CN111850121A

CN111850121A - Screening reagent for detecting DNA damage signal channel core molecule and application thereof

Info

Publication number: CN111850121A
Application number: CN202010666783.2A
Authority: CN
Inventors: 刘世海; 贺桂芳; 潘华政; 刘畅畅; 蔡铎
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2020-10-30

Abstract

The PCR reagent comprises PCR reaction primers for detecting related genes such as ABL1 gene, APEX1 gene, ATM gene, ATR gene and the like and reference genes and the like. The invention concentrates the core molecules related to the DNA damage signal path on a flat plate, reacts the core molecules regulated and controlled by the DNA damage signal path in cells by carrying out a real-time fluorescent quantitative PCR reaction, and uses the liver cancer Huh7 treated by drugs and the liver cancer Huh7 not treated as comparison examples to discuss the possible ways of the core signal molecules causing the DNA damage, thereby providing the most direct evidence for researching the regulation and control of key proteins; the invention can quickly and accurately find out the core signal molecule with DNA damage from the transcription level, and provides a powerful tool for the mechanism discussion of new targeted drugs, the development of targeted tumor inhibitors and the like.

Description

Screening reagent for detecting DNA damage signal channel core molecule and application thereof

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a screening reagent for detecting a DNA damage signal channel core molecule and application thereof.

Background

Due to various internal and environmental factors, every cell of our body produces thousands of DNA lesions every day. These lesions, if not properly repaired, may result in mutations and more extensive genomic aberrations that threaten cellular activity and ultimately lead to cancer. DNA lesions trigger a series of organized, multifaceted and synergistic responses within cells that collectively counteract this genotoxic stress to detect DNA damage, suggest its presence and mediate its repair, a series of defense responses known as DNA Damage Responses (DDR). DNA repair is critical in many biological processes, including the development of the immune system, meiotic recombination, and the prevention of a variety of human diseases. Genetic DNA repair defects can be catastrophic, leading to dysplasia, increased cancer propensity, immunodeficiency, neurodegenerative diseases, and premature aging.

The DNA repair process involves a series of signal transmission, similar to the conventional signal transduction pathway, including sensor, transmitter and effector molecules, which are respectively carried by different proteins and their modified forms. Unlike traditional signaling pathways, however, the activation of DNA repair signaling pathways typically starts with abnormal genomic DNA structures, and thus sensor molecules are proteins that recognize abnormal DNA structures and activate upstream kinases. The transmission molecules are mainly some proteins in a protein kinase cascade reaction, and the effector molecules are protein kinase substrates involved in DNA repair, and the substrate molecules are directly involved in DNA replication, DNA repair and cell cycle control. The development of new tools for targeting newly discovered targets in DNA damage response and studying these pathways will not only enhance our understanding of DNA damage, but will also provide effective drug targets for better understanding of human health and disease.

Disclosure of Invention

Aiming at how to determine the screening of the core molecules of the DNA damage signal channel in the prior art, the invention provides a screening reagent for detecting the core molecules of the DNA damage signal channel and application thereof, screens corresponding molecular biology core molecules, and can explain the change of the core molecules in drug treatment.

In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:

a screening reagent for detecting DNA damage signal channel core molecules comprises the following primers:

(1) the PCR reaction primers for detecting the ABL1 gene have the following primer sequences:

5'-ATCACGCCAGTCAACAGTC-3'(SEQ ID NO:1)；

5'-TACACCCTCCCTTCGTATC-3'(SEQ ID NO:2)；

(2) the PCR reaction primers for detecting the APEX1 gene have the following primer sequences:

5'-TTTCTTACGGCATAGGCGA-3'(SEQ ID NO:3)；

5'-ACAAACGAGTCAAATTCAGC-3'(SEQ ID NO:4)；

(3) the PCR reaction primer for detecting the ATM gene has the following primer sequence:

5'-TGATCTTGTGCCTTGGCTA-3'(SEQ ID NO:5)；

5'-ATGGTGTACGTTCCCCATG-3'(SEQ ID NO:6)；

(4) the PCR reaction primer for detecting the ATR gene has the following primer sequence:

5'-CCCTTGAATACAGTGGCCT-3'(SEQ ID NO:7)；

5'-CCTTGAAAGTACGGCAGTT-3'(SEQ ID NO:8)；

(5) the primer sequence of the PCR reaction primer for detecting the ATRIP gene is as follows:

5'-TGAACGAGCAAATAAACTGG-3'(SEQ ID NO:9)；

5'-AGGCTTGTATCCTGACTCC-3'(SEQ ID NO:10)；

(6) The primer sequence of the PCR reaction primer for detecting the ATRX gene is as follows:

5'-GTCACTGCATGTAACAGCG-3'(SEQ ID NO:11)；

5'-GGCACAATTAGTGCGGAATA-3'(SEQ ID NO:12)；

(7) PCR reaction primers for detecting the BARD1 gene have the following primer sequences:

5'-AGTGGCTCCTTGACAGAATC-3'(SEQ ID NO:13)；

5'-GTCTGGAGACTCTATTTGCTC-3'(SEQ ID NO:14)；

(8) the PCR reaction primer for detecting the BAX gene has the following primer sequence:

5'-CCGAGAGGTCTTTTTCCGA-3'(SEQ ID NO:15)；

5'-CAGCCCATGATGGTTCTGA-3'(SEQ ID NO:16)；

(9) the PCR reaction primers for detecting the BBC3 gene have the following primer sequences:

5'-ACCTCAACGCACAGTACGA-3'(SEQ ID NO:17)；

5'-GGAGTCCCATGATGAGATTG-3'(SEQ ID NO:18)；

(10) the PCR reaction primer for detecting the BLM gene has the following primer sequence:

5'-GACCTTGACACCTCTGACA-3'(SEQ ID NO:19)；

5'-GATTCAGCTCCTGCATACTC-3'(SEQ ID NO:20)；

(11) the PCR reaction primers for detecting BRCA1 gene have the following primer sequences:

5'-TGTTACAAATCACCCCTCAAG-3'(SEQ ID NO:21)；

5'-CCTGATACTTTTCTGGATGC-3'(SEQ ID NO:22)；

(12) the PCR reaction primers for detecting the BRIP1 gene have the following primer sequences:

5'-GAAACAGTCAAGAGTCATCGA-3'(SEQ ID NO:23)；

5'-CTGAGCAATCTGCTTGTGT-3'(SEQ ID NO:24)；

(13) PCR primers for detecting CDC25A gene have the following primer sequences:

5'-TCCTCTTTTTACACCCCAGTC-3'(SEQ ID NO:25)；

5'-CGGTTGTCAAGGTTTGTAGTT-3'(SEQ ID NO:26)；

(14) PCR primers for detecting CDC25C gene have the following primer sequences:

5'-AGTGGCCTATATCGCTCC-3'(SEQ ID NO:27)；

5'-CCTGGTTAGAATCTTCCTCC-3'(SEQ ID NO:28)；

(15) PCR primers for detecting CDK7 gene have the following primer sequences:

5'-GTATGGTGTAGGTGTGGAC-3'(SEQ ID NO:29)；

5'-GCAAAGGTATTCCAGGGAAA-3'(SEQ ID NO:30)；

(16) the PCR reaction primers for detecting the CDKN1A gene have the following primer sequences:

5'-GATGGAACTTCGACTTTGTC-3'(SEQ ID NO:31)；

5'-CACAAGGGTACAAGACAGT-3'(SEQ ID NO:32)；

(17) the PCR reaction primers for detecting the CHEK1 gene have the following primer sequences:

5'-CTTACTGCAATGCTCGCTG-3'(SEQ ID NO:33)；

5'-TGAGGGGTTTGTTGTACCAT-3'(SEQ ID NO:34)；

(18) the PCR reaction primers for detecting the CHEK2 gene have the following primer sequences:

5'-TATCTGCCTTAGTGGGTATCC-3'；(SEQ ID NO:35)；

5'-TGTCGTAAAACGTGCCTTT-3'；(SEQ ID NO:36)；

(19) the PCR reaction primers for detecting the CIB1 gene have the following primer sequences:

5'-ACGGCTTAGTGCGTCTGA-3'；(SEQ ID NO:37)；

5'-AAGTCTGGAGAACGGGAGA-3'；(SEQ ID NO:38)；

(20) the PCR reaction primers for detecting the CRY1 gene have the following primer sequences:

5'-TGGACAACCGCCTCTAACT-3'；(SEQ ID NO:39)；

5'-CCAGTGAAGGGACTCCATAT-3'；(SEQ ID NO:40)；

(21) the PCR reaction primers for detecting the CSNK2A2 gene have the following primer sequences:

5'-AAAGCTGCGACTGATAGATTG-3'；(SEQ ID NO:41)；

5'-AGGCTACACGAACATTGTACT-3'；(SEQ ID NO:42)；

(22) The PCR reaction primers for detecting the DDB1 gene have the following primer sequences:

5'-TTGACAGCGAAGTACAATGC-3'；(SEQ ID NO:43)；

5'-ATGGGCTCGCGTAATGATG-3'；(SEQ ID NO:44)；

(23) the PCR reaction primers for detecting the DDB2 gene have the following primer sequences:

5'-CTTCATCAAAGGGATTGGAG-3'；(SEQ ID NO:45)；

5'-TGAGGAGGCGTAAAACTGG-3'；(SEQ ID NO:46)；

(24) the PCR reaction primers for detecting the DDIT3 gene have the following primer sequences:

5'-AACGGCTCAAGCAGGAAAT-3'；(SEQ ID NO:47)；

5'-TCACCATTCGGTCAATCAGA-3'；(SEQ ID NO:48)；

(25) the PCR reaction primers for detecting the ERCC1 gene have the following primer sequences:

