技术领域technical field
本发明涉及基于聚合酶链反应（PCR）和DNA测序技术的分子生物学方法，尤其涉及一种用于雄激素受体（AR）基因全编码区突变检测的扩增和测序引物及其试剂盒。The present invention relates to a molecular biology method based on polymerase chain reaction (PCR) and DNA sequencing technology, in particular to an amplification and sequencing primer and kit for detecting mutations in the full coding region of the androgen receptor (AR) gene .
背景技术Background technique
雄激素不敏感综合征(AIS)是一类引起性发育异常（DSD）的X染色体隐形遗传病，发病机制与AR基因突变密切相关。患者核型为46,XY，雄激素靶器官上的雄激素受体缺陷而导致雄激素的正常效应全部或部分丧失。根据临床表现，可分为两种亚型：完全型雄激素不敏感综合征(CAIS)和不完全型雄激素不敏感综合征(PAIS)。 Androgen insensitivity syndrome (AIS) is a type of X-chromosome recessive genetic disease causing abnormal sex development (DSD), and its pathogenesis is closely related to AR gene mutation. The patient's karyotype is 46,XY, and the androgen receptor on the androgen target organ is defective, resulting in the loss of all or part of the normal effects of androgen. According to clinical manifestations, it can be divided into two subtypes: complete androgen insensitivity syndrome (CAIS) and incomplete androgen insensitivity syndrome (PAIS).
编码雄激素受体蛋白的雄激素受体基因位于Xq11-12，雄激素受体基因总长75～90kb，包括7个内含子与8个外显子。AR属于核受体超家族成员，由910～919个氨基酸组成，分子量为110～114kDa，由第1外显子编码的转录激活区、第2和第3外显子编码的DNA结合区以及第4—8外显子编码的配体结合区三个功能结构域组成。在基因水平，已发现的雄激素受体基因突变大致有以下几种：①基因点突变导致氨基酸代替或终止密码提前出现或正常剪切位点突变 ②核苷酸缺失或插入导致移码突变 ③外显子1中CAG重复序列长度延长或缩短等等。截止2013年4月更新的AR基因突变引起的CAIS、PAIS、MAIS以及癌症（包括前列腺癌、肝癌、乳腺癌）有1000多种。雄激素受体基因突变测序检测，可为诊断雄激素不敏感综合征提供重要的诊断依据，对研究雄激素受体的结构和功能、雄激素不敏感综合征的发病机制间的联系提供技术分析的基础，具有较重要的临床意义。 The androgen receptor gene encoding the androgen receptor protein is located at Xq11-12, and the total length of the androgen receptor gene is 75-90 kb, including 7 introns and 8 exons. AR is a member of the nuclear receptor superfamily, consisting of 910-919 amino acids, with a molecular weight of 110-114 kDa, and is encoded by the transcription activation region encoded by exon 1, the DNA binding region encoded by exon 2 and 3, and the DNA binding region encoded by exon 2. The ligand-binding region encoded by exons 4-8 consists of three functional domains. At the gene level, the androgen receptor gene mutations that have been discovered roughly fall into the following categories: ① Point mutations in the gene lead to early appearance of amino acid substitutions or stop codons, or mutations in normal splicing sites② Nucleotide deletions or insertions lead to frameshift mutations ③ The length of the CAG repeat sequence in exon 1 is extended or shortened, etc. As of April 2013, there are more than 1,000 types of CAIS, PAIS, MAIS and cancers (including prostate cancer, liver cancer, and breast cancer) caused by AR gene mutations. Androgen receptor gene mutation sequencing detection can provide an important diagnostic basis for the diagnosis of androgen insensitivity syndrome, and provide technical analysis for the study of the structure and function of androgen receptor and the relationship between the pathogenesis of androgen insensitivity syndrome The basis has more important clinical significance.
目前，还未开发出检测全面、结果准确的雄激素受体基因突变测序检测试剂盒，用于临床诊断。本发明基于聚合酶链反应（PCR）和DNA测序技术，通过设计、筛选得到一套理想的对雄激素受体基因全编码区进行扩增和测序的引物，达到全面检测雄激素受体基因是否发生突变的目的，进行准确的基因分型。 At present, no comprehensive and accurate test kit for the detection of androgen receptor gene mutation sequencing has been developed for clinical diagnosis. Based on polymerase chain reaction (PCR) and DNA sequencing technology, the present invention obtains a set of ideal primers for amplifying and sequencing the entire coding region of the androgen receptor gene through design and screening, so as to comprehensively detect whether the androgen receptor gene is For mutation purposes, accurate genotyping is performed.
发明内容Contents of the invention
本发明的目的是克服现有分子诊断技术中检测雄激素受体突变类型多、检测不全面的缺陷，提供一套优化引物用于PCR扩增和测序，能在相同条件下一次性进行11个PCR，顺利扩增和测序雄激素受体基因的全编码区。The purpose of the present invention is to overcome the defects of many types of androgen receptor mutations and incomplete detection in the existing molecular diagnostic technology, and provide a set of optimized primers for PCR amplification and sequencing, which can perform 11 mutations at one time under the same conditions. PCR, the full coding region of the androgen receptor gene was successfully amplified and sequenced.
