任选9场

任选9场

多聚酶链反应

时间:2019-10-12 11:33    浏览:

多聚酶链反应

任选9场 SUIZHEDNAFENZIDEFAZHAN,1985NIANMEIGUOCetusGONGSIRENLEIYICHUANBUMullisDENGJIANLILEYIZHONGCHENGWEIDUOJUMEILIANFANYING(polymerase chain reaction, PCR)DEDNAKUOZENGXINJISHU。GAIJISHUSHIDUITEYIXINGDNAPIANDUANJINXINGFEIXIBAOYILAIXINGDETIWAIKUOZENG。TADEWENSHI,BIAOZHIZHESHENGWUXUELINGYUJINGLILEYICIZHANXINDEJISHUGEMING,YINCIBEI“Science”ZAZHIYUWEI1989NIANZUIYOUYINGXIANGDEJISHUFAMING,YEYOURENJIANGPCRJISHULIEWEI20SHIJI90NIANDAISHIDASHENGWUXUEREDIANZHIGUAN。ZHEIZHONGFANGFAJINJINIANLAIZAIFENZISHENGWUXUEHELINCHUANGYIXUEJISHOUYIXUELINGYUZHONGYIDELEXUNSUDEFAZHAN,ZAIWEISHENGWUXUEZHENDUANHEKEYANFANGMIANYIBEIYINGYONG,BINGJUYOUJUDADEQIANZAIYINGYONGJIAZHI。

(YI) PCRDEJIBENYUANLI PCRKUOZENGFANGDADNADEJIBENYUANLIHEJIBENGUOCHENGRUXIA:JIANGHANYOUSUOXUFANGDAFENXIXULIEDEDNAJIAREBIANXINGWEIDANLIAN,RANHOUJIARUYIDUIYUSUOFANGDAFENXIDEDNALIANGDUANLINJINXULIEHUBUDEGUAJUHEGANSUANPIANDUANZUOWEIYINWU,JIZUODUANYINWUHEYOUDUANYINWU。ZHEIDUIYINWUBIXUJIANGSUOXUFANGDAFENXIDEDNAPIANDUANBAOKUOZAIQIFANWEINEI。YINWUKEGENJUYIZHIDNAXULIEYONGDNAZIDONGHECHENGYIHECHENG,YIBANZHIXU20GEJIANJIDUI,DANBUNENGGENGSHAO,FOUZEZAI68℃JIUBUNENGBAOCHIYUDNADANLIANDEHUBUJIEHE,ZAIYOUDNADUOJUMEIHE4ZHONGSANLINSUANTUOYANGHEGAN(dATP、dTTP、dCTP、dGTPHUOdNTP)CUNZAIXIA,YIFANGDAFENXIDEDNADANLIANWEIMOBAN,AN5′DAO3′FANGXIANGJIANGYINWUYANZHANG,ZIDONGHECHENGXINDEDNALIAN,SHIDNAZHONGXINBIANCHENGSHUANGLIAN。YINCI,ZAIFANYINGZHONGZUO、YOUYINWUBUJINQIZHIDAOZUOYONG,ERQIEQIDAOTEYIXINGXIANZHIFANGDAHECHENGDNAPIANDUANFANWEIDAXIAODEZUOYONG。JIANGHECHENGDEDNASHUANGLIANZAICIJIAREBIANXING,JIXUZHONGFUYISHANGDEDNADUOJUMEIFANYING,ZUO、YOUYINWUZHIJIANZHEIDUANDNADELIANGJIUKECHENGZHISHUBEISHUZENGJIA。RUZHONGFUJINXINGZHEIYIFANYINGnCI,ZECONGLILUNSHANGJIANG,PCRFANGDADEDNABEISHUJIUWEI2nBEI,ZHEIYANGDNADELIANGJIUKEZAISHUXIAOSHINEITEYIDIHUODEJISHIWANBEIDEFANGDA。T4DNADUOJUMEIHEDACHANGGANJUNDNADUOJUMEIIDEklenowPIANDUANDOUKEYONGYUPCR,DANBIJIAOSHIYANDEJIEGUOXIANSHIT4FANGDACHANSHENGDEDNAYUMOBANYIZHI,ERYONGklenowPIANDUANFANGDADECHANWUNEICHUHANYOUYUMOBANYIZHIDEDNAWAI,HAIHUNYOUFEITEYIDEDIFENZIPIANDUAN,YINERT4BIklenowGENGSHIYONGYUPCR。1988NIANErlichDENGCONGNAIGAOWENDESHUISHENGSHIREJUN(Thermus aquaticus)ZHONGTIQULEYIZHONGJIANCHENGWEITaqDENAIGAOWENDNADUOJUMEI。YOUYUTaqDUOJUMEIJIHUBUSHOUJIAREBIANXINGDEYINGXIANG,YINERYONGZHEIZHONGMEIJINXINGPCRSHENGQULEMEICIJIAREBIANXINGHOUZHONGXINJIARUDNADUOJUMEIDEBUZHOU,SHIZHONGFUFANYINGNENGLIANXUJINXING。