5'-TTGGCGACGTAATTCCCGA-3'；(SEQ ID NO:49)；

5'-CTGCTGGGGATCTTTCAC-3'；(SEQ ID NO:50)；

(26) the PCR reaction primers for detecting the ERCC2 gene have the following primer sequences:

5'-TCGATGGGAAATGCCACA-3'；(SEQ ID NO:51)；

5'-TCATCCAGGTTGTAGATGC-3'；(SEQ ID NO:52)；

(27) the primer sequence of the PCR reaction primer for detecting the EXO1 gene is as follows:

5'-CTCGTGGCTCCCTATGAA-3'；(SEQ ID NO:53)；

5'-GGAGATCCGAGTCCTCTGTA-3'；(SEQ ID NO:54)；

(28) the primer sequence of the PCR reaction primer for detecting the FANCA gene is as follows:

5'-GCACACAGTATGTTCTCCC-3'；(SEQ ID NO:55)；

5'-TGTACGTGAAGATGCCACA-3'；(SEQ ID NO:56)；

(29) the primer sequence of the PCR reaction primer for detecting the FANCD2 gene is as follows:

5'-TTCGCCAGTTGGTGATGGA-3'；(SEQ ID NO:57)；

5'-GGAAGCCTGTAACCGTGA-3'；(SEQ ID NO:58)；

(30) the primer sequence of the PCR reaction primer for detecting the FANCG gene is as follows:

5'-AGGCTCTATCAGCAACTGG-3'；(SEQ ID NO:59)；

5'-AACTGCGGGGCTTTGGA-3'；(SEQ ID NO:60)；

(31) the PCR reaction primers for detecting the FEN1 gene have the following primer sequences:

5'-TCTTGATGCACCCAGTGAG-3'；(SEQ ID NO:61)；

5'-AGCCGCAGCATAGACTTTG-3'；(SEQ ID NO:62)；

(32) the PCR reaction primers for detecting the GADD45A gene have the following primer sequences:

5'-CCTGATCCAGGCGTTTT-3'；(SEQ ID NO:63)；

5'-ATCCATGTAGCGACTTTCC-3'；(SEQ ID NO:64)；

(33) the PCR reaction primers for detecting the GADD45G gene have the following primer sequences:

5'-AGATCCATTTTACGCTGATCC-3'；(SEQ ID NO:65)；

5'-CCTCGCAAAACAGGCTGA-3'；(SEQ ID NO:66)；

(34) the PCR reaction primers for detecting the H2AX gene have the following primer sequences:

5'-AACGACGAGGAGCTCAACAA-3'；(SEQ ID NO:67)；

5'-GCGGGCCCTCTTAGTACTCC-3'；(SEQ ID NO:68)；

(35) the PCR reaction primers for detecting the HUS1 gene have the following primer sequences:

5'-AATGCCAGGGCTTTGAAAAT-3'；(SEQ ID NO:69)；

5'-ACAATGCGGCTACTGCTT-3'；(SEQ ID NO:70)；

(36) the PCR reaction primers for detecting the LIG1 gene have the following primer sequences:

5'-CAGTTCCCCATCAGGGATT-3'；(SEQ ID NO:71)；

5'-TCTGTGAGGCTTTCTTTCG-3'；(SEQ ID NO:72)；

(37) the primer sequence of the PCR reaction primer for detecting the MAPK12 gene is as follows:

5'-ATGAGAAGCTAGGCGAGGA-3'；(SEQ ID NO:73)；

5'-AGCGTGGATATACCTCAGC-3'；(SEQ ID NO:74)；

(38) The PCR reaction primers for detecting the MBD4 gene have the following primer sequences:

5'-GGCAAAATGGCAATACCTG-3'；(SEQ ID NO:75)；

5'-TCTGCGGTTCTTGCTACCT-3'；(SEQ ID NO:76)；

(39) the PCR reaction primers for detecting the MCPH1 gene have the following primer sequences:

5'-TAGTCACCCCTGACCAAAA-3'；(SEQ ID NO:77)；

5'-CAGCCTCGGCATGATAG-3'；(SEQ ID NO:78)；

(40) the PCR reaction primer for detecting MDC1 gene has the following primer sequence:

5'-GTGGGAGCCTTAATGGTAC-3'；(SEQ ID NO:79)；

5'-TCCCTCAGACGGTGACT-3'；(SEQ ID NO:80)；

(41) the PCR reaction primers for detecting MLH1 gene have the following primer sequences:

5'-CAAACCCCTGTCCAGTCA-3'；(SEQ ID NO:81)；

5'-TGGGAGTTCAAGCATCTCC-3'；(SEQ ID NO:82)；

(42) the PCR reaction primers for detecting MLH3 gene have the following primer sequences:

5'-ACGTATGTTCCCGATTTTGTC-3'；(SEQ ID NO:83)；

5'-CTTCAGAGCTGATATAGCCAC-3'；(SEQ ID NO:84)；

(43) the PCR reaction primers for detecting MPG gene have the following primer sequences:

5'-AATGGCACAGAACTCCGAG-3'；(SEQ ID NO:85)；

5'-ATGCAGAAGTACATGCCGT-3'；(SEQ ID NO:86)；

(44) the PCR reaction primers for detecting the MRE11A gene have the following primer sequences:

5'-GGGCAGATGCACTTTGT-3'；(SEQ ID NO:87)；

5'-AAGCAAAACCGGACTAATGTC-3'；(SEQ ID NO:88)；

(45) the PCR reaction primers for detecting the MSH2 gene have the following primer sequences:

5'-GTCAGAGCCCTTAACCTTTTT-3'；(SEQ ID NO:89)；

5'-AGAGGCTGCTTAATCCACT-3'；(SEQ ID NO:90)；

(46) the PCR reaction primers for detecting the MSH3 gene have the following primer sequences:

5'-TACCAGCTATCTTCTGTGCAT-3'；(SEQ ID NO:91)；

5'-CTCTGTTTGCTCGGACAA-3'；(SEQ ID NO:92)；

(47) the PCR reaction primer for detecting NBN gene has the following primer sequence:

5'-GGTGGGGAAGCTAGGTTGA-3'；(SEQ ID NO:93)；

5'-ACCGCCAATCCAATTTCTG-3'；(SEQ ID NO:94)；

(48) the PCR reaction primer for detecting NTHL1 gene has the following primer sequence:

5'-TACCCCGTCGGTTTCTG-3'；(SEQ ID NO:95)；

5'-GATGTCCCCACCGTAGT-3'；(SEQ ID NO:96)；

(49) PCR primers for detecting the OGG1 gene have the following primer sequences:

5'-CTCCCACTTCCAAGAGGT-3'；(SEQ ID NO:97)；

5'-GATGAGCCGAGGTCCAAAA-3'；(SEQ ID NO:98)；

(50) the PCR reaction primers for detecting the PARP1 gene have the following primer sequences:

5'-CTGAGCTTCGGTGGGATG-3'；(SEQ ID NO:99)；

5'-TGGCATACTCTGCTGCAAA-3'；(SEQ ID NO:100)；

(51) the PCR reaction primer for detecting the PCNA gene has the following primer sequence:

5'-CACTAAGGGCCGAAGATAAC-3'；(SEQ ID NO:101)；

5'-CAGCATCTCCAATATGGCTG-3'；(SEQ ID NO:102)；

(52) the PCR reaction primers for detecting the PMS1 gene have the following primer sequences:

5'-CTTACGGTTTTCGTGGAGAA-3'；(SEQ ID NO:103)；

5'-GCAGCCGTTCTTGTTGTAA-3'；(SEQ ID NO:104)；

(53) the PCR reaction primers for detecting the PMS2 gene have the following primer sequences:

5'-TTGCCGACCTAACTCAGGT-3'；(SEQ ID NO:105)；

5'-GATGCGTGGCAGGTAGA-3'；(SEQ ID NO:106)；

(54) The PCR reaction primer for detecting the PNKP gene has the following primer sequence:

5'-AAGCGTATGCGGAAGTCAA-3'；(SEQ ID NO:107)；

5'-CGTCCCGTCCAGATCAA-3'；(SEQ ID NO:108)；

(55) the PCR reaction primers for detecting the PPM1D gene have the following primer sequences:

5'-TGGTCATCATTCGGGGCA-3'；(SEQ ID NO:109)；

5'-ATCCTTCGGGTCATCCTGA-3'；(SEQ ID NO:110)；

(56) the PCR reaction primers for detecting PPP1R15A gene have the following primer sequences:

5'-GTGCCAACCCAGTGATGA-3'；(SEQ ID NO:111)；

5'-GACACCTGTAGCAGGAGTG-3'；(SEQ ID NO:112)；

(57) the PCR reaction primer for detecting the PRKDC gene has the following primer sequence:

5'-GCTGGCTTGCGCCTATT-3'；(SEQ ID NO:113)；

5'-GGCACACCACTTTAACAAGA-3'；(SEQ ID NO:114)；

(58) the primer sequence of the PCR reaction primer for detecting the RAD1 gene is as follows:

5'-TATGCCAGGGACTTTAACTG-3'；(SEQ ID NO:115)；

5'-GGACTTCACTCGTCATATCC-3'；(SEQ ID NO:116)；

(59) the primer sequence of the PCR reaction primer for detecting the RAD17 gene is as follows:

5'-AAGCCCGAGGATATGCTCA-3'；(SEQ ID NO:117)；

5'-CAGGCAATTTTCCCGATACT-3'；(SEQ ID NO:118)；

(60) the primer sequence of the PCR reaction primer for detecting the RAD18 gene is as follows:

5'-GAGATCGCTCCAGATCCCT-3'；(SEQ ID NO:119)；

5'-CGCAAACAGGACAATCCA-3'；(SEQ ID NO:120)；

(61) the primer sequence of the PCR reaction primer for detecting the RAD21 gene is as follows:

