CN100362110C - 瘦素和瘦素受体基因多态性检测芯片及其制备方法和用途 - Google Patents
瘦素和瘦素受体基因多态性检测芯片及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种瘦素和瘦素受体基因多态性检测芯片及其制备方法和用途。在载体上设有如下探针:1 25 CCC ATC CAA AAA GTC2 25 CCC ATC CAG AAA GTC3 25 CCC ATC CAC AAA GTC4 25 CCC ATC CAT AAA GTC5 85 TAT CCA AAA CAA CTT TCC6 85 TAT CCA AAG CAA CTT TCC7 85 TAT CCA AAC CAA CTT TCC8 85 TAT CCA AAT CAA CTT TCC9 109 TGA AGG AAA GAC ATT TGT10 109 TGA AGG AAG GAC ATT TGT11 109 TGA AGG AAC GAC ATT TGT12 109 TGA AGG AAT GAC ATT TGT13 223 ATT TTC CAG TCA CCT CT14 223 ATT TTC CGG TCA CCT CT15 223 ATT TTC CCG TCA CCT CT16 223 ATT TTC CTG TCA CCT CT本发明的优点是:实现了对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性进行检测,具有快速、简捷、高通量的特点,有助于肥胖、糖尿病等易感者的筛选,推动基因诊断和治疗的发展,具有重要的社会和经济效益。
Description
技术领域
本发明涉及一种瘦素和瘦素受体基因多态性检测芯片及其制备方法和用途。
背景技术
瘦素是人和动物肥胖基因编码的一种多靶器官、多功能的分泌型产物,并与瘦素运转蛋白结合,通过与定位于中枢和外周多种组织及其多种形式的瘦素受体结合发挥生物学效应,作为一种机体代谢状态的信号,影响机体生理功能及代谢通路,进而调节体内脂质沉积和能量平衡。肥胖、糖耐量异常和糖尿病、血脂异常、高血压、高胰岛素血症和胰岛素抵抗以及高瘦素血症等因素密切相关。而瘦素受体的结构和功能的改变直接影响着瘦素的生物学功能。
至今为止,已发现多种瘦素瘦素受体基因多态性,如瘦素CAA25CAG、瘦素受体Asn85Ser、Lys109Arg和Gln223Arg等,这些多态性导致基因产物发生变化,而这些变化与糖尿病、高血压、肥胖等多种疾病相关。
日本学者研究发现肥胖患者中瘦素25CAG变异发生率较正常对照者明显增多,认为瘦素25CAG变异可以作为一个新的肥胖易感的遗传标志。[1]日本学者认为瘦素受体在胰岛素抵抗的发病机制中起着修饰基因的作用[2]。Van等学者认为Lys109Arg变异对瘦素受体功能有修饰作用,研究发现肥胖者携带Arg109等位基因比非携带者瘦素水平明显升高[3],瘦素受体基因Lys109Arg多态性可能导致肥胖表型的差异[4],并对体重增加的易感性增加[5]。Gln223Arg变异影响外周和中枢瘦素与瘦素受体的结合能力。Stefan N研究发现Arg223纯合体24小时能量消耗和体力劳动强度低于Gln223纯合体、而腹部皮下脂肪细胞体积明显大于Gln223纯合体。[6]Chiu Kc等通过多因素方差分析发现Gln223Arg基因多态性与胰岛素敏感指数和葡萄糖清除率独立相关。[7]日本学者发现LEPR Arg223Gln基因型携带者血清胆固醇和LDL-C明显升高,而Arg223基因型携带者血清HDL-C水平降低,并对HMG-CoA还原酶抑制剂的反应减弱[8]。
用于SNPs做基因分析是特指检测人类基因组中大量存在的单核苷酸变异,这与疾病易感性和毒物易感性有关[9]。SNPs不仅是人类种族和个体差异的标记,亦是决定不同人群种族和个体差异的标记,可以成为寻找疾病相关基因,进行疾病诊断、预防和药物筛选的基础。用于SNPs检测的常规方法有等位基因特异性寡核苷酸(ASO)杂交,限制性内切酶法(RFLP),位点特异性引物PCR(PCR-SSP)和直接测序等。基因芯片技术的发展为新的SNPs的检测方法提供了可能性[10]。寡核苷酸芯片技术是新近发展起来的一种基因检测技术,它可将大量的寡核苷酸探针有规律地排列在一张玻片上,这些探针可以与信号显示物质标记的样品DNA的扩增序列的互补序列进行相结合,通过对信号物质进行检测,对杂交结果进行计算机软件处理分析,从而获得杂交信号的强度及其分布模式图。
