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pcr瀹為獙鍘熺悊鍙婃濂廮涓婃捣鎴峰疄鍖昏嵂绉戞妧鏈夐檺鍏徃
鏂伴椈濯掍綋
鎮ㄥ綋鍓嶇殑浣嶇疆锛欬a href="/index.aspx">棣栭〉>鏂伴椈濯掍綋
  • pcr瀹為獙鍘熺悊鍙婃濂廃/li>
  • 2019-10-14 14:37:08
  • 瀹為獙鏂规硶鍘熺悊

    鍩烘湰鍘熺悊绫讳技浜嶞/span>DNA鐨勫ぉ鐒跺鍒惰繃绋嬶紝鍏剁壒寮傛€т緷璧栦簬涓庨澏搴忓垪涓ょ浜掕ˉ鐨勫鏍歌嫹閰稿紩鐗┿€侟/span>

     

     

    PCR鐢卞彉鎬?/span>--閫€鐏?/span>--寤朵几涓変釜鍩烘湰鍙嶅簲姝ラ鏋勬垚锛欬/span>

     

    鈶犳ā鏉军span>DNA鐨勫彉鎬э細妯℃澘DNA缁忓姞鐑嚦94鈩冨乏鍙充竴瀹氭椂闂村悗锛屼娇妯℃澘DNA鍙岄摼鎴栫粡PCR鎵╁褰㈡垚鐨勫弻閾近/span>DNA瑙g锛屼娇涔嬫垚涓哄崟閾撅紝浠ヤ究瀹冧笌寮曠墿缁撳悎锛屼负涓嬭疆鍙嶅簲鍋氬噯澶囷紱

     

     

    鈶℃ā鏉军span>DNA涓庡紩鐗╃殑閫€鐏?/span>(澶嶆€?/span>)锛氭ā鏉军/span>DNA缁忓姞鐑彉鎬ф垚鍗曢摼鍚庯紝娓╁害闄嶈嚦55鈩冨乏鍙筹紝寮曠墿涓庢ā鏉军/span>DNA鍗曢摼鐨勪簰琛ュ簭鍒楅厤瀵圭粨鍚堬紱

     

     

    鈶㈠紩鐗╃殑寤朵几锛欬span>DNA妯℃澘--寮曠墿缁撳悎鐗╁湪Taq閰剁殑浣滅敤涓嬶紝浠?/span>dNTP涓哄弽搴斿師鏂欙紝闈跺簭鍒椾负妯℃澘锛屾寜纰卞熀閰嶅涓庡崐淇濈暀澶嶅埗鍘熺悊锛屽悎鎴愪竴鏉℃柊鐨勪笌妯℃澘DNA 閾句簰琛ョ殑鍗婁繚鐣欏鍒堕摼銆侟/span>

     

     

    閲嶅寰幆鍙樻€?/span>--閫€鐏?/span>--寤朵几涓夎繃绋嬶紝灏卞彲鑾峰緱鏇村鐨勨€滃崐淇濈暀澶嶅埗閾锯€濓紝鑰屼笖杩欑鏂伴摼鍙堝彲鎴愪负涓嬫寰幆鐨勬ā鏉裤€傛瘡瀹屾垚涓€涓惊鐜渶2锝濣/span>4鍒嗛挓锛 2锝濣/span>3灏忔椂灏辫兘灏嗗緟鎵╃洰鐨勫熀鍥犳墿澧炴斁澶у嚑鐧句竾鍊嶃€侟/span>

     

     

    瀹為獙鏉愭枡 妯℃澘DNA

    璇曞墏銆佽瘯鍓傜洅 dNTPTaq DNA鑱氬悎閰惰捀棣忔按PCR缂撳啿娑插紩鐗╂隘鍖栭晛

    浠櫒銆佽€楁潗 PCR浠Щ娑叉灙PCR鏉胯杽澹佺绂诲績绠$蹇冪鐩扅/span>

    瀹為獙姝ラ

    涓€銆佹爣鍑嗙殑PCR鍙嶅簲浣撶郴

     

     

    10脳鎵╁缂撳啿娑 銆€   10 ul

     

     

     

     

    4绉岦/span>dNTP娣峰悎鐗 銆€ 鍚凕/span>200 umol/L

     

     

     

     

    寮曠墿     鍚凕/span>10锝濣/span>100 pmol

     

     

     

     

    妯℃澘DNA 0.1锝濣/span>2 ug

     

     

     

     

    Taq DNA鑱氬悎閰 銆€  2.5 u

     

     

     

     

    Mg2+ 1.5 mmol/L

     

     

     

     

    鍔犲弻鎴栦笁钂告按鑷颤/span>  100 ul

     

     

     

     