5'-GCCTGCACATGACGATATG-3'；(SEQ ID NO:121)；

5'-TGGTTGGCATTGGTTCAAC-3'；(SEQ ID NO:122)；

(62) the primer sequence of the PCR reaction primer for detecting the RAD50 gene is as follows:

5'-AAGTGTGCAGAAATTGACCGA-3'；(SEQ ID NO:123)；

5'-ACGTACCTGCCGAAGTGT-3'；(SEQ ID NO:124)；

(63) the primer sequence of the PCR reaction primer for detecting the RAD51 gene is as follows:

5'-GAGCGTTCAACACAGACC-3'；(SEQ ID NO:125)；

5'-TGGCACTGTCTACAATAAGC-3'；(SEQ ID NO:126)；

(64) the primer sequence of the PCR reaction primer for detecting the RAD51B gene is as follows:

5'-AGAGTCTGCATTTAGTGCTG-3'；(SEQ ID NO:127)；

5'-CAGGTGAGTTCCCGATAAAGA-3'；(SEQ ID NO:128)；

(65) the primer sequence of the PCR reaction primer for detecting the RAD9A gene is as follows:

5'-ATTGACTCTTACATGATCGCC-3'；(SEQ ID NO:129)；

5'-CCAGGTGAAAGGGAAATG-3'；(SEQ ID NO:130)；

(66) the PCR reaction primers for detecting the RBBP8 gene have the following primer sequences:

5'-AAATATCCGGCAGCAGAATCT-3'；(SEQ ID NO:131)；

5'-GCTGTTATCGGTGAATCTGG-3'；(SEQ ID NO:132)；

(67) the PCR reaction primers for detecting the REV1 gene have the following primer sequences:

5'-TTCCGCTGAGGAATTGAGAA-3'；(SEQ ID NO:133)；

5'-GAGAGGAGTCGTCCAGCT-3'；(SEQ ID NO:134)；

(68) the primer sequence of the PCR reaction primer for detecting the RNF168 gene is as follows:

5'-CAACGTGGAACTGTGGAC-3'；(SEQ ID NO:135)；

5'-AGGTTTACTGAGCAGACGAA-3'；(SEQ ID NO:136)；

(69) the primer sequence of the PCR reaction primer for detecting the RNF8 gene is as follows:

5'-CGCGTCTGGAACCTTTAA-3'；(SEQ ID NO:137)；

5'-AGGACCTGCTAATTCATCCAA-3'；(SEQ ID NO:138)；

(70) The PCR reaction primers for detecting the RPA1 gene have the following primer sequences:

5'-TGGACCATTTGTGCTCGT-3'；(SEQ ID NO:139)；

5'-TCGTCAACCAGTTCTAGGG-3'；(SEQ ID NO:140)；

(71) the PCR reaction primers for detecting the SIRT1 gene have the following primer sequences:

5'-GTGTCATAGGTTAGGTGGTG-3'；(SEQ ID NO:141)；

5'-GCCAATTCTTTTTGTGTTCGT-3'；(SEQ ID NO:142)；

(72) the PCR reaction primers for detecting the SMC1A gene have the following primer sequences:

5'-TGGCGCAGGAGTATGACAA-3'；(SEQ ID NO:143)；

5'-CCGTACTACCTCATCCTTC-3'；(SEQ ID NO:144)；

(73) the PCR reaction primers for detecting the SUMO1 gene have the following primer sequences:

5'-GACCAGGAGGCAAAACCTT-3'；(SEQ ID NO:145)；

5'-ATTCATTGGAACACCCTGTCT-3'；(SEQ ID NO:146)；

(74) the primer sequence of the PCR reaction primer for detecting the TOPBP1 gene is as follows:

5'-GAGTGTGCCAAGAGATGGA-3'；(SEQ ID NO:147)；

5'-GCTTCTGGTCTAGGTTCTG-3'；(SEQ ID NO:148)；

(75) the primer sequence of the PCR reaction primer for detecting the TP53 gene is as follows:

5'-CAGCTTTGAGGTGCGTGTT-3'；(SEQ ID NO:149)；

5'-CCTTTCTTGCGGAGATTCTC-3'；(SEQ ID NO:150)；

(76) the primer sequence of the PCR reaction primer for detecting the TP53BP1 gene is as follows:

5'-GAGCAGTTACCTCAGCCAA-3'；(SEQ ID NO:151)；

5'-AGGGAATGTGTAGTATTGCCT-3'；(SEQ ID NO:152)；

(77) the primer sequence of the PCR reaction primer for detecting the TP73 gene is as follows:

5'-ACGAGGACACGTACTACCT-3'；(SEQ ID NO:153)；

5'-TGCCGATAGGAGTCCACC-3'；(SEQ ID NO:154)；

(78) the PCR reaction primer for detecting the UNG gene has the following primer sequence:

5'-GGCAGTGCCATTGATAGGA-3'；(SEQ ID NO:155)；

5'-TGTCTACATCCAAAGAACCCT-3'；(SEQ ID NO:156)；

(79) the PCR reaction primer for detecting the XPA gene has the following primer sequence:

5'-CAGGACCTGTTATGGAATTTG-3'；(SEQ ID NO:157)；

5'-CTTCTTGACTACCCCAAACTT-3'；(SEQ ID NO:158)；

(80) the PCR reaction primer for detecting the XPC gene has the following primer sequence:

5'-ATCGTGGGAGCCATCGTAA-3'；(SEQ ID NO:159)；

5'-TCACCATCGCTGCACATTT-3'；(SEQ ID NO:160)；

(81) the PCR reaction primers for detecting the XRCC1 gene have the following primer sequences:

5'-CTTTGGCTTGAGTTTTGTAC-3'；(SEQ ID NO:161)；

5'-CTCCTTCACACGGAACTG-3'；(SEQ ID NO:162)；

(82) the PCR reaction primers for detecting the XRCC2 gene have the following primer sequences:

5'-CTGGATAGACCGCGTCAAT-3'；(SEQ ID NO:163)；

5'-TCGAGAGGCATGAGAAGG-3'；(SEQ ID NO:164)；

(83) the PCR reaction primers for detecting the XRCC3 gene have the following primer sequences:

5'-CCCATTCCGCTGTGAATTT-3'；(SEQ ID NO:165)；

5'-GTTAGCCCAGGTTATGCC-3'；(SEQ ID NO:166)；

(84) the PCR reaction primers for detecting the XRCC6 gene have the following primer sequences:

5'-TGCTTCTGCCTAGCGATAC-3'；(SEQ ID NO:167)；

5'-AACCTGGATCATCAAACCGT-3'；(SEQ ID NO:168)；

(85) the PCR reaction primers for detecting the ACTB gene have the following primer sequences:

5'-GTCTGCCTTGGTAGTGGATAAT-3'；(SEQ ID NO:169)；

5'-TCGAGGACGCCCTATCATG-3'；(SEQ ID NO:170)；

(86) The PCR reaction primers for detecting the B2M gene have the following primer sequences:

5'-CCAAGGAAGGCGTCTAAGG-3'；(SEQ ID NO:171)；

5'-CTTTCGAGCGCAACCACTTT-3'；(SEQ ID NO:172)；

(87) the PCR reaction primers for detecting the GAPDH gene have the following primer sequences:

5'-GGAGCGAGATCCCTCCAAAA-3'；(SEQ ID NO:173)；

5'-GGCTGTTGTCATACTTCTCATG-3'；(SEQ ID NO:174)；

(88) the PCR reaction primers for detecting the HPRT1 gene have the following primer sequences:

5'-CCTGGCGTCGTGATTAGTGA-3'；(SEQ ID NO:175)；

5'-AGACGTTCAGTCCTGTCCATA-3'；(SEQ ID NO:176)；

(89) the PCR reaction primers for detecting the RPLP0 gene have the following primer sequences:

5'-AGCCCAGAACACTGGTCT-3'；(SEQ ID NO:177)；

5'-ACTCAGGATTTCAATGGTGC-3'。(SEQ ID NO:178)。

the invention also provides application of the screening reagent in preparing a detection reagent for detecting the cell line DNA damage signal channel core molecule.

Further: the detection reagent adopts a real-time fluorescent quantitative PCR method for detection.

Further: the reaction system of the real-time fluorescent quantitative PCR is as follows: 10 mul of Taq polymerase reaction mixture, 0.1-0.2 mul of upstream primer, 0.1-0.2 mul of downstream primer, 25-100ng of cDNA template, and sterile distilled water to make up to 20 mul.

Further: the Taq polymerase is TaKaRa

Premix Ex Taq polymerase.

Further: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 5 seconds, 59 ℃ for 20 seconds, 40 cycles. The dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.

The PCR reaction instrument is LightCycler480 of Roche. The 89 pairs of primers were placed in a 96-well plate for real-time fluorescent quantitative PCR detection. The change of the target core molecule of the liver cancer drug can be obtained through one reaction.

Compared with the prior art, the invention has the advantages and positive effects that: the invention provides a reagent for detecting molecular change of a DNA damage signal path, which can be used for rapidly detecting genes related to DNA damage at a transcription level through real-time fluorescence quantification, and analyzing and researching core molecules related to DNA damage and action mechanisms on the basis, thereby rapidly and accurately finding a core regulation signal path of a tumor-related disease target drug, and providing a powerful tool for screening anticancer drugs, mechanism discussion of new targeted drugs and the like.

The invention determines 84 genes related to a DNA damage signal path through a large number of creative experiments, and the invention focuses on the combination of primers, and experiments prove that the conditions of primer dimer and non-specific amplification during PCR amplification can be avoided only through proper primer combination, thereby ensuring the effective amplification of all target genes. The primer dimer often appears in the combination of any primer during the specific PCR, which affects the failure of the target gene amplification, or the non-specific amplification, which greatly affects the effectiveness of the PCR result. Under the same experimental condition, PCR detection of 89 genes (84 of which are detection genes and 5 of which are reference genes) is carried out simultaneously, so that not only is primer design required, but also the optimal amplification condition is required to be well grasped, which is a technical difficulty. The most central is primer design, the invention obtains the best amplification condition through multiple pcr and adjustment, and ensures that the amplification efficiency of each gene is very high without nonspecific amplification.