1.Ohshiro Y,Ueda K,Nishi M,Ishigame M,Wakasaki H,Kawashima H,Furuta H,SasakiH,Sanke T,Takasu N,Nanjo K.A polymorphic marker in the leptin gene associatedwith Japanese morbid obesity.J Mol Med.2000;78(9):516-20
2.van Rossum CT,Hoebee B,van Baak MA,et al.Genetic variation in the leptinreceptor gene,leptin,and weight gain in young Dutch adults.Obes Res.2003Mar:11(3):377-86
3.Takahashi-Yasuno A,Masuzaki H,Miyawaki T,et al.Association of Ob-R genepolymorphism and insulin resistance in Japanese men.Metabolism.2004May;53(5):650-4
4.Liu YJ,Rocha-Sanchez SM,Liu PY,et al.Tests of linkage and/or association ofthe LEPR gene polymorphisms with obesity phenotypes in Caucasian nuclear families.Physiol Genomics.2004 Apr 13;17(2):101-6
5.van Rossum CT,Hoebee B,Seidell JC,et al.Genetic factors as predictors of weightgain in young adult Dutch men and women.Int J Obes Relat Metab Disord.2002;26(4):517-28
6.Stefan N,Vozarova B,Del Parigi A,Ossowski V,Thompson DB,Hanson RL,RavussinE,Tataranni PA.The Gln223Arg polymorphism of the leptin receptor in Pima Indians:influence on energy expernditure,physical activity and lipid metabolism.Int JObes Relat Metab Disord.2002 Dec;26(12):1629-32
7.Chiu KC,Chu A,Chuang LM,Saad MF.Association of leptin receptor polymorphismwith insul in resistance.Eur J Endocrinol.2004 May;150(5):725-9
8.Takahashi-Yasuno A,Masuzaki H,Miyawaki T,Ogawa Y,Matsuoka N,Hayashi T,HosodaK,Inoue G,Yoshimasa Y,Nakao K.Leptin receptor polymorphism is associated withserum lipid levels and impairment of cholesterol lowering effect by simvastatinin Japanese men.Diabetes Res Clin Pract.2003 Dec;62(3):169-75
9.Khner M K,Beerli P,Yamato J,et al.Usefulness of single nucleatide polymorphismdata for estimating population parameters.Genetics,2000,156:439-447
10.Hirschlhorn J N,Sklar P,Lindblad-Toh K,et al.SBE-TAGS:an array-based method forefficient single-nucleotide polymorphism genotyping.Proc Natl Acad SciUSA,2000,97:12164-12169