    浜屻€?/span>PCR寮曠墿璁捐

     

    PCR鍙嶅簲涓湁涓ゆ潯寮曠墿锛屽嵆5鈥茬寮曠墿鍜孅/span>3鈥插紩鐗┿€傝璁″紩鐗╂椂浠ヤ竴鏉狘/span>DNA鍗曢摼涓哄熀鍑嗭紙甯镐互淇℃伅閾句负鍩哄噯锛夛紝5鈥茬寮曠墿涓庝綅浜庡緟鎵╁鐗囨5鈥茬涓婄殑涓€灏忔DNA搴忓垪鐩稿悓锛汓/span>3鈥茬寮曠墿涓庝綅浜庡緟鎵╁鐗囨3鈥茬鐨勪竴灏忔DNA搴忓垪浜掕ˉ銆侟/span>

     

    1.   寮曠墿璁捐鐨勫熀鏈師鍒橖/span>

     

    锛圏/span>1锛  寮曠墿闀垮害锛欬/span>15-30 bp锛屽父鐢ㄤ负20 bp宸﹀彸銆侟/span>

     

    锛圏/span>2锛 寮曠墿纰卞熀锛欬/span>G+C鍚噺浠?/span>40-60%涓哄疁锛孅/span>G+C澶皯鎵╁鏁堟灉涓嶄匠锛孅/span>G+C 杩囧鏄撳嚭鐜伴潪鐗瑰紓鏉″甫銆侟/span>ATGC鏈€濂介殢鏈哄垎甯冿紝閬垮厤5涓互涓婄殑鍢屽懁鎴栧槯鍟舵牳鑻烽吀鐨勬垚涓叉帓鍒椼€侟/span>

     

     

     

    锛圏/span>3锛 寮曠墿鍐呴儴涓嶅簲鍑虹幇浜掕ˉ搴忓垪銆侟/span>

     

     

     

     

    锛圏/span>4锛変袱涓紩鐗╀箣闂翠笉搴斿瓨鍦ㄤ簰琛ュ簭鍒楋紝灏ゅ叾鏄伩鍏岦/span>3 鈥茬鐨勪簰琛ラ噸鍙犮€侟/span>

     

     

     

     

    锛圏/span>5锛 寮曠墿涓庨潪鐗瑰紓鎵╁鍖虹殑搴忓垪鐨勫悓婧愭€т笉瑕佽秴杩嘃/span>70%锛屽紩鐗?/span>3鈥叉湯绔繛缁?/span>8涓⒈鍩哄湪寰呮墿澧炲尯浠ュ涓嶈兘鏈夊畬鍏ㄤ簰琛ュ簭鍒楋紝鍚﹀垯鏄撳鑷撮潪鐗瑰紓鎬ф墿澧炪€侟/span>

     

     

     

     

    锛圏/span>6锛夊紩鐗?/span>3鈥樼鐨勭⒈鍩猴紝鐗瑰埆鏄渶鏈強鍊掓暟绗簩涓⒈鍩猴紝搴斾弗鏍艰姹傞厤瀵癸紝鏈€浣抽€夋嫨鏄?/span>G鍜孅/span>C銆侟/span>

     

     

     

    锛圏/span>7锛 寮曠墿鐨凕/span>5 鈥茬鍙互淇グ銆傚闄勫姞闄愬埗閰朵綅鐐癸紝寮曞叆绐佸彉浣嶇偣锛岀敤鐢熺墿绱犮€佽崸鍏夌墿璐ㄣ€佸湴楂樿緵鏍囪锛屽姞鍏ュ叾瀹冪煭搴忓垪锛屽寘鎷捣濮嬪瘑鐮佸瓙銆佺粓姝㈠瘑鐮佸瓙绛夈€侟/span>

     

     

     

    2.   寮曠墿璁捐杞欢

     

    Primer Premier5.0 锛堣嚜鍔ㄦ悳绱級*銆?/span>Oligo6 锛堝紩鐗╄瘎浠凤級銆?/span>Vector NTI Suit銆?/span>DNAsis銆?/span>Omiga銆?/span>DNAstar銆?/span>Primer3 锛堝湪绾挎湇鍔★級銆侟/span>

     

    涓夈€佹ā鏉跨殑鍒跺

     

    锛圏/span>1锛堻/span>PCR鐨勬ā鏉垮彲浠ユ槸DNA锛屼篃鍙互鏄?/span>RNA銆侟/span>

     