The invention also provides a PCR method for detection, the experimental system can be carried out on any real-time fluorescence quantitative PCR instrument, the experimental operation is simple and convenient, the cost is low, the result repeatability is good, the unnecessary detection of the downstream can be reduced, and the method is an important means for researching the screening action mechanism of the target drug.

Other features and advantages of the present invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

Drawings

FIG. 1 shows the total RNA extraction of the differential expression group of liver cancer cell Huh7 treated by SR-4835 and untreated liver cancer cell Huh7, wherein 1: SR-4835-treated Huh7 cells; 2: hepatoma cell Huh 7.

FIG. 2 shows the real-time fluorescent quantitative PCR detection of the expression of the DNA damage signaling pathway molecule of Huh7 hepatoma carcinoma cells treated by SR-4835.

FIG. 3 shows the expression of H2AFX protein following SR-4835 treatment of Huh7 cells.

FIG. 4 shows the detection of H2AFX protein expression after SR-4835 treatment of Huh7 cells.

FIG. 5 shows that CCK-8 detects the effect of SR-4835 target molecule H2AFX on liver cancer cell proliferation.

FIG. 6 shows Edu staining to detect the effect of DNA repair target core molecule on liver cancer Huh7 cell proliferation.

FIG. 7 shows the cloning formation to detect the effect of DNA repair core molecules on the proliferation of hepatoma cells.

FIG. 8 shows the effect of flow cytometry on the apoptosis of hepatoma cells by DNA repair target molecule H2 AFX.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and the detailed description.

Example 1

The invention is exemplified by human liver cancer Huh7 and liver cancer cell Huh7 processed by SR-4835, but the invention is not limited to the two cells, and the detection reagent can be applied to liver cancer cell lines such as SMMC7721 and HCCLM3, and can also detect DNA damage signals of other tumors such as clinical liver cancer tissues and tissues beside the cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer and the like.

The change of the core molecules in the treated cell line is detected by culturing a human liver cancer cell line Huh7, then treating the cell line with SR-4835(100nM) for 24 hours and using the screening reagent of the DNA damage signal path.

The human liver cancer Huh7 used in the present invention was purchased from ATCC company of USA, Shanghai cell bank, DMEM medium, fetal bovine serum were purchased from Gibco company of USA, RNAiso for extracting total RNA, reverse transcription kit,

the Premix Ex Taq kit was purchased from TaKaRa.

The core molecule for detecting the DNA damage signal path target by using the screening reagent comprises the following specific steps:

First, culture and passage of human liver cancer Huh7 cell line

Adding 1mL of 0.2% pancreatin solution into a 60mm cell culture plate, slightly shaking to ensure that the pancreatin is fully contacted with cells, incubating in a carbon dioxide incubator at 37 ℃ for 2min, observing most cells floating under a mirror, adding 4mL of DMEM medium containing 10% FBS, uniformly blowing, adding into a 15mL centrifugal tube, centrifuging for 6min at 2000g, discarding supernatant, adding corresponding culture solution with proper volume according to cell types, and repeatedly blowing and beating for several times by using a 10mL pipette to enable the cells to be free. In this process, air bubbles are prevented from being generated, so as not to influence the cell count. Taking 20 mu L of cell suspension, dripping into a cell counting plate, and counting the total number of cells in four big grids at four corners according to a cell counting method. When counting, only cells with intact cell morphology are counted, and the number of stacked cells is counted as 1 cell.

The calculation formula is as follows:

cell concentration (one/mL) ═ 4 big grid cell count × 10⁴X dilution factor/4.

Diluting to 5 × 10 with corresponding culture solution according to cell type⁶Adding 2mL of cell dilution into 60mm cell culture dish, placing at 37 deg.C and 5% CO₂The cells are cultured in a cell culture box, the cells are attached to the wall after 0.5h-1h generally, the cells begin to grow after a plurality of hours, and the cells become monolayer cells after 24h-48 h.

Huh7 cells were treated with SR-4835 (final concentration of 100nM) added to the medium, changed medium, and then incubated with controls for 24 hours.

Second, extraction of Total RNA

1) Taking CO₂After sucking out the culture medium from a disk of cells in a 60mm plate in logarithmic phase in an incubator, washing the disk of cells for 2 times by using sterile 1 XPBS, adding 1mL of RNAasso on the surfaces of the cells, repeatedly sucking the cells uniformly by using a gun head, and transferring the cells into a sterile tube.

2) Incubate at room temperature for 5min to solubilize the nucleoprotein. Add 0.2mL phenol/chloroform (1:1), shake vigorously for 15sec, incubate at room temperature for 2-3 min.

3) Cells were stratified by centrifugation at 12000g for 15min at 4 ℃.

4) Transferring the upper layer liquid to a centrifuge tube without RNase, adding 0.5mL isopropanol, reversing and mixing uniformly for 3 times, and incubating for 10min at room temperature.

5) Centrifugation at 12000g for 10min at 4 ℃ resulted in the formation of a bottom pellet of RNA.

6) The supernatant was discarded and the pellet was washed with 1mL of RNase-free 75% ethanol. Centrifuge at 7000g for 5min at 4 ℃.

8) The RNA was air dried for about 5-10 min. It should not be dried too much.

9) RNA was dissolved in 30-50. mu.L DEPC water.

The results are shown in FIG. 1, and it can be seen from the electrophoresis in FIG. 1 that lane 1 is Huh7, lane 2 is normal liver cell, the total RNA band is complete, and it is suitable for downstream reverse transcription and other experiments.

Synthesis of cDNA

1) The following reaction mixture was added to a pre-cooled tube:

1-5 mug of total RNA; oligo (dT) (0.5. mu.g/. mu.l) 1. mu.L; diluting to 12 μ L with RNase-free water;

2) mixing, and centrifuging for 3-5 sec;

3) acting at 70 deg.C for 5min, ice-cooling for 30sec, and centrifuging for 3-5 sec;

4) the tube was ice-cooled and the following components were added: 5 × Reaction buffer4 μ L; 1 muL of RNase inhibitor (20U/muL); dNTP mix (10mM) 2. mu.L;

5) mixing, and centrifuging for 3-5 sec;

6) adding 1 μ L M-MuLV Reverse Transcriptase (20U/. mu.L) in water bath at 37 deg.C for 5min, and mixing;

7) acting at 37 deg.C for 60 min. The reaction was terminated by incubation at 70 ℃ for 10min and downstream testing was performed on ice.

Four, real-time fluorescent quantitative PCR

Taking the synthesized cDNA as a template, and carrying out real-time fluorescence quantitative PCR reaction according to the following system:

premix Ex Taq 10. mu.L; upstream primer 0.1-0.2 μmol; downstream primer 0.1-0.2 μmol; 25-100ng of cDNA template; DW made up the volume to 20. mu.L.

The following program was set up on the LightCycler480 instrument from roche: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 ℃ for 5 seconds, 59 ℃ for 20 seconds, 40 cycles. The dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.

The screening reagent for detecting the DNA damage signal path is the screening reagent (comprising 84 primers of detection genes and 5 primers of reference genes) in the invention content, and the sequence is shown as SEQ ID NO. 1-SEQ ID NO. 178.

And (3) placing the primers on a 96-well plate to perform PCR reaction, wherein each pair of primers is placed in any one well, and 89 pairs of primers are placed in 89 wells in total.

Fifth, data analysis

And calculating the expression difference of the target core gene of the liver cancer drug by using the Ct value of each gene. Compared with a control Huh7, the expression of CDK7, CHEK1, CHEK2 and the like is obviously reduced, and the expression of H2AFX and the like is obviously increased in the Huh7 cells of the SR-4835 treatment group, and the existing research shows that the genes of CDK7, H2AFX and the like have obvious correlation with DNA damage repair, so that the screening reagent can be used for rapidly and effectively screening molecules related to DNA damage.

Sixthly, detecting the expression of the DNA damage response factor H2AFX by using Western blot

1. Collecting protein samples

Adherent cells were lysed using RIPA lysate as required for the experiment, and protease inhibitors, phosphatase inhibitors or deacetylase inhibitor cocktail were additionally added to prevent degradation of proteins.

After the protein samples are collected, the protein concentration of each protein sample needs to be determined in order to ensure that the loading amount of each protein sample is consistent. Protein concentration was detected using the BCA protein concentration assay kit.

2. Electrophoresis

Preparing polyacrylamide gel, 12% of separation gel and 5% of lamination gel.

100ml of Bio-Rad 10 Xelectrophoresis buffer was dissolved in 900ml of distilled water to prepare 1L of electrophoresis buffer, which was prepared as it is.

Prepared as lx buffer (19 × 100ml of stock buffer solution of transmembrane buffer +200ml of methanol (analytically pure) +700ml of deionized water); 100ml 10xTBS buffer from Biochemical company was dissolved in 900ml distilled water to prepare 1L of 1 xTBS buffer, and 1ml Tween-20 was added to prepare L x TBST buffer.

0.5ml of skimmed milk and 9.5ml of TBST solution are prepared into a confining liquid containing 5 percent of skimmed milk, and the confining liquid is prepared for use.

0.5ml of skimmed milk and 9.5ml of TBST are prepared into an antibody diluent containing 5% of skimmed milk, wherein the primary antibody diluent contains 0.002g of sodium azide, and the secondary antibody diluent does not contain, so that the antibody diluent is prepared for use.

And putting the prefabricated gel into an electrophoresis device, immersing the electrophoresis device in lx electrophoresis liquid, and checking to determine that no liquid leakage exists.