本发明第二个目的是提供一种配套此引物进行雄激素受体基因全编码区突变检测的特定的PCR反应程序，达到快速扩增的目的。The second object of the present invention is to provide a specific PCR reaction program for detecting mutations in the entire coding region of the androgen receptor gene with the primers, so as to achieve the purpose of rapid amplification.
本发明第三个目的是提供一种对临床标本进行雄激素受体进行序列分析的荧光PCR扩增试剂盒。The third object of the present invention is to provide a fluorescent PCR amplification kit for sequence analysis of androgen receptor in clinical samples.
本发明的技术方案概述如下：Technical scheme of the present invention is summarized as follows:
用于雄激素受体基因全编码区突变检测的扩增和测序引物，包括第一外显子的扩增和测序引物为SEQ ID NO.1-SEQ ID NO.9，第二外显子的扩增和测序引物为SEQ ID NO.10和SEQ ID NO.11，第三外显子的扩增和测序引物为SEQ ID NO.12和SEQ ID NO.13，第四外显子的扩增和测序引物为SEQ ID NO.14-SEQ ID NO.16，第五外显子的扩增和测序引物为SEQ ID NO.17和SEQ ID NO.18，第六外显子的扩增和测序引物为SEQ ID NO.19和SEQ ID NO.20，第七外显子的扩增和测序引物为SEQ ID NO.21-SEQ ID NO.23，第八外显子的扩增和测序引物为SEQ ID NO.24和SEQ ID NO.25。Amplification and sequencing primers for the detection of mutations in the full coding region of the androgen receptor gene, including the amplification and sequencing primers of the first exon are SEQID NO.1-SEQ ID NO.9, the amplification and sequencing primers of the second exon are SEQ ID NO.10 and SEQ ID NO.11, the amplification and sequencing primers of the third exon are SEQID NO.12 and SEQ ID NO.13, the amplification and sequencing primers of the fourth exon are SEQ ID NO.14-SEQ ID NO.16, the amplification and sequencing primers of the fifth exon are SEQ ID NO .17 and SEQID NO.18, the amplification and sequencing primers of the sixth exon are SEQ ID NO.19 and SEQ ID NO.20, the amplification and sequencing primers of the seventh exon are SEQ ID NO.19ID NO.21-SEQ ID NO.23, the amplification and sequencing primers of the eighth exon are SEQ ID NO.24 and SEQ ID NO.25.
配套扩增雄激素受体外显子的PCR反应，为95℃预变性3min 后， 94℃变性15s ，60℃退火15s ，72℃延伸35s，进行35个循环，然后保存在25℃；及时取出进行测序。The PCR reaction for amplifying the exons of the androgen receptor is pre-denatured at 95°C for 3 minutes, denatured at 94°C for 15s, annealed at 60°C for 15s, and extended at 72°C for 35s for 35 cycles, and then stored at 25°C; take it out in time Perform sequencing.
一种用于对雄激素受体基因全编码区突变检测的扩增和测序试剂盒：An amplification and sequencing kit for detecting mutations in the full coding region of the androgen receptor gene:
试剂A：由2mL缓冲液组成，包含2×SYBR® Green I 染料，0.2mM dNTPs（包括dATP、dCTP、dGTP、dTTP）、80mM Tris-HCl、80mM KCl、40mM (NH4)2SO4、6mM MgSO4、100U Taq DNA聚合酶；Reagent A: Consists of 2mL buffer containing 2×SYBR® Green I dye, 0.2mM dNTPs (including dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4)2SO4, 6mMMgSO4, 100U Taq DNA polymerase;
试剂E1-A：由针对AR第一外显子E1-A的测序引物30μL水溶液，包含4μM SEQ ID NO.1；Reagent E1-A: 30 μL aqueous solution of sequencing primers targeting the first exon E1-A of AR, containing 4 μM SEQ ID NO.1;
试剂E1-B：由针对AR第一外显子E1-B的测序引物30μL水溶液，包含4μM SEQ ID NO.3；Reagent E1-B: 30 μL aqueous solution of sequencing primers targeting the first exon E1-B of AR, containing 4 μM SEQ ID NO.3;
试剂E1-C：由针对AR第一外显子E1-C的测序引物30μL水溶液，包含4μM SEQ ID NO.7；Reagent E1-C: 30 μL aqueous solution of sequencing primers targeting the first exon E1-C of AR, containing 4 μM SEQ ID NO.7;
试剂E1-D：由针对AR第一外显子E1-D的测序引物30μL水溶液，包含4μM SEQ ID NO.8；Reagent E1-D: 30 μL aqueous solution of sequencing primers targeting the first exon E1-D of AR, containing 4 μM SEQ ID NO.8;
试剂E2：由针对AR第二外显子E2的测序引物30μL水溶液，包含4μM SEQ ID NO.11；Reagent E2: 30 μL aqueous solution of sequencing primers targeting AR second exon E2, containing 4 μM SEQ ID NO.11;
试剂E3：由针对AR第三外显子E3的测序引物30μL水溶液，包含4μM SEQ ID NO.12；Reagent E3: 30 μL aqueous solution of sequencing primers targeting the third exon E3 of AR, containing 4 μM SEQ ID NO.12;