    锛圏/span>2锛夋ā鏉跨殑鍙栨潗涓昏渚濇嵁PCR鐨勬墿澧炲璞★紝鍙互鏄梾鍘熶綋鏍囨湰濡傜梾姣掋€佺粏鑿屻€佺湡鑿岀瓑銆備篃鍙互鏄梾鐞嗙敓鐞嗘爣鏈缁嗚優銆佽娑层€佺緤姘寸粏鑳炵瓑銆傛硶鍖诲鏍囨湰鏈夎鏂戙€佺簿鏂戙€佹瘺鍙戠瓑銆侟/span>

     

    锛圏/span>3锛夋爣鏈鐞嗙殑鍩烘湰瑕佹眰鏄櫎鍘绘潅璐紝骞堕儴鍒嗙函鍖栨爣鏈腑鐨勬牳閰搞€傚鏁版牱鍝侀渶瑕佺粡杩嘃/span>SDS鍜岃泲鐧介叾K澶勭悊銆傞毦浠ョ牬纰庣殑缁嗚弻锛屽彲鐢ㄦ憾鑿岄叾鍔燑/span>EDTA澶勭悊銆傛墍寰楀埌鐨勭矖鍒迭/span>DNA锛岀粡閰氥€佹隘浠挎娊鎻愮函鍖栵紝鍐嶇敤涔欓唶娌夋穩鍚庣敤浣淈/span>PCR鍙嶅簲妯℃澘銆侟/span>

     

    鍥涖€?/span>PCR鍙嶅簲鏉′欢鐨勬帶鍒迭/span>

     

    1.  PCR鍙嶅簲鐨勭紦鍐叉恫鎻愪緵鍚堥€傜殑閰哥⒈搴︿笌鏌愪簺绂诲瓙 銆

     

    2.  闀佺瀛愭祿搴︽€婚噺搴旀瘮dNTPs鐨勬祿搴﹂珮锛屽父鐢?/span>1.5 mmol/L銆侟/span>

     

     

    3.  搴曠墿娴撳害dNTP浠ョ瓑鎽╁皵娴撳害閰嶅埗锛孅/span>20锝濣/span>200 umol/L銆侟/span>

     

     

    4.  TaqDNA鑱氬悎閰迭/span>2.5U(100 ul锛夈€侟/span>

     

     

    5.  寮曠墿 娴撳害涓€鑸负0.1 锝濣/span>0.5 umol/L銆侟/span>

     

     

    6.  鍙嶅簲娓╁害

     

     

     

    锛圏/span>1锛夊彉鎬ф俯搴﹀拰鏃堕棿95鈩冿紝30 s銆侟/span>

     

     

     

    锛圏/span>2锛夐€€鐏俯搴﹀拰鏃堕棿 浣庝簬寮曠墿Tm鍊稽/span>5 鈩冨乏鍙筹紝涓€鑸湪45锝濣/span>55鈩冦€侟/span>

     

     

     

    锛圏/span>3锛夊欢浼告俯搴﹀拰鏃堕棿72鈩冿紝1 min/kb(10 kb鍐咃級銆侟/span>

     

     

     

    锛圏/span>4锛堻/span>Tm鍊稽/span>=4(G+C) +2(A+T)

     

     

    7.  寰幆娆℃暟 锛氫竴鑸负25 锝濣/span>30娆°€傚惊鐜暟鍐冲畾PCR鎵╁鐨勪骇閲忋€傛ā鏉垮垵濮嬫祿搴︿綆锛屽彲澧炲姞寰幆鏁颁互渚胯揪鍒版湁鏁堢殑 鎵╁閲忋€備絾寰幆鏁板苟涓嶆槸鍙互鏃犻檺澧炲姞鐨勩€備竴鑸惊鐜暟涓裹/span>30涓乏鍙筹紝寰幆鏁拌秴杩嘃/span>30涓互鍚庯紝DNA鑱氬悎閰舵椿鎬ч€愭笎杈惧埌楗卞拰锛屼骇鐗╃殑閲忎笉鍐嶉殢寰幆鏁扮殑澧炲姞鑰屽鍔狅紝鍑虹幇浜嗘墍璋撶殑鈥滃钩鍙版湡鈥濄€侟/span>

     

     

     

    浜斻€?/span>PCR鐨勫惊鐜弬鏁?/span>

     

    1.  棰勫彉鎬э紙Initial denaturation锛堻/span>

     

    妯℃澘DNA瀹屽叏鍙樻€т笌PCR閰剁殑瀹屽叏婵€娲诲PCR鑳藉惁鎴愬姛鑷冲叧閲嶈锛屽缓璁姞鐑椂闂村弬鑰冭瘯鍓傝鏄庝功锛屼竴鑸湭淇グ鐨凕/span>Taq閰舵縺娲绘椂闂翠负涓ゅ垎閽熴€侟/span>

     

    2.  寰幆涓殑鍙樻€ф楠|/span>

     