The power supply is switched on, and the electrophoresis is carried out for 15min at a constant voltage of 80V.

Add 20. mu.g of protein to the loading well and electrophoresis marker to the very edge of the lane.

The power is switched on, and the electrophoresis is carried out for 30min at a constant voltage of 80V and then for 1h at 120V.

And finishing electrophoresis when the lowest color band on the prefabricated glue is 1-2cm away from the lower edge of the glue, and preparing to transfer the film.

3. Rotary film

A PVDF membrane of the same size as the gel was cut out and soaked in anhydrous methanol for 10 seconds.

And sealing the rotary template and then placing the rotary template into a rotary film groove. Adding about 1000ml of pre-cooled membrane transferring liquid with 4 ℃ into the membrane transferring groove, and putting an ice bag into the membrane transferring groove to maintain the low temperature of the whole process.

The electrophoresis tank was placed in ice cubes. And (5) rotating the membrane for 90min at a constant current of 0.2A.

4. Sealing of

Immersing PVDF membrane in 5% skimmed milk, and shaking for 90min at room temperature.

5. Hybridization of

The blocked PVDF membrane was washed with PBST and immersed in primary antibody overnight in a shaker at 4 ℃ for 30min the next day at room temperature. PBST was washed 3 times for 15min each. The secondary antibody was immersed for 1h and washed 3 times with PBST, 15min each time.

6. Development

According to the ECL instruction, the solution A and the solution B are added into a 15ml centrifuge tube according to the proportion of 1:1, mixed by turning upside down for several times, and kept standing for 5min in a dark place. The ECL mixed solution is uniformly dripped on the membrane (preferably 1000ul of mixed solution per membrane), and the membrane is sealed by the preservative film and fixed in a dark box. The film was quickly covered on top of the ECL film in a dark room, the cassette was closed and exposed to light according to the intensity of the fluorescence seen (1s-30 min).

Taking out the film, immediately and completely immersing the film into the developing solution for 1min, rinsing the film for 30s with clear water, immersing the film into the fixing solution, completely fixing the film after about 30s, rinsing the film with clear water, drying the film, calibrating the size of the molecular weight of the strip according to the position of a Marker, and analyzing and scanning the film.

Research has shown that H2AFX plays an important role in DNA damage signaling pathway, and H2AFX can be used as a marker of DNA Double Strand Break (DSB) reaction. It is controlled by physiological processes in DNA damage repair, and may also respond during replication collapse or in response to ionizing radiation. H2AFX contributes to nucleosome formation, chromatin remodeling and DNA repair, and can also be used in vitro as a method of detection of double strand breaks in dsDNA. Therefore, Western blot is adopted to detect the expression of H2AFX under the treatment of SR-4835, and the result is shown in figure 3, and the expression of H2AFX is obviously improved after Huh7 liver cancer cells are treated by SR-4835. The result further proves that the DNA repair related protein specificity obtained by adopting the screening reagent is suitable for screening the drug sensitivity target of the tumor cells.

Seventhly, detecting the expression of the H2AFX protein by immunofluorescence

1. Sample preparation

Cells can be directly cultured by using a 24-well plate (a glass slide special for laser confocal is added in a culture hole), after the cells adhere to the wall, SR-4835(100nM) is added for incubation, and then the subsequent operations such as fixation and the like are carried out after the preset time.

2. Fixing

The cells can be fixed by using immunostaining fixative (P0098) produced in Biyun day, and after fixation, the cells can be washed twice with PBS for 5 minutes each time.

3. Sealing of

Adding the immunostaining sealing liquid, and sealing for 60 minutes.

All steps from the closing are carried out, and the moisture retention of the sample is necessarily required to avoid the drying of the sample, otherwise, a higher background is easily generated.

4. Primary antibody incubation

Primary antibodies were diluted with immunostaining primary antibody diluent at the appropriate 1:50 ratio, according to the instructions for primary H2 AFX.

The blocking solution was sucked up using a mini bench vacuum pump, diluted primary antibody was immediately added and incubated on a side-shaking table for one hour at room temperature with slow shaking.

Recovering the primary antibody. Adding an immunostaining washing solution, and slowly shaking and washing for 5 minutes on a side shaking table. After the washing solution was completely absorbed, the washing solution was added thereto and washed for 5 minutes. The total number of washes was 3. If the result background is high, the washing time can be prolonged and the number of washing can be increased appropriately.

5. Incubation with secondary antibody

Diluting the fluorescently-labeled secondary antibody with an immunofluorescent staining secondary antibody diluent according to the proportion of 1: 200.

The wash was drained with a mini bench vacuum pump, diluted secondary antibody was added immediately and incubated on a side-shaking table for one hour at room temperature with slow shaking.

And (5) recovering the secondary antibody. The immunostaining wash PBST was added and washed on a side-shaking table for 5 minutes with slow shaking. After the washing solution was completely absorbed, the washing solution was added thereto and washed for 5 minutes. The total number of washes was 3. If the result background is high, it is possible to appropriately prolong the washing time and increase the number of washing.

6. Protein detection

For immunofluorescent staining, it is now possible to observe directly under a fluorescent microscope. If a better immune effect is observed under a fluorescent microscope, the downstream experiment is continued.

7. Counterdyeing

Cells were incubated with 0.1-1. mu.g/mL DAPI (DNA staining) for 1 min, after which the cells were rinsed with PBS.

8. Sealing sheet

The coverslips were closed with a drop of mounting medium and sealed with nail polish to avoid drying and movement of the sample under the microscope. Stored at-20 ℃ protected from light.

9. Evaluation of

Examination under a confocal laser microscope, the results were recorded and the marked cells were finally imaged.

Results as shown in fig. 4, examination of H2AFX protein expression in Huh7 cells using confocal microscopy resulted in a significant increase in H2AX foci formation 24H after SR-4835 treatment. The expression of the H2AFX protein is obtained based on the DNA damage repair screening reagent, and the effectiveness and feasibility of the DNA damage signal path screening reagent are further verified.

Eighth, CCK-8 tests the effect of H2AFX on Huh7 cell proliferation

Transfection was performed using 6 well cell culture plates using Invitrogen's Lipofectamine 2000 instructions.

1. The day before transfection, 6 well plates were seeded at 4 × 105/ml cell Huh7 density. During transfection, the cells are fused to 90-95%.

2. Solution 1: 240ul serum-free medium +10ul lipofectamine 2000 per well (total volume 250ul) (incubation 5min)

3. Solution 2: 200ul serum free medium +4ug pcDNA-H2AFX plasmid (designated H2AFX) per well brought the total volume to 250 ul. Meanwhile, a blank plasmid of pGenesil-1 was used as a control and named sh-RNA.

4. Mix solution 1 and solution 2 and let stand at room temperature for 20 min.

5. At the same time, 2ml of serum-free medium was added after the cells in the 6-well plate were washed twice with serum-free medium.

6. The mixture of solution 1 and solution 2 was added drop-wise into the wells, the plates were shaken and mixed gently. 5% CO at 37 deg.C₂And preserving the heat for 5-6 hours.

7. After 6 hours, the whole medium containing serum was changed, and the cells were digested at 37 ℃ in 5% CO2 for 24 hours and added to a 96-well plate at 2000 cells per 100. mu.l per well. Cultures were incubated and given 100nM microliters of SR-4835 treatment as required for the experiment.

8. Add 10. mu.l of CCK-8 solution per well. Wells to which corresponding amounts of cell culture fluid and CCK-8 solution were added but no cells were added were used as blanks. If there is a fear that the drug used will interfere with the detection, a well to which the corresponding amounts of the cell culture solution, the drug and the CCK-8 solution are added but no cells are added is set as a blank.

9. Incubation was continued in the cell incubator for 24,48,72 hours.

10. Absorbance was measured at 450nm, and the measurement results were introduced into GraphPad for calculation.

The result is shown in figure 5, the growth of the group transfected with H2AFX or SR-4835(100nM) is significantly inhibited (P <0.01), further, the inhibition effect of the over-expressed treatment group of SR-4835 and H2AFX on the growth of liver cancer is further illustrated, and the result further verifies the effectiveness of the screening reagent in screening DNA damage core molecules.

Ninthly, detecting the influence of DNA damage core molecules on liver cancer cell proliferation by adopting Edu fluorescence method

(one) EdU labelling and fixation, washing and permeabilization of cultured cells

a. Appropriate numbers of cells were cultured in 6-well plates. After the cells were cultured overnight and returned to normal, treatment with H2AFX over-expression vector or SR-4835(100nM) was performed.

b. Preparing 2X EdU working solution: EdU (10mM) was diluted 1:500 in cell culture to give 2 XEdU working solution (20. mu.M).

c. 2 XEdU working solution (20. mu.M) preheated at 37 ℃ was added in equal volume to the 6-well plate so that the final concentration of EdU in the 6-well plate became 1X.

d. Incubation of the cells was continued for 2 hours. This time is about 10% of the cell cycle.

After completion of edu labeling of cells, the culture medium was removed and 1ml of a fixing solution (PBS solution containing 10% paraformaldehyde) was added and fixed at room temperature for 15 minutes.

f. The fixative is removed and the cells are washed 3 times with 1ml of wash solution per well, 3-5 minutes each.

g. The wash solution was removed and each well was incubated with 1ml of the permeate (0.3% Triton X-100 in PBS) for 10-15 minutes at room temperature.

h. The permeate was removed and the cells were washed 1-2 times with 1ml of wash solution per well, 3-5 minutes each time.

(II) EdU detection

a. Preparing a Click Additive Solution: dissolving a tube of Click Additive in 1.3ml of deionized water, and uniformly mixing until the Click Additive is completely dissolved to obtain the Click Additive Solution. After preparation, the mixture can be properly packaged and stored at the temperature of 20 ℃ below zero.

b. A Click reaction solution was prepared with reference to Table 1. Note that: please strictly follow the component sequence and volume in table 1 to prepare Click reaction solution, otherwise the Click reaction may not be effectively performed; meanwhile, the Click reaction solution should be used within 15 minutes after preparation.