试剂E4：由针对AR第四外显子E4的测序引物30μL水溶液，包含4μM SEQ ID NO.16；Reagent E4: 30 μL aqueous solution of sequencing primers targeting the fourth exon E4 of AR, containing 4 μM SEQ ID NO.16;
试剂E5：由针对AR第五外显子E5的测序引物30μL水溶液，包含4μM SEQ ID NO.18；Reagent E5: 30 μL aqueous solution of sequencing primers targeting the fifth exon E5 of AR, containing 4 μM of SEQ ID NO.18;
试剂E6：由针对AR第五外显子E6的测序引物30μL水溶液，包含4μM SEQ ID NO.19；Reagent E6: 30 μL aqueous solution of sequencing primers targeting the fifth exon E6 of AR, containing 4 μM of SEQ ID NO.19;
试剂E7：由针对AR第五外显子E7的测序引物30μL水溶液，包含4μM SEQ ID NO.23；Reagent E7: 30 μL aqueous solution of sequencing primers targeting AR fifth exon E7, containing 4 μM SEQ ID NO.23;
试剂E8：由针对AR第五外显子E8的测序引物30μL水溶液，包含4μM SEQ ID NO.24；Reagent E8: 30 μL aqueous solution of sequencing primers targeting AR fifth exon E8, containing 4 μM SEQ ID NO.24;
包被引物的PCR反应管：PCR reaction tubes coated with primers:
一号反应管：含有针对AR第一外显子E1-A的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.1和1.5×10-2nmol SEQ ID NO.2；Reaction tube No. 1: containing the amplification primer mixture for the first exon E1-A of AR, including 1.5×10-2 nmol of SEQ ID NO.1 and 1.5×10-2 nmol of SEQ ID NO.2;
二号反应管：含有针对AR第一外显子E1-B的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.3和1.5×10-2nmol SEQ ID NO.4；Reaction tube No. 2: containing the amplification primer mixture for the first exon E1-B of AR, including 1.5×10-2 nmol of SEQ ID NO.3 and 1.5×10-2 nmol of SEQ ID NO.4;
三号反应管：含有针对AR第一外显子E1-C的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.5和1.5×10-2nmol SEQ ID NO.6；Reaction tube No. 3: containing the amplification primer mixture for the first exon E1-C of AR, including 1.5×10-2 nmol of SEQ ID NO.5 and 1.5×10-2 nmol of SEQ ID NO.6;
四号反应管：含有针对AR第一外显子E1-D的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.8和1.5×10-2nmol SEQ ID NO.9；Reaction tube No. 4: containing the amplification primer mixture for the first exon E1-D of AR, including 1.5×10-2 nmol of SEQ ID NO.8 and 1.5×10-2 nmol of SEQ ID NO.9;
五号反应管：含有针对AR第二外显子E2的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.10和1.5×10-2nmol SEQ ID NO.11；Reaction tube No. 5: containing the amplification primer mixture for AR second exon E2, including 1.5×10-2 nmol of SEQ ID NO.10 and 1.5×10-2 nmol of SEQ ID NO.11;
六号反应管：含有针对AR第三外显子E3的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.12和1.5×10-2nmol SEQ ID NO.13；Reaction tube No. 6: containing the amplification primer mixture for exon E3 of AR, including 1.5×10-2 nmol of SEQ ID NO.12 and 1.5×10-2 nmol of SEQ ID NO.13;
七号反应管：含有针对AR第三外显子E4的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.14和1.5×10-2nmol SEQ ID NO.15；Reaction tube No. 7: containing the amplification primer mixture for exon E4 of AR, including 1.5×10-2 nmol of SEQ ID NO.14 and 1.5×10-2 nmol of SEQ ID NO.15;
八号反应管：含有针对AR第三外显子E5的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.17和1.5×10-2nmol SEQ ID NO.18；Reaction tube No. 8: containing the amplification primer mixture for exon E5 of AR, including 1.5×10-2 nmol of SEQ ID NO.17 and 1.5×10-2 nmol of SEQ ID NO.18;
九号反应管：含有针对AR第三外显子E6的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.19和1.5×10-2nmol SEQ ID NO.20；Reaction tube No. 9: containing the amplification primer mixture for exon E6 of AR, including 1.5×10-2 nmol of SEQ ID NO.19 and 1.5×10-2 nmol of SEQ ID NO.20;
十号反应管：含有针对AR第三外显子E7的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.21和1.5×10-2nmol SEQ ID NO.22；Reaction tube No. 10: containing the amplification primer mixture for exon E7 of AR, including 1.5×10-2 nmol of SEQ ID NO.21 and 1.5×10-2 nmol of SEQ ID NO.22;
十一号反应管：含有针对AR第三外显子E8的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.24和1.5×10-2nmol SEQ ID NO.25。Reaction tube No. 11: containing the amplification primer mixture for exon E8 of AR, including 1.5×10-2 nmol of SEQ ID NO.24 and 1.5×10-2 nmol of SEQ ID NO.25.