    寰幆涓竴鑸?/span>95鈩冿紝30绉掕冻浠ヤ娇鍚勭闈迭/span>DNA搴忓垪瀹屽叏鍙樻€э紝鍙兘鐨勬儏鍐典笅鍙 缂╃煭璇ユ楠ゆ椂闂达紝鍙樻€ф椂闂磋繃闀挎崯瀹抽叾娲绘€э紝杩囩煭闈跺簭鍒楀彉鎬т笉褰诲簳锛屾槗閫犳垚鎵╁澶辫触銆侟/span>

     

    3.  寮曠墿閫€鐏紙Primer annealing锛堻/span>

     

    閫€鐏俯搴﹂渶瑕佷粠澶氭柟闈㈠幓鍐冲畾锛屼竴鑸牴鎹紩鐗╃殑Tm鍊间负鍙傝€冿紝鏍规嵁鎵╁鐨勯暱搴﹂€傚綋涓嬭皟浣滀负閫€鐏俯搴︺€傜劧鍚庡湪姝ゆ瀹為獙鍩虹涓婂仛鍑洪浼般€侟/span>

     

    閫€鐏俯搴﹀PCR鐨勭壒寮傛€ф湁杈冨ぇ褰卞搷銆侟/span>

     

    4.  寮曠墿寤朵几

     

    寮曠墿寤朵几涓€鑸湪72鈩冭繘琛岋紙Taq閰舵渶閫傛俯搴︼級銆備絾鍦ㄦ墿澧為暱搴﹁緝鐭笖閫€鐏俯搴﹁緝楂樻椂锛屾湰姝ラ鍙渷鐣?/span>

     

    寤朵几鏃堕棿闅忔墿澧炵墖娈甸暱鐭€屽畾锛屼竴鑸帹鑽愬湪1000 bp浠ヤ笂锛屽惈Pfu鍙婂叾琛嶇敓鐗╃殑琛嶇敓璁惧畾涓裹/span>1 min/kbp銆侟/span>

     

    5.  寰幆鏁?/span>

     

    澶у鏁?/span>PCR鍚?/span>25-40寰幆锛岃繃澶氭槗浜х敓闈炵壒寮傛墿澧炪€侟/span>

     

    6.  鏈€鍚庡欢浼伏/span>

     

    鍦ㄦ渶鍚庝竴涓惊鐜悗锛屽弽搴斿湪72鈩冪淮鎸?/span>5-15鍒嗛挓锛庝娇寮曠墿寤朵几瀹屽叏锛屽苟浣垮崟閾句骇鐗╅€€鐏垚鍙岄摼銆侟/span>

     

    鍏€?/span>PCR姝ラ

     

    1.  DNA鍙樻€?/span>

     

    锛圏/span>90鈩傸/span>-96鈩冿級锛氬弻閾近/span>DNA妯℃澘鍦ㄧ儹浣滅敤涓嬶紝 姘㈤敭鏂锛屽舰鎴愬崟閾近/span>DNA銆侟/span>

     

    2.  閫€鐏?/span>

     

    锛圏/span>25鈩傸/span>-65鈩冿級锛氱郴缁熸俯搴﹂檷浣庯紝寮曠墿涓嶞/span>DNA妯℃澘缁撳悎锛屽舰鎴愬眬閮ㄥ弻閾俱€侟/span>

     

    3.  寤朵几

     

    锛圏/span>70鈩傸/span>-75鈩冿級锛氬湪Taq閰讹紙鍦?/span>72鈩冨乏鍙筹紝娲绘€ф渶浣筹級鐨勪綔鐢ㄤ笅锛屼互dNTP涓哄師鏂欙紝浠庡紩鐗╃殑5鈥茬鈫扅/span>3鈥茬寤朵几锛屽悎鎴愪笌妯℃澘浜掕ˉ鐨凕/span>DNA閾俱€侟/span>

     

    涓冦€?/span>PCR妫€娴婞/span>

     

    PCR鍙嶅簲鎵╁鍑轰簡楂樼殑鎷疯礉鏁帮紝涓嬩竴姝ユ娴嬪氨鎴愪簡鍏抽敭銆傝崸鍏夌礌锛堟捍鍖栦箼閿紝EB锛夋煋鑹插嚌鑳剁數娉虫槸鏈€甯哥敤鐨勬娴嬫墜娈点€傜數娉虫硶妫€娴嬬壒寮傛€ф槸涓嶅お楂樼殑锛屽洜姝ゅ紩鐗╀袱鑱氫綋绛夐潪鐗瑰紓鎬х殑鏉備氦浣撳緢瀹规槗寮曡捣璇垽銆備絾鍥犱负鍏剁畝鎹锋槗琛岋紝鎴愪负浜嗕富娴佹娴嬫柟娉曘€傝繎骞存潵浠ヨ崸鍏夋帰閽堜负浠h〃鐨勬娴嬫柟娉曪紝鏈夐€愭笎鍙栦唬鐢垫吵娉曠殑瓒嬪娍銆侟/span>