TABLE 1 preparation of Click reaction solution

Click Reaction Buffer	430μl
		CuSO4	20μl
Azide 555	1μl
		Click Additive Solution	50μl
Total volume	500μl

c. And removing the washing liquid in the previous step.

d. 0.5ml of Click reaction was added to each well and the plate was gently shaken to ensure that the reaction mixture covered the sample uniformly.

e. Incubate for 30 minutes at room temperature in the dark.

f. The Click reaction solution was aspirated and washed 3 times with washing solution for 3 to 5 minutes each.

g. The nuclei were stained as follows. The maximum excitation wavelength of Azide 555 was 555nm and the maximum emission wavelength was 565 nm.

(III) staining of cell nuclei

To examine the proportion of cell proliferation, nuclear staining using DAPI is considered. High-content screening instruments also generally require staining of the nuclei.

Preparation of 1X DAPI solution: DAPI (1000X) was diluted with PBS at a ratio of 1: 1000.

b. After the washing solution was aspirated, 1ml of 1 XDAPI solution was added to each well, and the wells were incubated for 10 minutes at room temperature in the dark.

c. The 1 XDAPI solution was aspirated.

d. Washing with washing solution for 3 times, each for 3-5 min.

e. Fluorescence detection can then be performed. DAPI is blue fluorescence with a maximum excitation wavelength of 346nm and a maximum emission wavelength of 460 nm.

(IV) interpretation of results

The result is shown in figure 6, the SR-4835 is adopted to treat Huh7 cells, DNA repair core molecule H2AFX is over-expressed in Huh7 cells, then Edu staining is adopted to detect the influence of the core molecule on the proliferation of liver cancer cells, and the result shows that after the SR-4835 is added for treatment for 24 hours, the H2AFX is over-expressed in Huh7 cells, the proliferation of the Huh7 cells is obviously inhibited, which indicates that the target molecules screened by the screening reagent have obvious targeting for screening tumor drugs and testing the action mechanism of the tumor drugs.

Ten, detecting the influence of DNA damage target molecule H2AFX on Huh7 cell proliferation by adopting clone formation experiment

(one) Experimental procedure

1. And (2) transfecting each group of cells with an H2AFX overexpression vector, treating the cells with an SR-4835 medicament for 24 hours, taking liver cancer Huh7 cells in a logarithmic growth phase, digesting the cells with 0.25% trypsin respectively, blowing the cells into single cells, and suspending the cells in a DMEM culture solution containing 10% fetal calf serum for later use.

2. Diluting the cell suspension by gradient multiple, respectively inoculating each group of cells into a dish containing 10mL of 37 ℃ pre-warming culture solution at the gradient density of 50 cells, 100 cells and 200 cells per dish, and slightly rotating to uniformly disperse the cells. Placing at 37 ℃ with 5% CO₂And culturing for 2-3 weeks in a cell culture box with saturated humidity.

3. It was frequently observed that when macroscopic colonies appeared in the culture dish, the culture was terminated. The supernatant was discarded and carefully rinsed 2 times with PBS. Cells were fixed for 15 minutes by adding 5mL of 4% paraformaldehyde. Then removing the fixing solution, adding a proper amount of GIMSA, dyeing for 10-30 minutes by using the dyeing solution, then slowly washing off the dyeing solution by using running water, and drying in air.

4. The plate was inverted and overlaid with a piece of transparent film with a grid, and the clones were counted directly with the naked eye.

(II) interpretation of results

The results are shown in fig. 7, the results are that the DNA repair core molecule H2AFX is overexpressed in Huh7 cells by processing Huh7 cells with SR-4835, and then the influence of the detection core molecule formed by plate cloning on the proliferation of liver cancer cells is detected, and the results show that the proliferation of Huh7 cells is significantly inhibited after H2AFX is overexpressed in Huh7 cells by adding SR-4835 for 24 hours, which indicates that the target molecules screened by the PCR detection reagent of the present invention have significant effectiveness for screening tumor drugs and testing the action mechanism of tumor drugs.

Eleven, detecting the influence of the target molecule BCL2 of the liver cancer SR-4835 on the apoptosis of the liver cancer cells by adopting a flow cytometer

Collecting cells: taking a proper amount of Huh7 cells in the logarithmic growth phase, inoculating the cells into a 6-well plate, treating the cells for a corresponding time under corresponding conditions (such as the above various treatment groups), collecting the cells, and taking care to combine the supernatant and the digested cells;

and (3) cleaning cells: washing 2 times with pre-cooled PBS;

grouping: each experiment is divided into a non-dyeing group, a single-dyeing Annexin V-PE group, a single-dyeing 7-AAD group and a 7-AAD and Annexin V-PE double-dyeing group, and the treatment groups are subjected to double dyeing from low to high;

dyeing: the 4 Xbinding buffer was diluted to 1 Xbuffer with PBS, the residual PBS in the centrifuge tube was aspirated, 100. mu.L of 1 Xbinding buffer was added to each tube, the cells were blown with a pipette to resuspend them thoroughly, and the dye was added in the dark. Adding Annexin V-PE or 7-AAD 5 mu L into a single-dyeing group, adding Annexin V-PE and 7-AAD 5 mu L into a double-dyeing group, and gently mixing by using a pipette gun;

And (3) computer detection: after incubation for 15 minutes at room temperature in the dark, 300. mu.L of binding buffer was added and mixed well, and the cell suspension was transferred to a 5mL flow tube in the dark for 1h on-machine detection on a FACSCalibur flow cytometer.

The results are shown in fig. 8, compared with the control group, the H2AFX over-expression group and the SR-4835(100nM) added group significantly improved apoptosis of Huh7 hepatoma cells. The result further shows that the DNA damage screening reagent has effectiveness on screening targets of tumor drugs.

The invention concentrates the genes closely related to the DNA damage repair core molecule on a flat plate, reacts the survival state of cells by carrying out a real-time fluorescent quantitative PCR reaction, discusses the possible ways of the DNA damage repair core molecule in various treatments of tumor cells, and provides the most direct evidence for researching the regulation and control of key protein; the invention can quickly and accurately find the target core molecule of DNA damage from the transcription level, and provides a powerful tool for screening anti-cancer drugs, discussing the mechanism of new target drugs and the like.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