本发明优点：Advantages of the present invention:
提供一种用于雄激素受体基因全编码区突变检测的扩增和测序引物及其试剂盒。其优点在于：1）提供了一套可进行快速PCR的引物和优化的测序引物，一次性扩增所有雄激素受体基因所有8个外显子的序列用于后续测序；2）反应体系中带荧光染料，可以直接通过荧光定量PCR观察扩增结果，避免电泳带来的不便和污染；3）可以发现雄激素受体第一外显子内的CAG重复序列，能提供更多的生物信息；4）引物直接包被在反应管中，避免引物配置困难。通过优化实验反应体系和反应条件，组成简单、快速、灵敏、特异的检测试剂盒，可用于对临床标本的检测，可减少人力和物力，提供更多的实验信息。Provided are an amplification and sequencing primer and a kit for detecting mutations in the entire coding region of the androgen receptor gene. Its advantages are: 1) It provides a set of primers for rapid PCR and optimized sequencing primers to amplify the sequences of all 8 exons of all androgen receptor genes at one time for subsequent sequencing; 2) in the reaction system With fluorescent dyes, the amplification results can be directly observed by fluorescent quantitative PCR, avoiding the inconvenience and pollution caused by electrophoresis; 3) The CAG repeat sequence in the first exon of the androgen receptor can be found, which can provide more biological information ; 4) The primers are directly coated in the reaction tube, avoiding the difficulty of primer configuration. By optimizing the experimental reaction system and reaction conditions, a simple, rapid, sensitive, and specific detection kit can be formed, which can be used for the detection of clinical specimens, can reduce manpower and material resources, and provide more experimental information.
附图说明Description of drawings
图1为人雄激素受体片段扩增的荧光值。Figure 1 is the fluorescence value of human androgen receptor fragment amplification.
图2为人雄激素受体片段测序结果。Figure 2 shows the sequencing results of human androgen receptor fragments.
具体实施方式Detailed ways
下述实施例中所用方法如无特别说明均为常规方法。The methods used in the following examples are conventional methods unless otherwise specified.
本发明的扩增和测序引物核苷酸序列为：Amplification and sequencing primer nucleotide sequence of the present invention is:
实施例1Example 1
一套用于雄激素受体扩增和测序用的特异性引物，包括第一外显子的扩增和测序引物为SEQ ID NO.1-SEQ ID NO.9，第二外显子的扩增和测序引物为SEQ ID NO.10和SEQ ID NO.11，第三外显子的扩增和测序引物为SEQ ID NO.12和SEQ ID NO.13，第四外显子的扩增和测序引物为SEQ ID NO.14-SEQ ID NO.16，第五外显子的扩增和测序引物为SEQ ID NO.17和SEQ ID NO.18，第六外显子的扩增和测序引物为SEQ ID NO.19和SEQ ID NO.20，第七外显子的扩增和测序引物为SEQ ID NO.21-SEQ ID NO.23，第八外显子的扩增和测序引物为SEQ ID NO.24和SEQ ID NO.25。A set of specific primers for the amplification and sequencing of the androgen receptor, including the amplification of the first exon and the sequencing primers are SEQID NO.1-SEQ ID NO.9, the amplification and sequencing primers of the second exon are SEQ ID NO.10 and SEQ ID NO.11, the amplification and sequencing primers of the third exon are SEQID NO.12 and SEQ ID NO.13, the amplification and sequencing primers of the fourth exon are SEQ ID NO.14-SEQ ID NO.16, the amplification and sequencing primers of the fifth exon are SEQ ID NO .17 and SEQID NO.18, the amplification and sequencing primers of the sixth exon are SEQ ID NO.19 and SEQ ID NO.20, the amplification and sequencing primers of the seventh exon are SEQ ID NO.19ID NO.21-SEQ ID NO.23, the amplification and sequencing primers of the eighth exon are SEQ ID NO.24 and SEQ ID NO.25.
