item 12: F12-AGGTGCTTGGGAATGTG, R12-CACCTGTAATCCCAGCACT;
item 13: F13-CCAAAGTGCTGGGATTACA; R13-TGCGTAGTTGTGCTGATG
Item 14: F14-CACATCAGCACAACTACGC, R14-TGGACGGACAGGATGTATGC;
item 15: F15-CATACATCCTGTCCGTCCA, R15-AGACTCAGCCAAGGTTGTGA;
item 16: F16-CCTTGGCTGAGTCTTGAGA, R16-AATAAAATAACTGGCA;
(5) overlapping PCR amplification: the overlapping PCR amplification is divided into two steps, specifically:
the first step of PCR amplification: carrying out PCR amplification on the DNA extracted in the step (2) by using the specific primers of the 16 MYC genes in the step (4), thereby amplifying DNA fragments of the MYC genes and obtaining 16 amplification products;
the PCR amplification conditions are as follows: pre-denaturation: 93-95 ℃ for 4-6 min; denaturation: 93-95 ℃ for 30-45 s; annealing: 50-60 ℃ for 30 s-1 min; extension: at 72 ℃ for 40 s-1 min; final extension: 72 ℃ for 7-10 min; and (3) storage: 4 ℃; wherein, the process of denaturation, annealing and extension is circulated for 30-40 times;
the second step of PCR amplification: taking 2 mu L of each 16 amplification products amplified by the PCR in the first step, fully mixing the 16 amplification products with 19 mu L of forward primers and 19 mu L of reverse primers, and carrying out PCR amplification reaction so as to amplify DNA fragments with the full length of 5279 bp;
the forward primer is F1-AGAAGGGCAGGGC TTCTCA in the step (4), and the reverse primer is R16-AATAAAATAACTGGCA in the step (4);
the conditions of the PCR amplification reaction are as follows: pre-denaturation: 95 deg.C for 4 min; denaturation: 95 ℃ for 15 s; annealing: 56 ℃ for 30 s; extension: 72 ℃ for 6 min; final extension: 72 ℃ for 10 min; and (3) storage: 4 ℃; wherein, the process of denaturation, annealing and extension is circulated for 35 times;
(6) purification of DNA fragment: adding magnetic beads, vortexing for 10min, and removing liquid; adding ethanol, mixing, removing internal liquid, adding ethanol again, mixing, and removing internal liquid; adding elution buffer solution, and vortexing in a vortexer at 2000rpm for 1min to maintain the concentration of DNA fragment at 100 ng/. mu.L;
repeating the purified DNA fragment for 1 time to obtain a MYC target gene;
(7) determination of DNA concentration: quantifying MYC target genes, measuring the DNA concentration of the MYC target genes, keeping the DNA concentration at 100 ng/mu L, and storing;
(8) gene detection: and carrying out detection comparison and data analysis on MYC target genes.
2. The method for obtaining MYC gene in human tissue by two-step overlapped PCR method as claimed in claim 1, wherein in step (1), the thickness of the slice of the human tissue sample is 8-12 μm.
3. The method for obtaining MYC genes in human tissues by using the two-step overlapping PCR method as claimed in claim 1, wherein in the step (1), the centrifugal operation is performed in a centrifuge, and the rotation speed of the centrifuge is 8000 rpm-14000 rpm.
4. The method for obtaining MYC gene in human tissue by two-step overlap PCR method as claimed in claim 1, wherein in step (6), the volume of the magnetic bead is 0.45-0.60 times the volume of the DNA fragment.
5. The method for obtaining MYC gene in human tissue by two-step overlapped PCR method as claimed in claim 1, wherein in step (6), the amount of ethanol is 400-600 μ L, and the mass concentration is 70%.
6. The method for obtaining MYC gene in human tissue by two-step overlapped PCR method as claimed in claim 1, wherein in step (6), the amount of elution buffer is 50-100. mu.L.
7. The method for obtaining MYC genes in human tissues by using the two-step overlapping PCR method as claimed in claim 1, wherein in the step (6), the vortex operation after the magnetic beads are added is performed in a vortex instrument, and the rotation speed of the vortex instrument is 1800 rpm-2000 rpm.
8. The method for obtaining MYC gene in human tissue by two-step overlapping PCR method as claimed in claim 1, wherein in step (6), the vortex operation after adding elution buffer is performed in a vortex apparatus, and the rotation speed of the vortex apparatus is 2000 rpm.
9. The method for obtaining MYC gene in human tissue by two-step overlapped PCR method as claimed in claim 1, wherein in step (7), the preservation conditions are: storing at 4 deg.C for 2 days, or storing at-20 deg.C for 1 month.
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| CN102191336A (en) * | 2011-06-09 | 2011-09-21 | 广州益善生物技术有限公司 | MYC gene single nucleotide polymorphism (SNP) detection specific primer and liquid-phase chip |
| CN103764849A (en) * | 2011-06-27 | 2014-04-30 | 佛罗里达大学研究基金公司 | Method for genome complexity reduction and polymorphism detection |
| CN105238867A (en) * | 2015-11-05 | 2016-01-13 | 复旦大学 | Methane/ ammoxidation bacterial community structure analysis method based on two-step method |
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