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<400>20

gattcagctc ctgcatactc 20

<210>21

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

tgttacaaat cacccctcaa g 21

<210>22

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

cctgatactt ttctggatgc 20

<210>23

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

gaaacagtca agagtcatcg a 21

<210>24

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

ctgagcaatc tgcttgtgt 19

<210>25

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

tcctcttttt acaccccagt c 21

<210>26

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

cggttgtcaa ggtttgtagt t 21

<210>27

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>27

agtggcctat atcgctcc 18

<210>28

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

cctggttaga atcttcctcc 20

<210>29

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

gtatggtgta ggtgtggac 19

<210>30

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>30

gcaaaggtat tccagggaaa 20

<210>31

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

gatggaactt cgactttgtc 20

<210>32

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>32

cacaagggta caagacagt 19

<210>33

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>33

cttactgcaa tgctcgctg 19

<210>34

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>34

tgaggggttt gttgtaccat 20

<210>35

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>35

tatctgcctt agtgggtatc c 21

<210>36

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>36

tgtcgtaaaa cgtgccttt 19

<210>37

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>37

acggcttagt gcgtctga 18

<210>38

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>38

aagtctggag aacgggaga 19

<210>39

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

tggacaaccg cctctaact 19

<210>40

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

ccagtgaagg gactccatat 20

<210>41

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

aaagctgcga ctgatagatt g 21

<210>42

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

aggctacacg aacattgtac t 21

<210>43

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

ttgacagcga agtacaatgc 20

<210>44

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

atgggctcgc gtaatgatg 19

<210>45

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

cttcatcaaa gggattggag 20

<210>46

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

tgaggaggcg taaaactgg 19

<210>47

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>47

aacggctcaa gcaggaaat 19

<210>48

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

tcaccattcg gtcaatcaga 20

<210>49

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

ttggcgacgt aattcccga 19

<210>50

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

ctgctgggga tctttcac 18

<210>51

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

tcgatgggaa atgccaca 18

<210>52

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

tcatccaggt tgtagatgc 19

<210>53

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

ctcgtggctc cctatgaa 18

<210>54

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

ggagatccga gtcctctgta 20

<210>55

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

gcacacagta tgttctccc 19

<210>56

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>56

tgtacgtgaa gatgccaca 19

<210>57

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>57

ttcgccagtt ggtgatgga 19

<210>58

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>58

ggaagcctgt aaccgtga 18

<210>59

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>59

aggctctatc agcaactgg 19

<210>60

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>60

aactgcgggg ctttgga 17

<210>61

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>61

tcttgatgca cccagtgag 19

<210>62

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>62

agccgcagca tagactttg 19

<210>63

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>63

cctgatccag gcgtttt 17

<210>64

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>64

atccatgtag cgactttcc 19

<210>65

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>65

agatccattt tacgctgatc c 21

<210>66

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>66

cctcgcaaaa caggctga 18

<210>67

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>67

aacgacgagg agctcaacaa 20

<210>68

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>68

gcgggccctc ttagtactcc 20

<210>69

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>69

aatgccaggg ctttgaaaat 20

<210>70

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>70

acaatgcggc tactgctt 18

<210>71

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>71

cagttcccca tcagggatt 19

<210>72

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>72

tctgtgaggc tttctttcg 19

<210>73

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>73

atgagaagct aggcgagga 19

<210>74

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>74

agcgtggata tacctcagc 19

<210>75

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>75

ggcaaaatgg caatacctg 19

<210>76

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>76

tctgcggttc ttgctacct 19

<210>77

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>77

tagtcacccc tgaccaaaa 19

<210>78

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>78

cagcctcggc atgatag 17

<210>79

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>79

gtgggagcct taatggtac 19

<210>80

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>80

tccctcagac ggtgact 17

<210>81

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>81

caaacccctg tccagtca 18

<210>82

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>82

tgggagttca agcatctcc 19

<210>83

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>83

acgtatgttc ccgattttgt c 21

<210>84

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>84

cttcagagct gatatagcca c 21

<210>85

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>85

aatggcacag aactccgag 19

<210>86

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>86

atgcagaagt acatgccgt 19

<210>87

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>87

gggcagatgc actttgt 17

<210>88

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>88

aagcaaaacc ggactaatgt c 21

<210>89

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>89

gtcagagccc ttaacctttt t 21

<210>90

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>90

agaggctgct taatccact 19

<210>91

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>91

taccagctat cttctgtgca t 21

<210>92

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>92

ctctgtttgc tcggacaa 18

<210>93

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>93

ggtggggaag ctaggttga 19

<210>94

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>94

accgccaatc caatttctg 19

<210>95

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>95

taccccgtcg gtttctg 17

<210>96

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>96

gatgtcccca ccgtagt 17

<210>97

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>97

ctcccacttc caagaggt 18

<210>98

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>98

gatgagccga ggtccaaaa 19

<210>99

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>99

ctgagcttcg gtgggatg 18

<210>100

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>100

tggcatactc tgctgcaaa 19

<210>101

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>101

cactaagggc cgaagataac 20

<210>102

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>102

cagcatctcc aatatggctg 20

<210>103

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>103

cttacggttt tcgtggagaa 20

<210>104

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>104

gcagccgttc ttgttgtaa 19

<210>105

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>105

ttgccgacct aactcaggt 19

<210>106

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>106

gatgcgtggc aggtaga 17

<210>107

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>107

aagcgtatgc ggaagtcaa 19

<210>108

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>108

cgtcccgtcc agatcaa 17

<210>109

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>109

tggtcatcat tcggggca 18

<210>110

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>110

atccttcggg tcatcctga 19

<210>111

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>111

gtgccaaccc agtgatga 18

<210>112

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>112

gacacctgta gcaggagtg 19

<210>113

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>113

gctggcttgc gcctatt 17

<210>114

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>114

ggcacaccac tttaacaaga 20

<210>115

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>115

tatgccaggg actttaactg 20

<210>116

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>116

ggacttcact cgtcatatcc 20

<210>117

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>117

aagcccgagg atatgctca 19

<210>118

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>118

caggcaattt tcccgatact 20

<210>119

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>119

gagatcgctc cagatccct 19

<210>120

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>120

cgcaaacagg acaatcca 18

<210>121

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>121

gcctgcacat gacgatatg 19

<210>122

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>122

tggttggcat tggttcaac 19

<210>123

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>123

aagtgtgcag aaattgaccg a 21

<210>124

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>124

acgtacctgc cgaagtgt 18

<210>125

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>125

gagcgttcaa cacagacc 18

<210>126

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>126

tggcactgtc tacaataagc 20

<210>127

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>127

agagtctgca tttagtgctg 20

<210>128

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>128

caggtgagtt cccgataaag a 21

<210>129

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>129

attgactctt acatgatcgc c 21

<210>130

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>130

ccaggtgaaa gggaaatg 18

<210>131

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>131

aaatatccgg cagcagaatc t 21

<210>132

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>132

gctgttatcg gtgaatctgg 20

<210>133

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>133

ttccgctgag gaattgagaa 20

<210>134

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>134

gagaggagtc gtccagct 18

<210>135

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>135

caacgtggaa ctgtggac 18

<210>136

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>136

aggtttactg agcagacgaa 20

<210>137

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>137

cgcgtctgga acctttaa 18

<210>138

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>138

aggacctgct aattcatcca a 21

<210>139

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>139

tggaccattt gtgctcgt 18

<210>140

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>140

tcgtcaacca gttctaggg 19

<210>141

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>141

gtgtcatagg ttaggtggtg 20

<210>142

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>142

gccaattctt tttgtgttcg t 21

<210>143

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>143

tggcgcagga gtatgacaa 19

<210>144

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>144

ccgtactacc tcatccttc 19

<210>145

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>145

gaccaggagg caaaacctt 19

<210>146

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>146

attcattgga acaccctgtc t 21

<210>147

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>147

gagtgtgcca agagatgga 19

<210>148

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>148

gcttctggtc taggttctg 19

<210>149

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>149

cagctttgag gtgcgtgtt 19

<210>150

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>150

cctttcttgc ggagattctc 20

<210>151

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>151

gagcagttac ctcagccaa 19

<210>152

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>152

agggaatgtg tagtattgcc t 21

<210>153

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>153

acgaggacac gtactacct 19

<210>154

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>154

tgccgatagg agtccacc 18

<210>155

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>155

ggcagtgcca ttgatagga 19

<210>156

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>156

tgtctacatc caaagaaccc t 21

<210>157

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>157

caggacctgt tatggaattt g 21

<210>158

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>158

cttcttgact accccaaact t 21

<210>159

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>159

atcgtgggag ccatcgtaa 19

<210>160

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>160

tcaccatcgc tgcacattt 19

<210>161

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>161

ctttggcttg agttttgtac 20

<210>162

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>162

ctccttcaca cggaactg 18

<210>163

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>163

ctggatagac cgcgtcaat 19

<210>164

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>164

tcgagaggca tgagaagg 18

<210>165

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>165

cccattccgc tgtgaattt 19

<210>166

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>166

gttagcccag gttatgcc 18

<210>167

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>167

tgcttctgcc tagcgatac 19

<210>168

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>168

aacctggatc atcaaaccgt 20

<210>169

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>169

gtctgccttg gtagtggata at 22

<210>170

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>170

tcgaggacgc cctatcatg 19

<210>171

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>171

ccaaggaagg cgtctaagg 19

<210>172

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>172

ctttcgagcg caaccacttt 20

<210>173

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>173

ggagcgagat ccctccaaaa 20

<210>174

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>174

ggctgttgtc atacttctca tg 22

<210>175

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>175

cctggcgtcg tgattagtga 20

<210>176

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>176

agacgttcag tcctgtccat a 21

<210>177

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>177

agcccagaac actggtct 18

<210>178

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>178

actcaggatt tcaatggtgc 20

Claims

1. A screening reagent for detecting DNA damage signal channel core molecules is characterized by comprising the following primers:

5'- ATCACGCCAGTCAACAGTC -3'；

5'- TACACCCTCCCTTCGTATC -3'；

5'- TTTCTTACGGCATAGGCGA -3'；

5'- ACAAACGAGTCAAATTCAGC -3'；

5'-TGATCTTGTGCCTTGGCTA -3'；

5'- ATGGTGTACGTTCCCCATG -3'；

5'- CCCTTGAATACAGTGGCCT -3'；

5'- CCTTGAAAGTACGGCAGTT -3'；

5'- TGAACGAGCAAATAAACTGG -3'；

5'- AGGCTTGTATCCTGACTCC -3'；

5'- GTCACTGCATGTAACAGCG -3'；

5'- GGCACAATTAGTGCGGAATA -3'；

5'- AGTGGCTCCTTGACAGAATC -3'；

5'- GTCTGGAGACTCTATTTGCTC -3'；

5'- CCGAGAGGTCTTTTTCCGA -3'；

5'-CAGCCCATGATGGTTCTGA -3'；

5'- ACCTCAACGCACAGTACGA -3'；

5'- GGAGTCCCATGATGAGATTG -3'；

5'- GACCTTGACACCTCTGACA -3'；

5'- GATTCAGCTCCTGCATACTC -3'；

5'- TGTTACAAATCACCCCTCAAG -3'；

5'- CCTGATACTTTTCTGGATGC -3'；

5'- GAAACAGTCAAGAGTCATCGA -3'；

5'- CTGAGCAATCTGCTTGTGT-3'；

(13) PCR primers for detecting CDC25A gene have the following primer sequences:

5'- TCCTCTTTTTACACCCCAGTC -3'；

5'- CGGTTGTCAAGGTTTGTAGTT -3'；

(14) PCR primers for detecting CDC25C gene have the following primer sequences:

5'- AGTGGCCTATATCGCTCC -3'；

5'- CCTGGTTAGAATCTTCCTCC -3'；

(15) PCR primers for detecting CDK7 gene have the following primer sequences:

