绗簩绔?/p>
1.
鍚嶈瘝瑙ｉ噴锛氭牳閰稿唴鍒囬叾銆佹牳閰稿唴鍒囬檺鍒堕叾銆佸悓瑁傞叾銆佸悓灏鹃叾
銆佹牳閰稿鍒囬叾
銆佹湯绔劚姘?/p>
鏍歌嫹閰歌浆绉婚叾
绛旓細
鏍搁吀鍐呭垏閰讹細鏄竴绫讳粠澶氭牳鑻烽吀閾剧殑鍐呴儴鍌寲纾烽吀浜岄叝閿柇瑁傜殑閰躲�?/p>
鏍搁吀鍐呭垏闄愬埗閰讹細鏄竴绫昏兘澶熻瘑鍒弻閾?/p>
DNA
鍒嗗瓙涓殑鏌愮鐗瑰畾鏍歌嫹閰稿簭鍒楋紙
4
鈥?/p>
8bp
锛?/p>
锛屽苟
鐢辨澶勫垏鍓?/p>
DNA
鍙岄摼鐨勬牳閰稿唴鍒囬叾銆?/p>
鍚岃閰讹細璇嗗埆浣嶇偣鐨勫簭鍒楃浉鍚岀殑闄愬埗鎬у唴鍒囬叾銆?/p>
鍚屽熬閰讹細璇嗗埆鐨勫簭鍒椾笉鍚岋紝浣嗚兘鍒囧嚭鐩稿悓鐨勭矘鎬ф湯绔�?/p>
鏍搁吀澶栧垏閰讹細鏄竴绫讳粠澶氭牳鑻烽吀閾剧殑涓�澶村紑濮嬪偓鍖栭檷瑙ｆ牳鑻烽吀鐨勯叾銆?/p>
鏈鑴辨哀鏍歌嫹閰歌浆绉婚叾
锛氬彲浠ヤ笉闇�瑕佹ā鏉匡紝鍦ㄥ崟閾?/p>
DNA
鎴栫獊鍑虹殑鍙岄摼
DNA
3鈥?/p>
-OH
绔殢鏈?
娣诲姞
dNTPs
鐨勯叾
2.
闄愬埗鎬у唴鍒囨牳閰搁叾鐨勫懡鍚嶅師鍒欐槸浠�涔堬紵
绛旓細闄愬埗鎬у唴鍒囨牳閰搁叾鎸夊睘鍚嶅拰绉嶅悕鐩哥粨鍚堢殑鍘熷垯鍛藉悕鐨勶紝鍗筹細灞炲悕
+
绉嶅悕
+
鏍悕
+
搴忓彿锛?/p>
棣栧瓧姣嶏細鍙栧睘鍚嶇殑绗竴涓瓧姣嶏紝涓旀枩浣撳ぇ鍐欙紱
绗簩瀛楁瘝锛氬彇绉嶅悕鐨勭涓�涓瓧姣嶏紝鏂滀綋灏忓啓锛?/p>
绗笁瀛楁瘝锛?/p>
(1)
鍙栫鍚嶇殑绗簩涓瓧姣嶏紝鏂滀綋灏忓啓锛?/p>
(2)
鑻ョ鍚嶆湁璇嶅ご锛屼笖宸插懡鍚嶈繃闄愬埗閰讹紝鍒欏彇璇嶅ご鍚庣殑绗竴瀛楁瘝浠ｆ浛銆?/p>
绗洓瀛楁瘝锛氳嫢鏈夋牚鍚嶏紝鏍悕鍒欎綔涓虹鍥涘瓧姣嶏紝鏄惁澶у皬鍐欙紝鏍规嵁鍘熸潵鐨勬儏鍐佃�屽畾锛屼絾鐢?/p>
姝ｄ綋銆?/p>
椤哄簭鍙凤細鑻ュ湪鍚屼竴鑿屾牚涓垎绂讳簡鍑犵闄愬埗閰讹紝鍒欐寜鍏堝悗椤哄簭鍐犱互
I
銆佲叀銆佲參銆佲�?/p>
绛夛紝
鐢ㄦ浣撱�?/p>
3
锛庨儴鍒嗛叾鍒囧彲閲囧彇鐨勬帾鏂芥湁鍝簺锛?/p>
绛旓細
1
锛夌缉鐭繚娓╂椂闂?/p>
2
锛夐檷浣庡弽搴旀俯搴?/p>
3
锛夊噺灏戦叾鐨勭敤閲?/p>
4.