     

     

    灞曞紑

    娉ㄦ剰浜嬮」

    1.  鐢变簬PCR鍙嶅簲鐏垫晱搴﹀緢楂橈紝鍥犳瑕佺壒鍒富鎰忛槻姝↑/span>DNA姹℃煋鐨勫彂鐢熴€傛牱鍝侀棿鐨勭浉浜掓薄鏌撳彲鑳戒骇鐢熷亣闃虫€х粨鏋滐紝鐗瑰埆鏄皢PCR鎶€鏈簲鐢ㄥ埌涓村簥鐥呭師鑿屾劅鏌撶‘瀹氫腑銆侟/span>

     

     

     

    2.  濡傛灉鏄叕鐢ㄧ殑銆佹病鏈夊瘑鐮佷繚鎶ょ殑PCR浠紝缁忓父妫€鏌?/span>PCR浠笂鐨勭▼搴忔纭笌鍚︺€侟/span>

     

     

     

    3.  涓嶄娇鐢ㄨ繃閲忚瘯鍓傗€淈/span>less is usually better 锛圏/span>more specific锛夆€濄€侟/span>

     

     

     

    4.  璇曞墏璐洖鍚庡簲鍒嗚鎴愬皬浠戒娇鐢紝杩欐牱涓€鏃︽湁姹℃煋鍙戠敓锛屽彲浠ョ珛鍗充涪寮冩薄鏌撶殑璇曞墏锛屼笉浼氶€犳垚澶х殑鎹熷け锛涜瘯鍓傚垎瑁呬篃鏈夊姪浜庡噺灏戝弽澶嶅喕铻嶆鏁帮紙渚嬪dNTPs瀵瑰弽澶嶅喕铻嶆晱鎰燂級銆侟/span>

     

     

     

    5.  鍔犲畬鎵€鏈夎瘯鍓傚悗鐢ㄦ灙澶村惞鎵撳嚑娆℃垨绋嶅井娑℃棆鎴栬交寮圭澹佷互淇濊瘉鍏呭垎娣峰寑銆侟/span>

     

     

     

    6.  浣跨敤闃存€у鐓ф鏌ユ薄鏌撶殑鍙戠敓銆侟/span>

     

     

     

    7.  浣跨敤闃虫€у鐓э紙鑳借壇濂芥墿澧炵殑鏍锋湰锛夈€侟/span>

     

     

     

    8.  鐢垫吵鏃朵娇鐢?/span>DNA鍒嗗瓙閲忔爣鍑嗗搧锛堟寚绀烘槸鍚?/span>PCR澶辫触鎴栨潯甯﹁窇鍑哄嚌鑳舵垨鎷嶇収绯荤粺澶辫触锛夈€侟/span>

     

     

     

    9.  褰撴墿澧炲緢闀跨殑銆?/span>GC鍚噺楂樼殑妯℃澘鏃舵垨瀹规槗浜х敓浜岀骇缁撴瀯鐨勬ā鏉挎椂閫傞噺浣跨敤娣诲姞鍓傦紙glycerol, formamide, NMP浣垮彉鎬у拰閫€鐏俯搴﹂檷浣庡嚑搴︼紱glycerol涔熷彲璧峰埌绋冲畾鑱氬悎閰舵椿鎬х殑浣滅敤锛汓/span>DMSO 鍑忓皯浜岀骇缁撴瀯鐨勫彂鐢燂紝骞堕€氳繃鍑忓急闈炵壒寮傛€у紩鐗╃粨鍚堢殑绋冲畾鎬э紝鎻愰珮鍙嶅簲鐨勭壒寮傛€э級銆侟/span>

     

     

     

    10.  涓嶄娇鐢ㄥ甫鏈夎嚜鍔ㄩ櫎闇滃姛鑳界殑鍐扮瀛樺偍閰讹紙閬垮厤鍙嶅鍐昏瀺锛夛紝姣忔鍙栭叾鏃堕兘浣跨敤鏂扮殑鏋ご閰讹紝閰朵娇鐢ㄥ悗搴旂珛鍗虫斁鍥炲啺绠便€傞叾瑕佸湪鍔犲畬缂撳啿娑插悗鍐嶅姞锛岀洿鎺ュ皢閰跺姞鍏ユ按涓彲鑳藉鑷撮叾鍙樻€уけ娲汇€侟/span>

     

     

     