实施例2Example 2
一种用于对雄激素受体进行扩增和测序的试剂盒，由下列试剂组成：A kit for amplifying and sequencing the androgen receptor, consisting of the following reagents:
试剂A：由2mL缓冲液组成，包含2×SYBR® Green I 染料，0.2mM dNTPs（包括dATP、dCTP、dGTP、dTTP）、80mM Tris-HCl、80mM KCl、40mM (NH4)2SO4、6mM MgSO4、100U Taq DNA聚合酶；Reagent A: Consists of 2mL buffer containing 2×SYBR® Green I dye, 0.2mM dNTPs (including dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4)2SO4, 6mMMgSO4, 100U Taq DNA polymerase;
试剂E1-A：由针对AR第一外显子E1-A的测序引物30μL水溶液，包含4μM SEQ ID NO.1；Reagent E1-A: 30 μL aqueous solution of sequencing primers targeting the first exon E1-A of AR, containing 4 μM SEQ ID NO.1;
试剂E1-B：由针对AR第一外显子E1-B的测序引物30μL水溶液，包含4μM SEQ ID NO.3；Reagent E1-B: 30 μL aqueous solution of sequencing primers targeting the first exon E1-B of AR, containing 4 μM SEQ ID NO.3;
试剂E1-C：由针对AR第一外显子E1-C的测序引物30μL水溶液，包含4μM SEQ ID NO.7；Reagent E1-C: 30 μL aqueous solution of sequencing primers targeting the first exon E1-C of AR, containing 4 μM SEQ ID NO.7;
试剂E1-D：由针对AR第一外显子E1-D的测序引物30μL水溶液，包含4μM SEQ ID NO.8；Reagent E1-D: 30 μL aqueous solution of sequencing primers targeting the first exon E1-D of AR, containing 4 μM SEQ ID NO.8;
试剂E2：由针对AR第二外显子E2的测序引物30μL水溶液，包含4μM SEQ ID NO.11；Reagent E2: 30 μL aqueous solution of sequencing primers targeting AR second exon E2, containing 4 μM SEQ ID NO.11;
试剂E3：由针对AR第三外显子E3的测序引物30μL水溶液，包含4μM SEQ ID NO.12；Reagent E3: 30 μL aqueous solution of sequencing primers targeting the third exon E3 of AR, containing 4 μM SEQ ID NO.12;
试剂E4：由针对AR第四外显子E4的测序引物30μL水溶液，包含4μM SEQ ID NO.16；Reagent E4: 30 μL aqueous solution of sequencing primers targeting the fourth exon E4 of AR, containing 4 μM SEQ ID NO.16;
试剂E5：由针对AR第五外显子E5的测序引物30μL水溶液，包含4μM SEQ ID NO.18；Reagent E5: 30 μL aqueous solution of sequencing primers targeting the fifth exon E5 of AR, containing 4 μM of SEQ ID NO.18;
试剂E6：由针对AR第五外显子E6的测序引物30μL水溶液，包含4μM SEQ ID NO.19；Reagent E6: 30 μL aqueous solution of sequencing primers targeting the fifth exon E6 of AR, containing 4 μM of SEQ ID NO.19;
试剂E7：由针对AR第五外显子E7的测序引物30μL水溶液，包含4μM SEQ ID NO.23；Reagent E7: 30 μL aqueous solution of sequencing primers targeting AR fifth exon E7, containing 4 μM SEQ ID NO.23;
试剂E8：由针对AR第五外显子E8的测序引物30μL水溶液，包含4μM SEQ ID NO.24；Reagent E8: 30 μL aqueous solution of sequencing primers targeting AR fifth exon E8, containing 4 μM SEQ ID NO.24;
包被引物的PCR反应管：PCR reaction tubes coated with primers:
一号反应管：含有针对AR第一外显子E1-A的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.1和1.5×10-2nmol SEQ ID NO.2；Reaction tube No. 1: containing the amplification primer mixture for the first exon E1-A of AR, including 1.5×10-2 nmol of SEQ ID NO.1 and 1.5×10-2 nmol of SEQ ID NO.2;
二号反应管：含有针对AR第一外显子E1-B的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.3和1.5×10-2nmol SEQ ID NO.4；Reaction tube No. 2: containing the amplification primer mixture for the first exon E1-B of AR, including 1.5×10-2 nmol of SEQ ID NO.3 and 1.5×10-2 nmol of SEQ ID NO.4;
三号反应管：含有针对AR第一外显子E1-C的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.5和1.5×10-2nmol SEQ ID NO.6；Reaction tube No. 3: containing the amplification primer mixture for the first exon E1-C of AR, including 1.5×10-2 nmol of SEQ ID NO.5 and 1.5×10-2 nmol of SEQ ID NO.6;
四号反应管：含有针对AR第一外显子E1-D的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.8和1.5×10-2nmol SEQ ID NO.9；Reaction tube No. 4: containing the amplification primer mixture for the first exon E1-D of AR, including 1.5×10-2 nmol of SEQ ID NO.8 and 1.5×10-2 nmol of SEQ ID NO.9;
五号反应管：含有针对AR第二外显子E2的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.10和1.5×10-2nmol SEQ ID NO.11；Reaction tube No. 5: containing the amplification primer mixture for AR second exon E2, including 1.5×10-2 nmol of SEQ ID NO.10 and 1.5×10-2 nmol of SEQ ID NO.11;
六号反应管：含有针对AR第三外显子E3的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.12和1.5×10-2nmol SEQ ID NO.13；Reaction tube No. 6: containing the amplification primer mixture for exon E3 of AR, including 1.5×10-2 nmol of SEQ ID NO.12 and 1.5×10-2 nmol of SEQ ID NO.13;
七号反应管：含有针对AR第三外显子E4的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.14和1.5×10-2nmol SEQ ID NO.15；Reaction tube No. 7: containing the amplification primer mixture for exon E4 of AR, including 1.5×10-2 nmol of SEQ ID NO.14 and 1.5×10-2 nmol of SEQ ID NO.15;
八号反应管：含有针对AR第三外显子E5的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.17和1.5×10-2nmol SEQ ID NO.18；Reaction tube No. 8: containing the amplification primer mixture for exon E5 of AR, including 1.5×10-2 nmol of SEQ ID NO.17 and 1.5×10-2 nmol of SEQ ID NO.18;
九号反应管：含有针对AR第三外显子E6的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.19和1.5×10-2nmol SEQ ID NO.20；Reaction tube No. 9: containing the amplification primer mixture for exon E6 of AR, including 1.5×10-2 nmol of SEQ ID NO.19 and 1.5×10-2 nmol of SEQ ID NO.20;
十号反应管：含有针对AR第三外显子E7的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.21和1.5×10-2nmol SEQ ID NO.22；Reaction tube No. 10: containing the amplification primer mixture for exon E7 of AR, including 1.5×10-2 nmol of SEQ ID NO.21 and 1.5×10-2 nmol of SEQ ID NO.22;
十一号反应管：含有针对AR第三外显子E8的扩增引物混合组成，包含1.5×10-2nmol SEQ ID NO.24和1.5×10-2nmol SEQ ID NO.25。Reaction tube No. 11: containing the amplification primer mixture for exon E8 of AR, including 1.5×10-2 nmol of SEQ ID NO.24 and 1.5×10-2 nmol of SEQ ID NO.25.
