¹ØÓÚÑÇÁòËáÄÆ´¦ÀíºÍMSP¼×»ù»¯PCRµÄ·½·¨ÎÊÌâ - Çë½Ìhula (Aug/01/2004 )
ÒѾ°´ÕÕÄú½éÉܵķ½·¨È¥×ö£¬ÕýÔڵȴý½á¹û£¬ìþìý²»°²ÄÄ£¡ 
ºÜ´óÓÂÆøÀ´Ð´Õâ´ÎµÄÁôÑÔ£ºÊµÑéûÓгɹ¦£¬²»ÖªµÀÊÇÄÄÀï³öÁËÎÊÌ⣬ÒòΪÊÇÐÇÆÚÁùûÓÐÄܹ»
²â¾´¦ÀíºóDNAµÄŨ¶È£¬Ö±½Ó×÷ÁËPCR£¬Ã»ÓÐÄ¿µÄÌõ´ø£¨ÔÚ×ÏÍâµÆÏ¿´£¬ÖÜĩûÓÐÄܹ»ÓÃÉÏͼ
Ï
ñ·ÖÎöÒÇ£©¡£
DNA´¦Àí¹ý³Ì»ù±¾°´ÕÕÄú¸øµÄ·½°¸£¬¾ßÌå²½ÖèΪ£ºÈ¡»ùÒò×éDNA10΢Éý£¬Å¨¶ÈԼΪ100-200ng/ul£¬¼Ó40ulÃð¾ú´¿Ë®£¬»ìÔÈ£¬¼ÓÐÂÏÊÅäÖÃ3MÇâÑõ»¯ÄÆÈÜÒº5΢Éý£¬37ÉãÊ϶ȱ£ÎÂ25·ÖÖÓ£¬ÆÚ¼
äÅäÖÆ40.5%ÑÇÁòËáÇâÄÆºÍ10mMÇâõ«£¬ÆäÖÐÑÇÁòËáÇâÄÆÓÃ3MÇâÑõ»¯ÄƵ÷½ÚPHÖµÖÁ5¡£È¡³ö±£ÎÂÑùÆ·£¬¼ÓÈë1
0mMÇâõ«30ul£¨ÈÜÒºÑÕɫת»Æ£©£¬¼ÓÈë40.5%ÑÇÁòËáÇâÄÆ510΢Éý£¬Îý²Ö½°ü¹üÑùÆ·£¬ÇáÈá»ìÔÈ¡£55¶È±£ÎÂ16Сʱ¡£
ÓÃpromega¹«Ë¾DNA clean-upÊÔ¼ÁºÐ´¿»¯DNA£¬ÊÕ¼¯50ulÏ´ÍÑÒº£¬¼Ó5.5ulŨ¶ÈΪ3MµÄÇâÑõ»¯ÄÆ£¬»ìÔÈ£¬¼Ó8MÒÒËáï§ÈÜÒº
35ul£¬»ìÔÈ£¬¼ÓÔ¤ÀäÎÞË®ÒÒ´¼300ul£¬µßµ¹»ìÔÈ£¬ÖÃ-20¶È±ùÏä¹ýÒ¹¡£
È¡³öÑù±¾£¬13200rpmÀëÐÄ20min£¬ÆúÈ¥ÉÏÇ壬¼Ó70%ÒÒ´¼100ul£¬13200rpmÀëÐÄ2min£¬ÆúÈ¥ÉÏÇ壬ÁÀ¸ÉDNA£¬¼ÓÈëÃð¾ú´¿Ë®30ul£¬65ÉãÊ϶ÈÈܽâ5Ð
¡Ê±£¨ÖмäÓÐÈÕ³£¹¤×÷µ¢Îó£¬×ÉѯÆäËûÓоÑéµÄÈË£¬ËµÃ»ÓÐÎÊÌ⣩£¬Ö±½ÓÓøÃDNA½øÐÐPCR!