    11.   PCR浜х墿鐨勭數娉虫娴嬫椂闂达紝涓€鑸负48 h浠ュ唴锛屾湁浜涙渶濂戒簬褰撴棩鐢垫吵妫€娴嬶紝澶т簬48h鍚庡甫鍨嬩笉瑙勫垯鐢氳嚧娑堝け銆侟/span>

     

    鏀惰捣

    鍏朵粬

    涓€銆?/span>  PCR鍙嶅簲鐨勫叧閿幆鑺侟/span>

     

    1.  妯℃澘鏍搁吀鐨勫埗澶囥€侟/span>

     

    2.  寮曠墿鐨勮川閲忎笌鐗瑰紓鎬с€侟/span>

     

    3.  閰剁殑璐ㄩ噺銆侟/span>

     

    4.  PCR寰幆鏉′欢銆侟/span>

     

    浜屻€?/span> PCR甯歌闂鍙婂绛朁/span>

     

     

    1.  鍋囬槾鎬э紝涓嶅嚭鐜版墿澧炴潯甯?/span>

     

     

     

     

    锛圏/span>1锛夋ā鏉垮師鍥燑/span>

     

    鈶  妯℃澘涓惈鏈夋潅铔嬬櫧璐ㄣ€侟/span>

     

    鈶  妯℃澘涓惈鏈堻span>Taq閰舵姂鍒跺墏銆侟/span>

     

    鈶  妯℃澘涓泲鐧借川娌℃湁娑 鍖栭櫎鍑€锛岀壒鍒槸鏌撹壊浣撲腑鐨勭粍铔嬬櫧銆侟/span>

     

    鈶  鍦ㄦ彁鍙栧埗澶囨ā鏉挎椂涓㈠け杩囧锛屾垨鍚稿叆閰氥€侟/span>

     

    鈶  妯℃澘鏍搁吀鍙樻€т笉褰诲簳銆傚湪閰跺拰寮曠墿璐ㄩ噺濂芥椂锛屼笉鍑虹幇鎵╁甯︼紝鏋佹湁鍙兘鏄爣鏈殑娑堝寲澶 鐞嗭紝妯℃澘鏍搁吀鎻愬彇杩囩▼鍑轰簡姣涚梾锛屽洜鑰岃閰嶅埗鏈夋晥鑰岀ǔ瀹氱殑娑堝寲澶勭悊娑诧紝鍏剁▼搴忎害搴斿浐瀹氫笉瀹滈殢鎰忔洿鏀广€侟/span>

     

     

     

     

    锛圏/span>2锛夐叾澶辨椿

     

    鈶  闇€鏇存崲鏂伴叾锛屾垨鏂版棫涓ょ閰跺悓鏃朵娇鐢紝浠ュ垎鏋愭槸鍚﹀洜閰剁殑娲绘€т抚澶辨垨涓嶅鑰 瀵艰嚧鍋囬槾鎬с€侟/span>

     

     

     

    鈶  蹇樺姞Taq閰舵垨婧翠箼閿€侟/span>

     

     

     

     

    锛圏/span>3锛夊紩鐗?/span>

     

     

     

    鈶  寮曠墿璐ㄩ噺銆侟/span>

     

     

     

    鈶  寮曠墿鐨勬祿搴︺€侟/span>

     

     

     

    鈶  涓ゆ潯寮曠墿鐨勬祿搴︽槸鍚﹀绉般€侟/span>

     

    瑙e喅瀵圭瓥锛欬/span>

     

    鈶  閫夊畾涓€涓ソ鐨勫紩鐗╁悎鎴愬崟浣嶃€侟/span>

     

    鈶  寮曠墿鐨勬祿搴︿笉浠呰鐪婞span>OD鍊硷紝鏇磋娉ㄩ噸寮曠墿鍘熸恫鍋氱惣鑴傜硸鍑濊兌鐢垫吵锛屼竴瀹氳鏈夊紩鐗╂潯甯﹀嚭鐜帮紝鑰屼笖涓ゅ紩鐗╁甫鐨勪寒搴﹀簲澶т綋涓€鑷达紝濡備竴鏉″紩鐗╂湁鏉″甫锛屼竴鏉″紩鐗╂棤鏉″甫锛屾鏃跺仛PCR鏈夊彲鑳藉け璐ワ紝搴斿拰寮曠墿鍚堟垚鍗曚綅鍗忓晢瑙e喅銆傚涓€鏉″紩鐗╀寒搴﹂珮锛屼竴鏉′寒搴︿綆锛屽湪绋€閲婂紩鐗╂椂瑕佸钩琛″叾娴撳害銆侟/span>

     