实施例3Example 3
用本发明试剂盒进行实时荧光定量PCR（real-time FQ PCR）扩增雄激素受体的所有外显子。首先，按实验的标本个数计算各试剂的需要量，实验步骤如下：Carry out real-time fluorescent quantitative PCR (real-timeFQ PCR) amplifies all exons of the androgen receptor. First, calculate the required amount of each reagent according to the number of experimental specimens. The experimental steps are as follows:
1．扩增反应液配置：按顺序配置反应液（1个检测标本），加入水120μL、试剂A 150μL、DNA标本30μL，共300μL；混匀。1. Amplification reaction solution configuration: Prepare the reaction solution (1 test sample) in order, add 120 μL of water, 150 μL of reagent A, and 30 μL of DNA sample, a total of 300 μL; mix well.
2．各管按顺序加入25μL 的扩增反应液，盖好盖子。2. Add 25μL amplification reaction solution to each tube sequentially, and cover the cap well.
3．按实验设计的PCR程序进行扩增：为95℃预变性3min 后， 94℃变性1min ，60℃退火1mins ，72℃延伸35s，延伸期获取荧光值，进行35个循环，最后保存在25℃；及时取出进行测序。3. Amplify according to the PCR program designed for the experiment: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 35 seconds. During the extension period, the fluorescence value was obtained for 35 cycles, and finally stored at 25°C; Remove in time for sequencing.
4.荧光值在22-28个循环起跳，各组同时起跳认为是特异性扩增,见图1。4. Fluorescence value jumps at 22-28 cycles, and jumps at the same time in each group are considered as specific amplification, as shown in Figure 1.
实施例4Example 4
用本发明试剂盒配套的引物进行测序：Sequencing with the matching primers of the kit of the present invention:
1. PCR产物纯化（ExoI/SAP方法）：1. PCR product purification (ExoI/SAP method):
1.1配制纯化体系：核酸外切酶1（ExoI）6μL +虾解酶（SAP）12μL +水6μL，共24μL，混匀。1.1 Prepare the purification system: exonuclease 1 (ExoI) 6 μL + shrimp lytic enzyme (SAP) 12 μL + water 6 μL, a total of 24 μL, mix well.
1.2取配制的纯化体系2μL与每个扩增产物8μL混合（共11个），加入另个空白的反应板内。1.2 Mix 2 μL of the prepared purification system with 8 μL of each amplification product (11 in total), and add to another blank reaction plate.
1.3将反应板放在PCR仪以上，37℃ 30min，80℃ 20min，可在4℃冰箱内保存1周左右。1.3 Place the reaction plate on the PCR instrument, heat at 37°C for 30 minutes, 80°C for 20 minutes, and store it in a refrigerator at 4°C for about 1 week.
2．测序反应：2. Sequencing reaction:
2.1 10μL PCR纯化产物中每孔加入20μL水，充分混匀后，离心。2.1 Add 20 μL of water to each well of 10 μL PCR purified product, mix well, and centrifuge.
2.2 配制测序反应体系：取4.8μL BDT Ready Reaction Premix(2.5×)加21.6μL BDT 缓冲液(5×)加69.6μL Deionised water混合。每次取8μL上述混合液分别与E1-A、E1-B、E1-C、E1-D、E2、E3、E4、E5、E6、E7、E8各1μL充分混匀，快速离心。2.2 Prepare the sequencing reaction system: Take 4.8μL BDT ReadyReaction Premix (2.5×) plus 21.6 μL BDT buffer solution (5×) plus 69.6 μL Deionised water and mix. Take 8 μL of the above mixture each time and mix well with 1 μL each of E1-A, E1-B, E1-C, E1-D, E2, E3, E4, E5, E6, E7, and E8, and centrifuge quickly.