PCR·´Ó¦ÒýÎïŨ¶ÈΪ0.2uM£¬·´Ó¦Ìõ¼þΪ£º95¶È5·ÖÖÓ£¬ 95¶È45Ãë  56¶È45Ãë 72¶È45Ã룬40Ñ»·£¬72¶È7·ÖÖÓ¡£1.5%ÇíÖ¬ÌÇÄý½ºµçÓ¾¹Û²ì½á¹û¡£
Çë°ïæ·ÖÎöһϿÉÄÜ»áÊÇÔÚÄÄÀï³öÁËÎÊÌ⣿  ºÜ¶àºÜ¶àµÄ¸Ðл£¡
 ±¾×Å»³ÒÉÒ»Çеľ«Éñ£¬ÊÇ·ñÒýÎïµÄÎÊÌ⣿ÔʼÐòÁÐÊäÈëµÄÊÇ
taaaataaaata aaataacaca aaataaaata aaataaaata aaataaaata aaataataaa
ataaaataaa ataaaataaa ataaaataaa ataaagcaat ttcctttcct ctaagcggcc
tccacccctc tcccctgccc tgtgaagcgg gtgtgcaagc tccgggatcg cagcggtctt
agggaatttc cccccgcgat gtcccggcgc gccagttcgc tgcgcacact tcgctgcggt
cctcttcctg ctgtctgttt actccctagg ccccgctggg gacctgggaa agagggaaag
gcttccccgg ccagctgcgc ggcgactccg gggactccag ggcgcccctc tgcggccgac
µÃµ½ÎåÌ×ÒýÎï
Sequence Name: GSTP1
Sequence Length: 362
CpG island prediction results
Criteria used: Island size > 100, GC Percent > 50.0, Obs/Exp > 0.60
 1 CpG island(s) were found in your sequence
            Size	(Start - End)
 Island 1   189 bp	 (116 - 304)
Primer picking results for methylation specific PCR (MSP)
Ñ¡ÔñµÚÒ»Ì×£º   
   Primer            Start Size  Tm      GC%   'C's  Sequence
1  Left  M primer     208   27   69.54   70.37   6  GGCGCGTTAGTTCGTTGCGTATATTTC
   Right M primer     347   23   69.57   78.26   6  ACGCCCTAAAATCCCCGAAATCG
   Product size: 140, Tm: 57.4
   Left  U primer     209   28   57.03   71.43   7  GTGTGTTAGTTTGTTGTGTATATTTTGT
   Right U primer     347   25   65.94   80.00   6  ACACCCTAAAATCCCCAAAATCACC