发明内容
本发明的目的是提供一种瘦素和瘦素受体基因多态性检测芯片及其制备方法和用途。
瘦素和瘦素受体基因多态性检测芯片:在载体上设有如下探针:
1 25 CCC ATC CAAAA GTC
9 109 TGA AGG AAA GAC ATT TGT
14 223 ATT TTC CG TCA CCT CT
16 223 ATT TTC CG TCA CCT CT
瘦素和瘦素受体基因多态性检测芯片制备方法步骤为:
1)芯片载体的处理:玻片用铬酸洗液浸泡过夜,水洗,24~26%氨水中过夜,水洗,玻璃片浸入氨丙基三甲氧基硅烷的95%乙醇溶液中,用冰醋酸调节pH值至4~4.5,95%乙醇超声清洗,150~160℃烘干3~5小时,9~11%戊二醛处理成醛基化;
2)引物及探针的合成:针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计了一组探针,它的长度范围在15-18个碱基,(表1)针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计合成了3对引物,它的长度范围在19-24个碱基,(表2),每条引物中各有一条Cyp-3信号分子标记,合成后用浓氨水50~60℃脱保护,切割13~17小时,OPC柱纯化,紫外定量后真空干燥浓缩,-20℃保存备用;
3)基因芯片制备及后处理:将探针稀释至终浓度为100μmol/L;用750mol/LNACl,75mmol/L乙酸钠,5%glycerol,1%Ficoll and 0.1%SDS合成点样液;前述两者1∶1稀释,并取10μL至96孔板,用芯片点样仪点至醛基化玻片上,每点体积约为0.5nL,点心间距为500μm,点直径约为200μm,每条探针重复点2次,点样完毕后,室温放置24小时固相化。
瘦素和瘦素受体基因多态性检测芯片用于检测瘦素和瘦素受体基因多态性。
本发明将经过选择的探针点样于玻片上,分别与经PCR扩增的瘦素和瘦素受体基因片段进行杂交,一次性获得各位点的多态性情况,从而实现对瘦素和瘦素受体基因相关基因多态性进行快速、简捷、高通量检测和分型。有助于肥胖、糖尿病等易感者的筛选,可以作为疾病的预防、诊断和药物筛选的基础,推动基因诊断和治疗的发展。寡核苷酸芯片技术是新近发展起来的一种基因检测技术,它可将大量的寡核苷酸探针有规律地排列在一张玻片上,这些探针可以与信号显示物质标记的样品DNA的扩增序列的互补序列进行相结合,通过对信号物质进行检测,对杂交结果进行计算机软件处理分析,从而获得杂交信号的强度及其分布模式图。基于寡核苷酸芯片的方法为分析SNP提供了一种高通量的技术,为SNP或基因突变提供了一个高通量的检测平台,可用来平行分析已知基因在不同疾病样本中的突变或SNP位点,以获得疾病易感基因的遗传学改变,并作为线索加强临床上早预防、早发现、早诊断和早治疗,具有重要的社会和经济效益。
具体实施方式
本发明玻片上固相化的探针为寡核苷酸探针(表2),其序列根据瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性特征设计。
所说引物是针对瘦素和瘦素受体基因多态性特征设计合成引物(表2),用于上述基因的扩增。信号分子标记是针对瘦素和瘦素受体基因序列扩增的引物。信号分子标记是荧光分子。
表1:瘦素和瘦素受体基因多态性检测芯片的探针
5 85 TAT CCA AACAA CTT TCC
9 109 TGA AGG AAA GAC ATT TGT
表2:瘦素和瘦素受体基因多态性检测芯片的引物
基因 | 正向引物 | 反向引物(或荧光标记反向引物) |
Leptin | 5’-gcc cta tct ttt cta tgt cca-3’ | 5’-gtg tgt gaa atg tca ttg atc c-3’ |
Leptinreceptor | 5’-gag aca gct gtt gaa cct aag-3’ | 5’-aga cat cta ttt cat aca ggt atc-3’ |
Leptinreceptor | 5’-ctg tgc caa cag cca aac t-3’ | 5’-ccc ata ttt atg ggc tga act-3’ |
实施例1:寡核苷酸基因芯片的制备:
1)芯片载体的处理:波片用铬酸洗液浸泡过夜,水洗,24%氨水中过夜,水洗。玻璃片浸入氨丙基三甲氧基硅烷的95%乙醇溶液中,用冰醋酸调节pH值至4.0,95%乙醇超声清洗,15℃烘干3小时,9%戊二醛处理成醛基化。