任选9场 (ER) PCRDETEDIAN

1.MINGAN YOUYUPCRNENGJIANGYIDUANDNAFENZITEYIDIFANGDAYUE100WANBEI,YINERJIWEIMINGAN。PCRKEYONGYUJIANCEDNAHANLIANGJIWEIDEBIAOBEN(1ng DNA),SHENZHIKEJIANGDANGENTOUFASIZHONGSUOHANDEDANYIFENZIDNAFANGDAHOUJINXINGJIANCE。

    2. TEYI PCRBUJINMINGAN,ERQIETEYI。JUBAODAO,JIANGPCRFANGDAHOUDEDNAKELONGZUOXULIEFENXI,JIEGUOBIAOMING,PCRFANGDADEDNAXULIEYUYUANMOBANDNAXULIEYIZHI。

任选9场 3. JIANYIKUAISU PCRKEYONGYUZHIJIEJIANCECUZHIDEDNABIAOBEN,RUXIBAOLIEJIEYE、XUEXIBAODNATIQUYE,SHENGQULEDUIBIAOBENZHONGJIANCEDNADEJINGZHICHUNHUAGUOCHENG。PCRZAIYITIANNEIJINENGWANCHENG,RUSHANGXUZUODIANYONGFENXI、ZAJIAOHUOXULIEFENXIDENG,YIBANYEKEZAI1~3TIANNEIWANCHENG。

任选9场 4. KEJIANCEGUDINGBAOMAIDEBINGLIBIAOBENHUOBUFENDNAJIANGJIEBIAOBEN YINWEIPCRBINGBUYAOQIUSUOFANGDADEDNAWEIWANZHENGDEDAFENZI,SUOYIPCRDELINGYITEDIANSHIKEYONGYUJIANCEYIYONGFUMALINGUDINGHUOSHILABAOMAIGUODEBINGLIZUZHIBIAOBENHUOZHIYOU5μmBODEQIEPIAN,YENENGYONGYUJIANCEYIBUFENJIANGJIEDEDNABIAOBEN。

任选9场 5. ZHIJIEZUOXULIEFENXI PCRKETIGONGZUGOULIANGDETEYIDNAZUOXULIEFENXI,SHENGQULEYIWANGBIXUKELONGHOUZAIZUOXULIEFENXIDEGUOCHENG。ZAIPCRYINWUSHANGSHIXIANYONG32PZUOMODUANBIAOJI,FANGDAHOUDEDNAKEZHIJIEZUOMaxam-GilbertXULIEFENXI,HUOYONGSHUANGTUOYANGHEGANSUANZHONGDUANFAZUOXULIEFENXI。

