瑷傝臣淇℃伅锛�
鐢�(ch菐n)鍝佸悕绋� | 銆€鍌虫煋鎬ц兏鑶滆偤鐐庢斁绶氭】鑿�(APP)鏍搁吀妾㈡脯瑭﹀姂鐩� |
瑕�(gu墨)鏍� | 48T/鐩� |
椤炲垾 | 鐔掑厜-PCR娉� |
鐢�(ch菐n)鍝佸悕绋憋細鍌虫煋鎬ц兏鑶滆偤鐐庢斁绶氭】鑿�(APP)鏍搁吀妾㈡脯瑭﹀姂鐩�
瑕�(gu墨)鏍硷細48T/鐩�
鍒嗛锛氱啋鍏�-PCR娉�
鍎插瓨姊濅欢锛�-20鈩冮伩鍏変繚瀛橈紝閬垮厤鍙嶅京鍑嶈瀺銆�
閬嬭几锛氫綆婧€侀伩鍏夛紝蹇仦鍏嶈不閫佽波涓婇杸銆�
鐗归粸锛�
1.鍗抽枊鍗崇敤锛岀敤鎴跺彧闇€瑕佹彁渚涙ǎ鍝� DNA 妯℃澘锛屾搷浣滅啊鍠紝瀹氶噺婧栫⒑蹇€熴€�
2. 寮曠墿缍�(j墨ng)閬庡劒(y艒u)鍖栵紝鐗圭暟鎬у挤銆傞爯鏈熺殑 PCR 鐢�(ch菐n)鐗╅暦搴︾偤 560 bp銆�
3. PCR mix 涓惈涓婃ǎ鏌撴枡锛孭CR 鍚庡彲浠ョ洿鎺ヤ笂妯i浕娉炽€�
4. 鎻愪緵闄芥€у皪鐓э紝渚夸簬鍒嗘瀽瑭﹂绲�(ji茅)鏋溿€傛敞鎰忎簨闋咃細
1锛庡熀绀�(ch菙)绋嬪簭锛�
2锛庢摯澧炴韩搴﹀拰寤朵几婧害锛�
3锛庡弽鎳夋檪闁擄紱
4锛庡惊鐠�(hu谩n)娆℃暩(sh霉)锛�
5锛嶱CR 鍙嶆噳娑茬殑閰嶅埗锛�
6锛嶱CR鎶€琛�(sh霉)鐨勫熀鏈師鐞嗭紱
7锛嶱CR鐨勫弽鎳夊嫊鍔涘锛�
8锛嶱CR鎿村鐢�(ch菐n)鐗╋紱
9锛嶱CR鍙嶆噳楂旂郴鑸囧弽鎳夋浠躲€�

浣跨敤鍙婃晥鏋滐細
PCR鍙嶆噳鐗归粸
(1) 鐗圭暟鎬у挤
PCR鍙嶆噳鐨勭壒鐣版€ф焙瀹氬洜绱犵偤锛�
銆€銆€銆€鈶犲紩鐗╄垏妯℃澘DNA鐗圭暟姝g⒑鐨勭祼(ji茅)鍚堬紱
銆€銆€銆€鈶″牽鍩洪厤灏嶅師鍓囷紱
銆€銆€銆€鈶aq DNA鑱氬悎閰跺悎鎴愬弽鎳夌殑蹇犲鎬э紱
銆€銆€銆€鈶i澏鍩哄洜鐨勭壒鐣版€ц垏淇濆畧鎬с€�
鍏朵腑寮曠墿鑸囨ā鏉跨殑姝g⒑绲�(ji茅)鍚堟槸闂�(gu膩n)閸点€傚紩鐗╄垏妯℃澘鐨勭祼(ji茅)鍚堝強寮曠墿閺堢殑寤朵几鏄伒寰牽鍩洪厤灏嶅師鍓囩殑銆傝仛鍚堥叾鍚堟垚鍙嶆噳鐨勫繝瀵︽€у強Taq DNA鑱氬悎閰惰€愰珮婧€э紝浣垮弽鎳変腑妯℃澘鑸囧紩鐗╃殑绲�(ji茅)鍚�(寰╂€�)鍙互鍦ㄨ純楂樼殑婧害涓嬮€茶锛岀祼(ji茅)鍚堢殑鐗圭暟鎬уぇ澶у鍔狅紝琚摯澧炵殑闈跺熀鍥犵墖娈典篃灏辫兘淇濇寔寰堥珮鐨勬纰哄害銆傚啀閫氶亷閬告搰鐗圭暟鎬у拰淇濆畧鎬ч珮鐨勯澏鍩哄洜鍗€(q奴)锛屽叾鐗圭暟鎬х▼搴﹀氨鏇撮珮銆�
(2) 闈堟晱搴﹂珮
PCR鐢�(ch菐n)鐗╃殑鐢熸垚閲忔槸浠ユ寚鏁�(sh霉)鏂瑰紡澧炲姞鐨勶紝鑳藉皣鐨厠(pg=10-12g)閲忕礆鐨勮捣濮嬪緟娓ā鏉挎摯澧炲埌寰厠(ug=10-6g)姘村钩銆傝兘寰�100钀€嬬窗鑳炰腑妾㈠嚭涓€鍊嬮澏绱拌優锛涘湪鐥呮瘨鐨勬娓腑锛孭CR鐨勯潏鏁忓害鍙仈3鍊婻FU(绌烘枒褰㈡垚鍠綅)锛涘湪绱拌弻瀛镐腑zui灏忔鍑虹巼鐐�3鍊嬬窗鑿屻€�
(3) 绨′究銆佸揩閫�
PCR鍙嶆噳鐢ㄨ€愰珮婧殑Taq DNA鑱氬悎閰讹紝涓€娆℃€у湴灏囧弽鎳夋恫鍔犲ソ鍚庯紝鍗冲湪DNA鎿村娑插拰姘存荡閸嬩笂閫茶璁婃€�-閫€鐏�-寤朵几鍙嶆噳锛屼竴鑸湪2锝�4 灏忔檪瀹屾垚鎿村鍙嶆噳銆傛摯澧炵敘(ch菐n)鐗╀竴鑸敤闆绘吵鍒嗘瀽锛屼笉涓€瀹氳鐢ㄥ悓浣嶇礌锛岀劇鏀惧皠鎬ф薄鏌撱€佹槗鎺ㄥ唬銆�
(4) 灏嶆鏈殑绱斿害瑕佹眰浣�
涓嶉渶瑕佸垎闆㈢梾姣掓垨绱拌弻鍙婂煿椁�(y菐ng)绱拌優锛孌NA 绮楀埗鍝佸強绺絉NA鍧囧彲浣滅偤鎿村妯℃澘銆傚彲鐩存帴鐢ㄨ嚚搴婃鏈琛€娑层€侀珨鑵旀恫銆佹礂鍡芥恫銆佹瘺鐧�(f膩)銆佺窗鑳炪€佹椿PCR鎶€琛�(sh霉)姒傝珫銆