2)引物及探针的合成:本发明针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计了一组探针(见表1)。针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计合成了6条引物(见表2)。每条引物中各有一条Cyp-3荧光标记。合成后用浓氨水80℃脱保护,切割13小时,OPC柱纯化。紫外定量后真空干燥浓缩,-20℃保存备用。
3)基因芯片制备及后处理:将探针稀释至终浓度为100μmol/L,与点样液(750mmol/LNACl,75mmol/L乙酸钠,5%glycerol,1%Ficoll and 0.1%SDS)1∶1稀释,并取10μL至96孔板,用芯片点样仪(Cartisan)点至醛基化玻片上,每点体积约为0.5nL,点心间距为500μm,点直径约为200μm,每条探针重复点2次,点样完毕后,室温放置24小时固相化。
实施例2:核苷酸基因芯片的制备:
1)芯片载体的处理:波片用铬酸洗液浸泡过夜,水洗,26%氨水中过夜,水洗。玻璃片浸入氨丙基三甲氧基硅烷的95%乙醇溶液中,用冰醋酸调节pH值至4.5,95%乙醇超声清洗,160℃烘干5小时,11%戊二醛处理成醛基化。
2)引物及探针的合成:本发明针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计了一组探针(见表1)。针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计合成了6条引物(见表2)。每条引物中各有一条Cyp-3荧光标记。合成后用浓氨水80℃脱保护,切割15小时,OPC柱纯化。紫外定量后真空干燥浓缩,-20℃保存备用。
3)基因芯片制备及后处理:将探针稀释至终浓度为100μmol/L,与点样液(750mmol/LNACl,75mmol/L乙酸钠,5%glycerol,1%Ficoll and 0.1%SDS)1∶1稀释,并取10μL至96孔板,用芯片点样仪(Cartisan)点至醛基化玻片上,每点体积约为0.5nL,点心间距为500μm,点直径约为200μm,每条探针重复点2次,点样完毕后,室温放置24小时固相化。
实施例3核苷酸基因芯片的制备:
1)芯片载体的处理:波片用铬酸洗液浸泡过夜,水洗,25%氨水中过夜,水洗。玻璃片浸入氨丙基三甲氧基硅烷的95%乙醇溶液中,用冰醋酸调节pH值至4.2,95%乙醇超声清洗,155℃烘干4小时,10%戊二醛处理成醛基化。
2)引物及探针的合成:本发明针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计了一组探针(见表1)。针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计合成了6条引物(见表2)。每条引物中各有一条Cyp-3荧光标记。合成后用浓氨水80℃脱保护,切割15小时,OPC柱纯化。紫外定量后真空干燥浓缩,-20℃保存备用。
3)基因芯片制备及后处理:将探针稀释至终浓度为100μmol/L,与点样液(750mmol/LNACl,75mmol/L乙酸钠,5%glycerol,1%Ficoll and 0.1%SDS)1∶1稀释,并取10μL至96孔板,用芯片点样仪(Cartisan)点至醛基化玻片上,每点体积约为0.5 nL,点心间距为500μm,点直径约为200μm,每条探针重复点2次,点样完毕后,室温放置24小时固相化。
瘦素和瘦素受体基因多态性检测芯片的使用方法为:
1)载有寡核苷酸探针的玻片使用前用0.2%SDS和清水各洗两次,空气干燥后1%NaBH4溶液还原10min,0.2%SDS洗一次,水洗一次,空气干燥后用待用,完成将探针共价固相化于玻片表面。
2)为产生单链的目标片段采用不对称PCR扩增的方法,上游引物与下游引物(或荧光标记下游引物)的比例优化为1∶10。20μL反应体系中含有1.5mM MgCl2,dNTP各200μmol/L,正向引物0.1μmol,反向引物1μmol,DNA 50ng,1×PCR缓冲液和1U Taq酶。PCR扩增条件为:预变性94℃ 5min;变性94℃ 30sec,退火55℃ 30sec,延伸72℃ 30sec,共30循环;最后延伸72℃ 5min。PCR产物用2%琼脂糖凝胶电泳分析。