2.3每孔PCR产物稀释液取1μL至反应孔中（11孔），每孔加入9μL测序反应混合液，混匀，快速离心。2.3 Take 1 μL of the PCR product dilution solution from each well into the reaction wells (11 wells), add 9 μL of the sequencing reaction mixture to each well, mix well, and centrifuge quickly.
2.4将反应板放在PCR仪上，反应板顶部置PCR密封压力垫，以防止液体蒸发，关上PCR仪,运行测序反应：96℃ 10s后，进行25个循环为96℃ 10s，50℃ 10s，60℃ 2min，最后保存15℃并取出。2.4 Put the reaction plate on the PCR instrument, place the PCR sealing pressure pad on the top of the reaction plate to prevent the liquid from evaporating, close the PCR instrument, and run the sequencing reaction: after 96°C for 10s, perform 25 cycles of 96°C for 10s, 50°C for 10s, 60°C for 2 minutes, and finally stored at 15°C and taken out.
3．测序反应产物纯化：3. Sequencing reaction product purification:
3.1每孔测序反应孔内加入1μLEDTA、1μL NaAc和25μL无水乙醇，充分震荡混匀30s。室温放置15min，3000rpm离心30min. 3.1 Add 1 μL of LEDTA, 1 μL of NaAc and 25 μL of absolute ethanol to each sequencing reaction well, shake and mix well for 30 seconds. Place at room temperature for 15 minutes, then centrifuge at 3000rpm for 30 minutes.
3.2倒置反应板，调节转速，500rpm离心1min 3.2 Invert the reaction plate, adjust the rotation speed, and centrifuge at 500rpm for 1min
3.3每孔测序反应产物中加入50μL 70%乙醇，3000 rpm离心10min。 3.3 Add 50 μL of 70% ethanol to each well of the sequencing reaction product, and centrifuge at 3000 rpm for 10 min.
3.4倒置反应板，500rpm离心1min。 3.4 Invert the reaction plate and centrifuge at 500rpm for 1min.
3.5室温避光静置30min,充分挥发板内乙醇。 3.5 Stand at room temperature in the dark for 30 minutes to fully evaporate the ethanol in the plate.
3.6每孔内加入甲酰胺10μL，封板，放在PCR以上95℃ 2min，迅速放入-20℃冰箱内冷却3-5min. 3.6 Add 10 μL of formamide to each well, seal the plate, place it above PCR at 95°C for 2 minutes, and quickly put it in a -20°C refrigerator to cool for 3-5 minutes.
4．测序仪读板检测：使用ABI3500测序仪对测序结果进行检测。测序结果使相应的测序软件进行数据分析，见图2。4. Sequencer plate reading detection: use the ABI3500 sequencer to detect the sequencing results. The sequencing results were analyzed by corresponding sequencing software, as shown in Figure 2.
实施例5Example 5
测序结果与雄激素受体标准序列进行比对：The sequencing results were compared with the standard sequence of androgen receptor:
1．测序结果进行拼接；1. Sequencing results were spliced;
2．将雄激素受体标准测序和测序结果输入软件进行比对；2. Input the androgen receptor standard sequencing and sequencing results into the software for comparison;
3．核对突变性质并分析第一外显子CAG的重复频率。3. The mutation nature was checked and the repeat frequency of the first exon CAG was analyzed.
序列表sequence listing
<110> 温州医科大学附属第一医院<110> The First Affiliated Hospital of Wenzhou Medical University
<120> 人雄激素受体基因突变检测的试剂盒<120> Kit for detection of human androgen receptor gene mutation
<160> 25<160> 25
<170> PatentIn Version 2.1<170> Patent In Version 2.1
<210> 1<210> 1
<211> 19<211> 19
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(19)<222>(1)...(19)
<400> 1<400> 1
gcagcgacta ccgcatcat 19gcagcgacta ccgcatcat19
<210> 2<210> 2
<211> 18<211> 18
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(18)<222>(1)...(18)
<400> 2<400> 2
acaacgtgga tggggcag 18acaacgtgga tggggcag18
<210> 3<210> 3
<211> 20<211> 20
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(20)<222>(1)...(20)
<400> 3<400> 3
gagagaggtt gcgtcccaga 20gagagaggtt gcgtccccaga 20
<210> 4<210> 4
<211> 22<211> 22
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(22)<222>(1)...(22)
<400> 4<400> 4
cctttggtgt aacctccctt ga 22cctttggtgt aacctccctt ga 22
<210> 5<210> 5
<211> 21<211> 21
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(21)<222>(1)...(21)
<400> 5<400> 5
tgtaaggcag tgtcggtgtc c 21tgtaaggcag tgtcggtgtc c 21
<210> 6<210> 6
<211> 21<211> 21
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(21)<222>(1)...(21)
<400> 6<400> 6
ccatacaact ggccttcttc g 21ccatacaact ggccttcttc g 21
<210> 7<210> 7
<211> 19<211> 19
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(19)<222>(1)...(19)
<400> 7<400> 7
gaggcgttgg agcatctga 19gaggcgttgg agcatctga19
<210> 8<210> 8
<211> 20<211> 20
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(20)<222>(1)...(20)
<400> 8<400> 8
tggagaaccc gctggactac 20tggagaaccc gctggactac 20
<210> 9<210> 9
<211> 21<211> 21