5'- GTATGGTGTAGGTGTGGAC -3'；

5'- GCAAAGGTATTCCAGGGAAA -3'；

5'- GATGGAACTTCGACTTTGTC -3'；

5'- CACAAGGGTACAAGACAGT -3'；

5'- CTTACTGCAATGCTCGCTG -3'；

5'-TGAGGGGTTTGTTGTACCAT -3'；

5'- TATCTGCCTTAGTGGGTATCC -3'；

5'- TGTCGTAAAACGTGCCTTT -3'；

5'- ACGGCTTAGTGCGTCTGA -3'；

5'- AAGTCTGGAGAACGGGAGA -3'；

5'- TGGACAACCGCCTCTAACT -3'；

5'- CCAGTGAAGGGACTCCATAT -3'；

5'- AAAGCTGCGACTGATAGATTG -3'；

5'- AGGCTACACGAACATTGTACT -3'；

5'- TTGACAGCGAAGTACAATGC -3'；

5'- ATGGGCTCGCGTAATGATG0-3'；

5'- CTTCATCAAAGGGATTGGAG -3'；

5'- TGAGGAGGCGTAAAACTGG -3'；

5'- AACGGCTCAAGCAGGAAAT -3'；

5'- TCACCATTCGGTCAATCAGA -3'；

5'- TTGGCGACGTAATTCCCGA -3'；

5'- CTGCTGGGGATCTTTCAC -3'；

5'- TCGATGGGAAATGCCACA -3'；

5'- TCATCCAGGTTGTAGATGC -3'；

5'- CTCGTGGCTCCCTATGAA -3'；

5'- GGAGATCCGAGTCCTCTGTA -3'；

5'- GCACACAGTATGTTCTCCC -3'；

5'- TGTACGTGAAGATGCCACA -3'；

5'- TTCGCCAGTTGGTGATGGA -3'；

5'- GGAAGCCTGTAACCGTGA -3'；

5'- AGGCTCTATCAGCAACTGG-3'；

5'- AACTGCGGGGCTTTGGA-3'；

5'- TCTTGATGCACCCAGTGAG -3'；

5'- AGCCGCAGCATAGACTTTG -3'；

5'- CCTGATCCAGGCGTTTT -3'；

5'-ATCCATGTAGCGACTTTCC -3'；

5'- AGATCCATTTTACGCTGATCC -3'；

5'- CCTCGCAAAACAGGCTGA -3'；

5'- AACGACGAGGAGCTCAACAA-3'；

5'- GCGGGCCCTCTTAGTACTCC-3'；

5'- AATGCCAGGGCTTTGAAAAT -3'；

5'- ACAATGCGGCTACTGCTT -3'；

5'- CAGTTCCCCATCAGGGATT -3'；

5'- TCTGTGAGGCTTTCTTTCG -3'；

5'- ATGAGAAGCTAGGCGAGGA -3'；

5'- AGCGTGGATATACCTCAGC -3'；

5'- GGCAAAATGGCAATACCTG -3'；

5'- TCTGCGGTTCTTGCTACCT -3'；

5'- TAGTCACCCCTGACCAAAA-3'；

5'- CAGCCTCGGCATGATAG -3'；

5'- GTGGGAGCCTTAATGGTAC -3'；

5'-TCCCTCAGACGGTGACT-3'；

5'- CAAACCCCTGTCCAGTCA -3'；

5'- TGGGAGTTCAAGCATCTCC -3'；

5'- ACGTATGTTCCCGATTTTGTC -3'；

5'- CTTCAGAGCTGATATAGCCAC-3'；

5'- AATGGCACAGAACTCCGAG -3'；

5'- ATGCAGAAGTACATGCCGT -3'；

5'- GGGCAGATGCACTTTGT -3'；

5'- AAGCAAAACCGGACTAATGTC -3'；

5'- GTCAGAGCCCTTAACCTTTTT-3'；

5'- AGAGGCTGCTTAATCCACT-3'；

5'- TACCAGCTATCTTCTGTGCAT-3'；

5'- CTCTGTTTGCTCGGACAA-3'；

5'- GGTGGGGAAGCTAGGTTGA -3'；

5'- ACCGCCAATCCAATTTCTG -3'；

5'- TACCCCGTCGGTTTCTG -3'；

5'- GATGTCCCCACCGTAGT -3'；

5'- CTCCCACTTCCAAGAGGT -3'；

5'- GATGAGCCGAGGTCCAAAA -3'；

5'- CTGAGCTTCGGTGGGATG -3'；

5'- TGGCATACTCTGCTGCAAA -3'；

5'- CACTAAGGGCCGAAGATAAC -3'；

5'- CAGCATCTCCAATATGGCTG-3'；

5'- CTTACGGTTTTCGTGGAGAA -3'；

5'- GCAGCCGTTCTTGTTGTAA -3'；

5'- TTGCCGACCTAACTCAGGT -3'；

5'- GATGCGTGGCAGGTAGA -3'；

5'- AAGCGTATGCGGAAGTCAA -3'；

5'- CGTCCCGTCCAGATCAA-3'；

5'- TGGTCATCATTCGGGGCA -3'；

5'- ATCCTTCGGGTCATCCTGA -3'；

5'- GTGCCAACCCAGTGATGA -3'；

5'- GACACCTGTAGCAGGAGTG -3'；

5'- GCTGGCTTGCGCCTATT -3'；

5'- GGCACACCACTTTAACAAGA -3'；

5'- TATGCCAGGGACTTTAACTG -3'；

5'- GGACTTCACTCGTCATATCC -3'；

5'-AAGCCCGAGGATATGCTCA -3'；

5'- CAGGCAATTTTCCCGATACT -3'；

5'- GAGATCGCTCCAGATCCCT-3'；

5'- CGCAAACAGGACAATCCA-3'；

5'- GCCTGCACATGACGATATG -3'；

5'- TGGTTGGCATTGGTTCAAC -3'；

5'- AAGTGTGCAGAAATTGACCGA -3'；

5'-ACGTACCTGCCGAAGTGT -3'；

5'- GAGCGTTCAACACAGACC-3'；

5'- TGGCACTGTCTACAATAAGC -3'；

5'- AGAGTCTGCATTTAGTGCTG -3'；

5'- CAGGTGAGTTCCCGATAAAGA -3'；

5'- ATTGACTCTTACATGATCGCC -3'；

5'- CCAGGTGAAAGGGAAATG -3'；

5'- AAATATCCGGCAGCAGAATCT -3'；

5'- GCTGTTATCGGTGAATCTGG -3'；

5'- TTCCGCTGAGGAATTGAGAA -3'；

5'- GAGAGGAGTCGTCCAGCT -3'；

5'- CAACGTGGAACTGTGGAC -3'；

5'- AGGTTTACTGAGCAGACGAA -3'；

5'- CGCGTCTGGAACCTTTAA-3'；

5'- AGGACCTGCTAATTCATCCAA-3'；

5'-TGGACCATTTGTGCTCGT -3'；

5'- TCGTCAACCAGTTCTAGGG-3'；

5'- GTGTCATAGGTTAGGTGGTG -3'；

5'- GCCAATTCTTTTTGTGTTCGT -3'；

5'- TGGCGCAGGAGTATGACAA -3'；

5'- CCGTACTACCTCATCCTTC -3'；

5'- GACCAGGAGGCAAAACCTT -3'；

5'- ATTCATTGGAACACCCTGTCT -3'；

5'- GAGTGTGCCAAGAGATGGA-3'；

5'- GCTTCTGGTCTAGGTTCTG-3'；

5'- CAGCTTTGAGGTGCGTGTT -3'；

5'- CCTTTCTTGCGGAGATTCTC -3'；

5'- GAGCAGTTACCTCAGCCAA -3'；

5'- AGGGAATGTGTAGTATTGCCT -3'；

5'- ACGAGGACACGTACTACCT -3'；

5'- TGCCGATAGGAGTCCACC -3'；

5'- GGCAGTGCCATTGATAGGA -3'；

5'- TGTCTACATCCAAAGAACCCT -3'；

5'-CAGGACCTGTTATGGAATTTG-3'；

5'-CTTCTTGACTACCCCAAACTT -3'；

5'- ATCGTGGGAGCCATCGTAA -3'；

5'-TCACCATCGCTGCACATTT -3'；

5'- CTTTGGCTTGAGTTTTGTAC -3'；

5'- CTCCTTCACACGGAACTG -3'；

5'- CTGGATAGACCGCGTCAAT -3'；

5'-TCGAGAGGCATGAGAAGG-3'；

5'- CCCATTCCGCTGTGAATTT -3'；

5'-GTTAGCCCAGGTTATGCC -3'；

5'-TGCTTCTGCCTAGCGATAC -3'；

5'- AACCTGGATCATCAAACCGT -3'；

5'- GTCTGCCTTGGTAGTGGATAAT-3'；

5'- TCGAGGACGCCCTATCATG-3'；

5'- CCAAGGAAGGCGTCTAAGG-3'；

5'- CTTTCGAGCGCAACCACTTT-3'；

5'- GGAGCGAGATCCCTCCAAAA-3'；

5'- GGCTGTTGTCATACTTCTCATG-3'；

5'- CCTGGCGTCGTGATTAGTGA-3'；

5'- AGACGTTCAGTCCTGTCCATA-3'；

5'- AGCCCAGAACACTGGTCT-3'；

5'- ACTCAGGATTTCAATGGTGC-3'。

2. use of the screening reagent of claim 1 for the preparation of a detection reagent for detecting a cell line DNA damage signaling pathway core molecule.

3. The use of a screening reagent according to claim 2 for the preparation of a test reagent for the detection of a cell line DNA damage signaling pathway core molecule, wherein: the cell lines are human liver cancer cell lines, stomach cancer cell lines, breast cancer cell lines, pancreas cancer cell lines, lung cancer cell lines, kidney cancer cell lines, melanoma cell lines and brain glioma cell lines.

4. The use of a screening reagent according to claim 2 for the preparation of a test reagent for the detection of a cell line DNA damage signaling pathway core molecule, wherein: the detection reagent adopts a real-time fluorescent quantitative PCR method for detection.

5. The use of the screening reagent of claim 4 for the preparation of a test reagent for the detection of a cell line DNA damage signaling pathway core molecule, wherein: the reaction system of the real-time fluorescent quantitative PCR is as follows: 10 mul of Taq polymerase reaction mixture, 0.1-0.2 mul of upstream primer, 0.1-0.2 mul of downstream primer, 25-100ng of cDNA template, and sterile distilled water to make up to 20 mul.

6. The use of a screening reagent according to claim 5 in the preparation of a test reagent for the detection of a cell line DNA damage signaling pathway core molecule, wherein: the Taq polymerase is 2X SYBR Premix Ex Taq polymerase of TaKaRa company.

7. The use of the screening reagent of claim 4 for the preparation of a test reagent for the detection of a cell line DNA damage signaling pathway core molecule, wherein: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95 deg.C for 5 seconds, 59 deg.C for 20 seconds, and 40 cycles; the dissolution curve analysis was carried out at 95 ℃ for 15 seconds to 65 ℃ for 1 minute, and the temperature was decreased in a gradient manner at 4.4 ℃/sec.