3)Cy3-标记的不对称PCR产物与杂交液(750mmol/LNACl,75mmol/Lsodium acetate,0.1%SDS,1ug/ml鲑精DNA,2.5%甲酰铵)按2∶8混合,将10μL混合液转移至芯片的杂交区域。芯片置于杂交盒中在42℃水浴中保温1小时。杂交后取出芯片依次在洗液A(150mmol/LNACl,15mmol/L乙酸钠,0.2% SDS),洗液B(30mmol/LNACl,3mmol/L乙酸钠)和洗液C(15mmol/LNACl,1.5mmol/L乙酸钠)中各洗涤1分钟。待干后芯片用激光共聚焦扫描仪GenePix 4000(Axon Instrument)在激发波长532nm、发射波长570nm(Cy3)扫描,产生并分析精度为10μm的16位TIFF图象。
瘦素和瘦素受体基因多态性使用实例:
在浙江大学医学院附属第一医院接受体检180例成年人,根据脂肪肝的诊断标准,分成2组:(1)脂肪肝组117例,男77例,女40例,年龄47.97±13.46岁;(2)对照组63例,男32例,女31例,年龄55.13±10.92岁。同时测定身高、体重、臀围、腰围、腹壁脂肪厚度、体脂含量、血压。所有入选对象均签署知情同意书。试验对象进行空腹采血,部分血标本用于检测总蛋白、白蛋白、丙基氨酸转移酶、甘油三酯、高密度脂蛋白和血糖水平。部分血标本用Genomic DNA purification kit(Promega公司)提取基因组DNA,寡核苷酸微阵列技术检测瘦素与瘦素受体基因多态性。结果为:所有研究对象瘦素第25位氨基酸和瘦素受体85位氨基酸均为野生型,没有发现变异情况。脂肪性肝病组瘦素受体基因Arg223和Arg223Gln两种基因型分布分别为89例和28例;正常对照组分别为53例和10例;脂肪性肝病组瘦素受体基因Lys109、Lys109Arg和Arg109三种基因型分布分别为95例、21例和1例,正常对照组分别为49例、12例和10例。
测序验证:
测序采用CEQTM TDCS试剂盒(BECKMAN COULTERTM,USA),在CEQTM 2000XL测序仪(BECKMAN COULTERTM,USA)上进行。结果寡核苷酸微阵列技术与测序检测瘦素与瘦素受体基因多态性结果完全一致。
Claims (7)
1.一种瘦素和瘦素受体基因多态性检测芯片,其特征在于:在载体上设有如下探针:
1 瘦素基因CAA25CAG CCC ATC CAA AAA GTC
2 瘦素基因CAA25CAG CCC ATC CAG AAA GTC
3 瘦素基因CAA25CAG CCC ATC CAC AAA GTC
4 瘦素基因CAA25CAG CCC ATC CAT AAA GTC
5 瘦素受体基因Asn85Ser TAT CCA AAA CAA CTT TCC
6 瘦素受体基因Asn85Ser TAT CCA AAG CAA CTT TCC
7 瘦素受体基因Asn85Ser TAT CCA AAC CAA CTT TCC
8 瘦素受体基因Asn85Ser TAT CCA AAT CAA CTT TCC
9 瘦素受体基因Lys109Arg TGA AGG AAA GAC ATT TGT
10 瘦素受体基因Lys109Arg TGA AGG AAG GAC ATT TGT
11 瘦素受体基因Lys109Arg TGA AGG AAC GAC ATT TGT
12 瘦素受体基因Lys109Arg TGA AGG AAT GAC ATT TGT
13 瘦素受体基因Gln223Arg ATT TTC CAG TCA CCT CT
14 瘦素受体基因Gln223Arg ATT TTC CGG TCA CCT CT
15 瘦素受体基因Gln223Arg ATT TTC CCG TCA CCT CT
16 瘦素受体基因Gln223Arg ATT TTC CTG TCA CCT CT。
2.根据权利要求1所述的一种瘦素和瘦素受体基因多态性检测芯片,其特征在于:所说的载体为玻片。
3.一种如权利要求1所述瘦素和瘦素受体基因多态性检测芯片的制备方法,其特征在于:方法的步骤为:
1)芯片载体的处理:玻片用铬酸洗液浸泡过夜,水洗,24~26%氨水中过夜,水洗,玻片浸入氨丙基三甲氧基硅烷的95%乙醇溶液中,用冰醋酸调节pH值至4~4.5,95%乙醇超声清洗,150~160℃烘干3~5小时,9~11%戊二醛处理成醛基化;
2)引物及探针的合成:针对瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性位点设计了一组探针,它的长度范围在15-18个碱基,针对瘦素基因设计1对引物,针对瘦素受体基因设计合成2对引物,下游引物用Cyp-3荧光标记,合成后用浓氨水50~60℃脱保护,切割13~17小时,OPC柱纯化,紫外定量后真空干燥浓缩,-20~-80℃保存备用;