   Product size: 139, Tm: 57.1
¿´ÆðÀ´Ã»ÓдóµÄÎÊÌ⣬Óм¸¸öϸ½Ú£¬²»ÖªµÀÊÇÄãûÓÐд£¬»¹ÊÇûÓÐ×öµ½¡£
35ul£¬»ìÔÈ£¬¼ÓÔ¤ÀäÎÞË®ÒÒ´¼300ul£¬µßµ¹»ìÔÈ£¬ÖÃ-20¶È±ùÏä¹ýÒ¹¡£
ÕâÒ»²½¼ÓÈë£Î£á£Ï£ÈºóÓ¦¸ÃÔÚ£³£·£Ã·ÅÖã±£µ£í£é£î£¬È»ºóÔÙ¼ÓÒÒËáï§ÈÜÒº¡£
¡¡
×îºó³ÁµíºóÓÐûÓп´µ½³ÁµíÎ¡£
£Ð£Ã£Ò·´Ó¦Ìå»ýÊǶàÉÙ£¬¼ÓÁ˶àÉÙÌå»ýµÄÄ£°æ£Ä£Î£Á£¿
½¨Ò飺
Èç¹ûÔøÂ©µôÉÏÃæÄÇÒ»²½£¬µÃ´ÓÍ·À´¡£
Èç¹û£²£°£õ£ìµÄ£Ð£Ã£Ò·´Ó¦Ìå»ý£¬Óã²£õ£ìµÄ£Ä£Î£Á¡£
Êʵ±Ôö¼Ó£Ð£Ã£ÒÑ»·´ÎÊý¡£
Èç¹ûÊÖÍ·ÓÐÌÇÔ£¬×îºÃ¼ÓÒ»µã¡£
±ðÈËÓÐûÓд¦ÀíºÃµÄ£Ä£Î£Á£¬¿ÉÓÃ×öÑôÐÔ¶ÔÕÕ¡£
Ò»¶¨ÒªÈÈÆô¶¯£Ð£Ã£Ò£¡
×îºóÒ»²½£¶£µ£ÃÈܽâûÓбØÒª¡£
µÚ¶þ´Î¼ÓÈëÇâÑõ»¯Äƺó¾¹ýÁË37¶È±£ÎÂ15min£¬Ð´µÄʱºòÍü¼ÇÁË£¡
PCRÊÇÈÈÆô¶¯µÄ£¬·´Ó¦Ìå»ýÊÇ20ul£¬ÓÃÁË2ulÄ£°å¡£³ÁµíµÄʱºòûÓп´µ½³ÁµíÎÏÖÔÚÊÖ±ßû
ÓÐÌÇÔ£¬²»¹ýÒѾ¶©ÁË£¬ÒѾµÈÁ˺ܾà  ¡£
55¶È±£ÎÂ16СʱÄǸö¹ý³ÌÊÇ·ñÐèÒªÓÿóÎïÓ͸²¸Ç£¿ÔÚ±ðµÄÎÄÏ×ÉÏ¿´µ½ÓÐÕâô×öµÄ£¬µ«ÎÒûÓÐ
Õâô×ö¡£
ÎÒËùÔڵĻ·¾³Ã»ÓÐÈË×ö¹ý¼×»ù»¯Ïà¹ØµÄʵÑ飬Éí±ßҲûÓÐÈËÔÚ×öÏà¹ØÄÚÈÝ£¬ËùÒԱȽÏÃÔ㣬
»¹Çë¶àÖ¸½Ì£¡
ÎÒ»á¼ÌÐøÅ¬Á¦µÄ 
²»ÊDZØÐè
ÄãµÄ£Ð£Ã£Ò²úÎïÊǶà´ó£¿¡¡²»ÒªÌ«³¤£¬£²£°£°ÒÔÏ¡£
PCR²úÎïΪ150bp×óÓ񵀮¬¶Î£¬ÎÒÏëÖØÐ»»±ðµÄ»ùÒò×éDNAÀ´ÊÔһϣ¬ÔÚPCR¹ý³Ìͬʱ×öÒ°ÉúÐÍ
ÒýÎï¶ÔÕÕ£¬¿´ÊÇ·ñ»¯Ñ§´¦Àí¹ý³ÌÓÐÎÊÌâ¡£Èç¹ûÄúÓпգ¬ÄÜ·ñ°ïÎÒ¿´Ò»ÏÂÄÇÒ»×éÒýÎïÊÇ·ñÓÐʲ
Ã
´ÎÊÌ⣿
ÎÒÔÚ¿¼ÒÅ´«Ñ§Ñо¿Éú£¬
ÏëÇë½Ìһϣ¬¿¼ÌâÖеÄϸ°ûÉúÎïѧʵÑéÉè¼Æ
ÔÚ´ðÌâʱӦ¸Ã×¢ÒâÄÄЩµØ·½£¬ÓÐÄÄЩ±ØÐëµÄ²½Ö裿×îºÃ¸ø¸öÀýÌ⣡
Íò·Öлл£¡
ÎÒÔÚ¿¼ÒÅ´«Ñ§Ñо¿Éú£¬
ÏëÇë½Ìһϣ¬¿¼ÌâÖеÄϸ°ûÉúÎïѧʵÑéÉè¼Æ
ÔÚ´ðÌâʱӦ¸Ã×¢ÒâÄÄЩµØ·½£¬ÓÐÄÄЩ±ØÐëµÄ²½Ö裿×îºÃ¸ø¸öÀýÌ⣡
Íò·Öлл£¡