鍦ㄥ簭鍒?/p>
5'-CGAACATATGGAGT-3'
涓惈鏈変竴涓?/p>
6bp
鐨勨叀绫婚檺鍒舵�у唴鍒囨牳閰搁叾鐨勮瘑鍒簭鍒楋紝璇ヤ綅鐐圭殑搴忓垪鍙兘鏄粈涔?/p>
?
绛旓細鍥炴枃搴忓垪鏄細
5'-CATA
TG-3
锛?/p>
5
锛庝粈涔堟槸闄愬埗鎬у唴鍒囨牳閰搁叾鐨勬槦鍙锋椿鎬?/p>
?
鍙楀摢浜涘洜绱犲奖鍝?/p>
?
绛旓細鈪＄被闄愬埗閰惰櫧鐒惰瘑鍒拰鍒囧壊鐨勫簭鍒楅兘鍏锋湁鐗瑰紓鎬э紝浣嗘槸杩欑鐗瑰紓鎬у彈鐗瑰畾鏉′欢鐨勯檺
鍒讹紝鍗冲湪涓�瀹氱幆澧冩潯浠朵笅琛ㄧ幇鍑烘潵鐨勭壒寮傛�с�傛潯浠剁殑鏀瑰彉锛岄檺鍒堕叾鐨勭壒寮傛�у氨浼氭澗
鍔紝璇嗗埆鐨勫簭鍒楀拰鍒囧壊閮芥湁涓�浜涙敼鍙橈紝鏀瑰彉鍚庣殑娲绘�ч�氬父绉扮浜屾椿鎬э紝鑰屽皢杩欑鍥?/p>
鏉′欢鐨勬敼鍙樹細鍑虹幇绗簩娲绘�х殑閰剁殑鍙充笂瑙掑姞涓�涓槦鍙疯〃绀猴紝鍥犳绗簩娲绘�у張绉颁负鏄?/p>
娲绘�с�?/p>
姒傛嫭璧锋潵锛岃鍙戞槦娲绘�х殑鍥犵礌鏈夊涓嬪嚑绉嶏細
(1)
楂樼敇娌瑰惈閲?/p>
(>5
锛?/p>
, v
锛?/p>
v)
锛?/p>
(2)
闄愬埗鎬?/p>
鍐呭垏鏍搁吀閰剁敤閲忚繃楂?/p>
(>100U
锛?/p>
ugDNA)
锛?/p>
(3)
浣庣瀛愬己搴?/p>
(<25 mmol
锛?/p>
L)
锛?/p>
(4)
楂?/p>
pH(8
锛?/p>
0
浠ヤ笂
)
锛?/p>
(5)
鍚湁鏈夋満婧跺墏锛屽
DMSO
锛屼箼閱囩瓑锛?/p>
(6)
鏈夐潪
Mg2+
鐨勪簩浠烽槼绂诲瓙瀛樺湪
(
濡?/p>
Mn2+
锛?/p>
Cu2+
锛?/p>
C02+
锛?/p>
Zn2+
绛?/p>
)
銆?/p>
绗笁绔?/p>
1
锛?/p>
濡備綍灏嗛噹鐢熷瀷鐨?/p>
飦?/p>
鍣弻浣撴敼閫犳垚涓轰竴涓悊鎯崇殑杞戒綋
?
绛旓細鈶犲垹闄?/p>
飦?/p>
鍣弻浣撶殑闈炲繀闇�鍖猴紝鐣欏嚭鎻掑叆绌洪棿锛涘苟鍦ㄤ綑涓嬬殑闈炲繀椤诲尯鍐呭埗閫犻檺鍒堕叾鍒囩偣
鈶″紩杩涙煇浜涚獊鍙樿〃鍨嬶紝浣滀负閫夋嫨鏍囪
鈶㈢獊鍙樻煇浜涘熀鍥狅紝浣垮畠鎴愪负瀹夊叏杞戒綋
鈶ｅ垹闄?/p>
飦?/p>
DNA
蹇呴』鍖烘涓婂父鐢ㄧ殑闄愬埗閰跺垏鐐?/p>