3)基因芯片制备及后处理:将探针稀释至终浓度为100μmol/L,与点样液1∶1稀释,点样液的配方为:750mmol/L NACl,75mmol/L乙酸钠,5%glycerol,1%Ficoll and 0.1%SDS,并取10μL至96孔板,用芯片点样仪点至醛基化玻片上,每点体积为0.5nL,点心间距为500μm,点直径约为200μm,每条探针重复点2次,点样完毕后,室温放置24小时即可。
4.根据权利要求3所述的一种瘦素和瘦素受体基因多态性检测芯片的制备方法,其特征在于:所说的探针产:
1 瘦素基因CAA25CAG CCC ATC CAA AAA GTC
2 瘦素基因CAA25CAG CCC ATC CAG AAA GTC
3 瘦素基因CAA25CAG CCC ATC CAC AAA GTC
4 瘦素基因CAA25CAG CCC ATC CAT AAA GTC
5 瘦素受体基因Asn85Ser TAT CCA AAA CAA CTT TCC
6 瘦素受体基因Asn85Ser TAT CCA AAG CAA CTT TCC
7 瘦素受体基因Asn85Ser TAT CCA AAC CAA CTT TCC
8 瘦素受体基因Asn85Ser TAT CCA AAT CAA CTT TCC
9 瘦素受体基因Lys109Arg TGA AGG AAA GAC ATT TGT
10 瘦素受体基因Lys109Arg TGA AGG AAG GAC ATT TGT
11 瘦素受体基因Lys109Arg TGA AGG AAC GAC ATT TGT
12 瘦素受体基因Lys109Arg TGA AGG AAT GAC ATT TGT
13 瘦素受体基因Gln223Arg ATT TTC CAG TCA CCT CT
14 瘦素受体基因Gln223Arg ATT TTC CGG TCA CCT CT
15 瘦素受体基因Gln223Arg ATT TTC CCG TCA CCT CT
16 瘦素受体基因Gln223Arg ATT TTC CTG TCA CCT CT。
5.根据权利要求3所述的一种瘦素和瘦素受体基因多态性检测芯片的制备方法,其特征在于:所说引物是根据瘦素和瘦素受体基因特征设计,分别用于上述基因的扩增,所说的瘦素引物为:正向引物与反向引物其中,正向引物为:5’GCC CTA TCT TTT CTA TGT CCA-3’,反向引物为:5’-GTG TGT GAA ATG TCA TTG ATC C-3’;所说的瘦素受体引物1为:正向引物与反向引物,其中,正向引物为:5′-GAG ACA GCT GTT GAA CCT AAG-3’,反向引物为:5’-AGA CAT CTATTT CAT ACA GGT ATC-3’;所说的瘦素受体引物2为:正向引物与反向引物,其中,正向引物为:5’-CTG TGC CAA CAG CCA AAC T-3’,反向引物为:5’-CCC ATA TTT ATG GGC TGA ACT-3’。
6.根据权利要求3所述的的一种瘦素和瘦素受体基因多态性检测芯片的制备方法,其特征在于:所说的信号分子标记是荧光分子。
7.一种如权利要求1所述瘦素和瘦素受体基因多态性检测芯片的用途,其特征在于:用于检测瘦素基因CAA25CAG和瘦素受体基因Asn85Ser、Lys109Arg和Gln223Arg多态性。
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CN1184330C (zh) * | 2000-06-30 | 2005-01-12 | 中国科学院上海冶金研究所 | 乙肝病毒多态性检测芯片的制作方法及其应用 |
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基因芯片及其在突变检测中的应用. 李金恒等.医学研究生学报,第18卷第1期. 2005 * |
瘦素受体Gln223Arg基因多态性与不同葡萄糖耐量血清瘦素的相关性研究. 李剑等.军医进修学院学报,第22卷第4期. 2001 * |
瘦素受体基因变异研究进展. 杜宏等.中国医师杂志 2004增刊. 2004 * |
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