    鈶  寮曠墿搴旈珮娴撳害灏忛噺鍒嗚淇濆瓨锛岄槻姝㈠娆″喕铻嶆垨闀挎湡鏀惧啺绠卞喎钘忛儴鍒嗭紝瀵艰嚧寮曠墿鍙樿川闄嶈В澶辨晥銆侟/span>

     

    鈶  寮曠墿璁捐涓嶅悎鐞嗭紝濡傚紩鐗╅暱搴︿笉澶燂紝寮曠墿涔嬮棿褰㈡垚浜岃仛浣撶瓑銆侟/span>

     

     

     

     

    锛圏/span>4锛堻/span>Mg2+娴撳害

     

    鈶  娴撳害杩囬珮闄嶄綆PCR鎵╁鐨勭壒寮傛€с€侟/span>

     

     

     

    鈶  娴撳害杩囦綆褰卞搷PCR鎵╁浜ч噺鐢氳嚦浣军/span>PCR鎵╁澶辫触鑰屼笉鍑烘墿澧炴潯甯︺€侟/span>

     

     

     

     

    锛圏/span>5锛夊弽搴斾綋绉殑鏀瑰彉

     

    鈶  閫氬父杩涜PCR鎵╁閲囩敤鐨勪綋绉负20 ul銆?/span>30 ul銆?/span>50 ul鎴朁/span>100 ul锛屽簲鐢ㄥ澶т綋绉繘琛孅/span>PCR鎵╁锛屾槸鏍规嵁绉戠爺鍜屼复搴婃娴嬩笉鍚岀洰鐨勮€岃瀹欬/span>;

     

    鈶  鍦ㄥ仛灏忎綋绉20 ul 鍚庯紝鍐嶅仛澶т綋绉椂锛屼竴瀹氳妯$储鏉′欢锛屽惁鍒欏鏄撳け璐ャ€侟/span>

     

     

     

     

    锛圏/span>6锛夌墿鐞嗗師鍥燑/span>

     

    鈶  鍙樻€уPCR鎵╁鏉ヨ鐩稿綋閲嶈锛屽鍙樻€ф俯搴︿綆锛屽彉鎬ф椂闂寸煭锛屾瀬鏈夊彲鑳藉嚭鐜板亣闃存€с€侟/span>

     

    鈶  閫€鐏俯搴﹁繃浣庯紝鍙嚧闈炵壒寮傛€ф墿澧炶€岄檷浣庣壒寮傛€ф墿澧炴晥鐜囷紱閫€鐏俯搴﹁繃楂樺奖鍝嶅紩鐗╀笌妯℃澘鐨勭粨鍚堣€岄檷浣嶞span>PCR鎵╁鏁堢巼銆侟/span>

     

    鈶  鎵╁浠垨姘存憾閿呭唴鐨勫彉鎬с€侀€€鐏拰寤朵几娓╁害鏈夐棶棰樸€侟/span>

     

     

     

     

    锛圏/span>7锛夐澏搴忓垪鍙樺紓

     

    鈶  闈跺簭鍒楀彂鐢熺獊鍙樻垨缂哄け锛屽奖鍝嶅紩鐗╀笌妯℃澘鐗瑰紓鎬х粨鍚堛€侟/span>

     

    鈶  闈跺簭鍒楁煇娈电己澶变娇寮曠墿涓庢ā鏉垮け鍘讳簰琛ュ簭鍒椼€侟/span>

     

     

     

    2.  鍋囬槼鎬э紝鍑虹幇鐨凕/span>PCR鎵╁鏉″甫涓庣洰鐨勯澏搴忓垪鏉″甫涓€鑷达紝鏈夋椂鍏舵潯甯︽洿鏁撮綈锛屼寒搴︽洿楂樸€侟/span>

    锛圏/span>1锛夊紩鐗╄璁′笉鍚堥€侟/span>

     

    鈶  閫夋嫨鐨勬墿澧炲簭鍒椾笌闈炵洰鐨勬墿澧炲簭鍒楁湁鍚屾簮鎬с€侟/span>

     

    鈶  闈跺簭鍒楀お鐭垨寮曠墿澶煭銆侟/span>

    锛圏/span>2锛夐澏搴忓垪鎴栨墿澧炰骇鐗╃殑浜ゅ弶姹℃煋

     

    鈶  鏁翠釜鍩哄洜缁勬垨澶х墖娈电殑浜ゅ弶姹℃煋銆侟/span>

     