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(21)<222>(1)...(21)
<400> 9<400> 9
gcacgcagca gaaattagga g 21gcacgcagca gaaattagga g 21
<210> 10<210> 10
<211> 25<211> 25
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(25)<222>(1)...(25)
<400> 10<400> 10
agcttcacac taactaactt gagca 25agcttcacac taactaactt gagca 25
<210> 11<210> 11
<211> 23<211> 23
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(23)<222>(1)...(23)
<400> 11<400> 11
gcaagtacaa aaatttgctt cct 23gcaagtacaa aaatttgctt cct 23
<210> 12<210> 12
<211> 25<211> 25
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(25)<222>(1)...(25)
<400> 12<400> 12
tgaataatag tcatttatgc ctgca 25tgaataatag tcatttatgc ctgca 25
<210> 13<210> 13
<211> 22<211> 22
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(22)<222>(1)...(22)
<400> 13<400> 13
ctatgaaagg gtcagcctgt gt 22ctatgaaagg gtcagcctgt gt 22
<210> 14<210> 14
<211> 23<211> 23
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(23)<222>(1)...(23)
<400> 14<400> 14
gtttagagtc tgtgaccagg gag 23gtttagagtc tgtgaccagg gag 23
<210> 15<210> 15
<211> 22<211> 22
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(22)<222>(1)...(22)
<400> 15<400> 15
ctgagttaat gggcagaaaa gc 22ctgagttaat gggcagaaaa gc 22
<210> 16<210> 16
<211> 23<211> 23
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(23)<222>(1)...(23)
<400> 16<400> 16
gggagaatgg tgattttctt agc 23gggagaatgg tgattttctt agc 23
<210> 17<210> 17
<211> 24<211> 24
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(24)<222>(1)...(24)
<400> 17<400> 17
tcaccatata gtttgtgctt ttcc 24tcaccatata gtttgtgctt ttcc 24
<210> 18<210> 18
<211> 24<211> 24
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(24)<222>(1)...(24)
<400> 18<400> 18
acaactgcag atcaaatgag ctaa 24acaactgcag atcaaatgag ctaa 24
<210> 19<210> 19
<211> 21<211> 21
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(21)<222>(1)...(21)
<400> 19<400> 19
gctcttcttg gaaaacctgg c 21gctcttcttg gaaaacctgg c 21
<210> 20<210> 20
<211> 24<211> 24
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(24)<222>(1)...(24)
<400> 20<400> 20
gctggctttt ccctaataat gttt 24gctggctttt ccctaataat gttt 24
<210> 21<210> 21
<211> 24<211> 24
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(24)<222>(1)...(24)
<400> 21<400> 21
agcacacaga cttcaactaa cagg 24agcacacaga cttcaactaa cagg 24
<210> 22<210> 22
<211> 22<211> 22
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(22)<222>(1)...(22)
<400> 22<400> 22
ttcttcctgg accacactca aa 22ttcttcctgg accacactca aa 22
<210> 23<210> 23
<211> 22<211> 22
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(22)<222>(1)...(22)
<400> 23<400> 23
aagccaagta gatggttccc tg 22aagccaagta gatggttccc tg 22
<210> 24<210> 24
<211> 22<211> 22
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(22)<222>(1)...(22)
<400> 24<400> 24
aggaaacaaa aggctgaaag ac 22aggaaacaaa aggctgaaag ac 22
<210> 25<210> 25
<211> 24<211> 24
<212> DNA<212>dna
<213> 人工序列<213> Artificial sequence
<220><220>
<221> prim_bind<221>prim_bind
<222> (1)...(24)<222>(1)...(24)
<400> 25<400> 25
agtgcagagt tataacaggc agaa 24agtgcagagt tataacaggc agaa 24
| Application Number | Priority Date | Filing Date | Title |
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| CN201410223073.7ACN103993084B (en) | 2014-05-26 | 2014-05-26 | Amplification and sequencing primers and kits for detecting mutations in the full coding region of androgen receptor gene |
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| CN201410223073.7ACN103993084B (en) | 2014-05-26 | 2014-05-26 | Amplification and sequencing primers and kits for detecting mutations in the full coding region of androgen receptor gene |
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| CN103993084A CN103993084A (en) | 2014-08-20 |
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| CN108060232A (en)* | 2017-12-13 | 2018-05-22 | 安徽师范大学 | For expanding tortoise androgen receptor（AR）The RT-qPCR primers and method of gene |
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| CN101905788A (en)* | 2010-07-14 | 2010-12-08 | 中冶赛迪工程技术股份有限公司 | A self-locking bottom opening trough |
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