任选9场 6.KEFANGDARNA   PCRYENENGDUIRNAHUOcDNAJINXINGFANGDA,YOUXIEWAIXIANZISANZAIFENBUZAIYIDUANHENZHANGDEDNAZHONG,YINERNANYIJIANGZHENGDUANDNADAFENZIFANGDAHEZUOXULIEFENXI,DANRUYONGmRNAZUOWEIMOBAN,JIUKEJIANGWAIXIANZIJIZHONG,YICIJIUNENGYONGPCRWANCHENGDUIWAIXIANZIDEFANGDAHEXULIEFENXI。

任选9场 7.WUFANGSHEXING PCRFANGDAZUOYONGBENSHENJUYOUTEYIXING,YINERFANGDAHOUZHIYAOYONGDIANYONGCEDINGDAOXIANGYINGLIANGHEDAXIAODEDNAPIANDUAN,JIUNENGZHENGMINGBIAOBENZHONGCUNZAISUOJIANCEDEDNAXULIE。YOUYUPCRFANGDADEDNALIANGZUYISHIROUYANJIUNENGDUIDIANYONGHOUJINGXIUHUAYIDINGRANSEDEDNATIAODAIJINXINGGUANCHAHUOSHEYING,YINERPCRKEZUOWEIYIZHONGBUYONGTONGWEISUDEFEIFANGSHEJIANCEFANGFA,YIYUZAILINCHUANGJIANCEZHONGTUIGUANG。

任选9场 XUYAOZHICHUDESHI,SHANGSHUDEPCRDUIDNABINGDUDEZHENDUANXIANSHILEHENGAODEMINGANXING,CAOZUOYIHENJIANDAN,YIYUYINGYONG。DANDUIRNABINGDUDEZHENDUAN,JIUBIJIAOFUZA,ZAIJINXINGPCRZHIQIAN,SHOUXIANYINGJINXINGRNADEFENLI,SUIHOUZUOFANZHUANLU,JIZAIFANZHUANLUTIXIZHONGJIARUYITIAOYUmRNADUOJUAHUBUDEDUOJUTZUOYINWU,FANZHUANLUYUmRNAHUBUDEcDNALIAN,ZUOWEIHOUXUSHIYANDEJIANCEWU,CIJIWEIJIANCERNABINGDUYONGDERT-PCR。

在PCR的基础上,Sano于1992年又创建了免疫PCR,其原理与PCR是一致的,只是在某些环节上有差别。免疫PCR就是将抗原抗体反应与PCR结合在一起,构成的一种新技术。首先将待测的抗原吸附于固相载体上,经封闭和冲洗后,加入特异的抗体,此抗体常采用生物素标记,经孵育和冲洗后,加入作为连接分子的亲和素,随后,孵育和冲洗,再加用生物素标记的DNA,孵育和冲洗 ,去除游离的DNA分子,然后加入PCR扩增系统(用其代替ELISA的酶催化底物显色),使结合于固相载体上的DNA放大,再用琼脂糖凝胶电泳检测放大的PCR产物,经溴化乙啶染色和照像,记录PCR产物的电泳结果,通过底片上PCR产物的光密度,可以得出PCR的量,即代表固相上吸附的待检抗原量。将其与用标准抗原制备的标准曲线进行比较,就可以准确地得出待测抗原的实际量。免疫PCR中的DNA,只是一个做为指示分子的角色,用DNA聚合酶将结合于固相上的DNA特异放大,由此定量检测抗原。所以,免疫PCR的敏感性比ELISA高105倍,且ELISA产生的背景信号很弱,只能测到几百个分子的抗原,而免疫PCR在理论上可以检测到一个分子的抗原,因此,免疫PCR特别适用于检测一些含量特别少的抗原分子。需要指出的是,免疫PCR技术目前仍处于研究阶段,在技术上还须要继续完善与提高,配套试剂还须要研制,方能推广应用。除上述外,于近些年来又建立了一些新的PCR技术如:带尾式PCR、抛锚式PCR、套叠式PCR、竞争模板式PCR、复合式PCR、不对称式PCR、重组式PCR等。
                                                                                                                                                                                                                                                       南京卡米洛生物工程有限公司

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