    瑙e喅鏂规锛氭搷浣滄椂搴斿皬蹇冭交鏌旓紝闃叉灏嗛澏搴忓垪鍚稿叆鍔犳牱鏋唴鎴栨簠鍑虹蹇冪澶栥€傞櫎閰跺強涓嶈兘鑰愰珮娓╃殑鐗╄川澶栵紝鎵€鏈夎瘯鍓傛垨鍣ㄦ潗鍧囧簲楂樺帇娑堟瘨銆傛墍鐢ㄧ蹇冪鍙婃牱杩涙灙澶寸瓑鍧囧簲涓€娆℃€т娇鐢ㄣ€傚繀瑕佹椂锛屽湪鍔犳爣鏈墠锛屽弽搴旂鍜岃瘯鍓傜敤绱绾跨収灏勶紝浠ョ牬鍧忓瓨鍦ㄧ殑鏍搁吀銆侟/span>

     

    鈶  绌烘皵涓殑灏忕墖娈垫牳閰告薄鏌擄紝杩欎簺灏忕墖娈垫瘮闈跺簭鍒楃煭锛屼絾鏈変竴瀹氱殑鍚屾簮鎬с€傚彲浜掔浉鎷兼帴锛屼笌寮曠墿浜掕ˉ鍚庯紝鍙墿澧炲嚭PCR浜х墿锛岃€屽鑷村亣闃虫€х殑浜х敓銆侟/span>

     

    瑙e喅鏂规锛氬彲鐢ㄥ发寮廃/span>PCR鏂规硶鏉ュ噺杞绘垨娑堥櫎銆侟/span>

    3.  鍑虹幇闈炵壒寮傛€ф墿澧烇細PCR鎵╁鍚庡嚭鐜扮殑鏉″甫涓庨璁$殑澶у皬涓嶄竴鑷达紝鎴栧ぇ鎴栧皬锛屾垨鑰呭悓鏃跺嚭鐜扮壒寮傛€ф墿澧炲甫涓庨潪鐗瑰紓鎬ф墿澧炲甫銆侟/span>

     

    锛圏/span>1锛  寮曠墿涓庨澏搴忓垪涓嶅畬鍏ㄤ簰琛ユ垨寮曠墿鑱氬悎褰㈡垚浜岃仛浣撱€侟/span>

     

    锛圏/span>2锛 Mg2+绂诲瓙娴撳害杩囬珮銆侟/span>

     

    锛圏/span>3锛 閫€鐏俯搴﹁繃浣庛€侟/span>

    锛圏/span>4锛 PCR寰幆娆℃暟 杩囧鏈夊叧銆侟/span>

     

    锛圏/span>5锛 閰剁殑璐ㄥ拰閲忥紝寰€寰€涓€浜涙潵婧愮殑閰舵槗鍑虹幇闈炵壒寮傛潯甯﹁€屽彟涓€鏉ユ簮鐨勯叾鍒欎笉鍑虹幇锛岄叾閲忚繃澶氭湁鏃朵篃浼氬嚭鐜伴潪鐗瑰紓鎬ф墿澧炪€侟/span>

     

    鍏跺绛栨湁锛氬繀瑕佹椂閲嶆柊璁捐寮旤/span> 鐗┿€傚噺浣庨叾閲忔垨璋冩崲鍙︿竴鏉ユ簮鐨勯叾銆傞檷浣庡紩鐗╅噺锛岄€傚綋澧炲姞妯℃澘閲忥紝鍑忓皯寰幆娆狘/span> 鏁般€傞€傚綋鎻愰珮閫€鐏俯搴︽垨閲囩敤浜屾俯搴︾偣娉曪紙93鈩冨彉鎬э紝65鈩冨乏鍙抽€€鐏笌寤朵几锛夈€侟/span>

    4.  鍑虹幇鐗囩姸鎷栧甫鎴栨秱鎶瑰甫

    锛圏/span>1锛夊師鍥燑/span>

     

    鈶  閰堕噺杩囧銆侟/span>

     

    鈶  閰剁殑璐ㄩ噺宸€侟/span>

     

    鈶  dNTP娴撳害杩囬珮銆侟/span>

    鈶  Mg2+娴撳害杩囬珮銆侟/span>

     

    鈶  閫€鐏俯搴﹁繃浣庛€侟/span>

     

    鈶  寰幆娆℃暟杩囧寮曡捣銆侟/span>

     

    锛圏/span>2锛夊绛朁/span>

     

    鈶 鍑忓皯閰堕噺銆侟/span>

     

    鈶  璋冩崲鍙︿竴鏉ユ簮鐨勯叾銆侟/span>

     

    鈶  鍑忓皯dNTP鐨勬祿搴︺€侟/span>

     

    鈶  閫傚綋闄嶄綆Mg2+娴撳害銆侟/span>

     

    鈶  澧炲姞妯℃澘閲忋€侟/span>

     

    鈶  鍑忓皯寰幆娆℃暟銆侟/span>

  • 杩斿洖鍒楄〃