full2010.pdf - page 103

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°»ž„¦–r ¨³ª· ›¸„µ¦
„µ¦Ã‡¨œ ¨³«¹„¬µ‡»–¨´„¬–³ °Š¥¸œ CCH ‹µ„¥µŠ¡µ¦µ
Á˜¦¸¥¤ total RNA ‹µ„œÊÎ µ¥µŠ¡µ¦µµ¥¡´œ›»r RRIM600 š¸É Ážd—„¦¸—¨o ªž¦³¤µ– 5 že ˜µ¤ª· ›¸„µ¦ °ŠÁž¨ºÊ°Š
(2549) Â¥„ Poly (A+) ‹µ„ total RNA ץčo Oligotex mRNA Kit ‹µ„¦·¬´š Qiagen čo Poly (A+) Á¡·É¤ž¦·¤µ–¥¸œ
HbCCH šµŠ—o µœž¨µ¥ 5c ¨³ž¨µ¥ 3c ץčo gene specific primer (GSP) š¸É‹ÎµÁ¡µ³„´¨Îµ—´Á °Š TDFRMA69
—oª¥ SMARTTM RACE cDNA Amplification kit (Clontech, USA) ˜µ¤ª·›¸ „µ¦ °Š¦·¬´ šŸ¼oŸ¨·˜ Clontech
Laboratories (CA) šÎµ°·Á¨È„ڦ⦸Ž· °Š PCR product š¸Éŗo ‹µ„ 5c- RACE ¨³ 3c- RACE ĜÁ‹¨°³„µÃ¦ ˜´—
™ cDNA Ážjµ®¤µ¥Â¨³Â¥„°°„‹µ„Á‹¨°³„µÃ¦ ץčo Gel Elute Extraction kit ‹µ„¦· ¬´š 5 PRIME GmbH
‹µ„œ´Ê œ‹¹ŠœÎµ cDNA ޚε TA-Cloning ×¥ÁºÉ°¤Á oµ„´ pGEM“- T Easy vector ¨³Á‡¨ºÉ °œ¥oµ¥Á oµÅžÄœÁºÊ°
E. coli JM109 ˜µ¤ª·›¸„µ¦ °Š¦· ¬´šŸ¼oŸ¨·˜ (Promega, Madison, WI) ‡´—Á¨º°„ positive clone ŞÁ˜¦¸¥¤¡¨µ¤· —×¥
čo High speed plasmid mini kit ‹µ„¦·¬´š Geneaid (Taiwan R.O.C.) ¨³Á˜¦¸¥¤ sequencing reaction ץčo ABI
Prism Big Dye Terminator Cycle Sequencing ready Reaction Kits (Applied Biosystem) ª· Á‡¦µ³®r ¨Îµ—´ÁÃ—¥Äo
Á‡¦ºÉ°Š Automatic DNA sequencer (model 377, Perkin-Elmer, USA) Áž¦¸ ¥Áš¸¥¨Îµ—´„¦—°³¤·Ãœ °ŠÃž¦˜¸œ
HbCCH „´Ãž¦˜¸œ CCH °Š¡ºÂ¨³·ÉŠ¤¸¸ª·˜°ºÉœÇ ץčoަ„¦¤ BioEdit version 7.0.9 (Hall, 1999)
šÎ µœµ¥Ã‡¦Š¦oµŠ˜˜· ¥£¼¤·°ŠÃž¦˜¸œ HbCCH —oª¥ª·›¸ homology based modeling
ž¨¦®´ ¨Îµ—´Á °Š HbCCH Áž}œ¦®´ „¦—°³¤· Üץčo ަ„¦¤ ExPaSy-Translate tool
(http://www.expasy.ch/tools/dna.html) ¨³Äo¨Îµ—´„¦—°³¤·Ãœ °ŠÃž¦˜¸œ HbCCH šÎµœµ¥Ã‡¦Š¦o µŠ˜˜·¥£¼¤·
(tree dimensional structure) —oª¥ª·›¸ homology based modeling ץčoަ„¦¤ SWISS-MODEL (Arnold et al.,
2006)
„µ¦Â—Š°°„ °Š¥¸œ HbCCH ĜÁœºÊ °Á¥ºÉ°˜nµŠÇ °Š¥µŠ¡µ¦µ
Á˜¦¸ ¥¤ total RNA ‹µ„ÁœºÊ°Á¥ºÉ°Ä „oµœÄ ¨Îµ˜oœ ¨³¦µ„ °Š„¨oµ¥µŠ¡µ¦µ°µ¥»ž¦³¤µ– 3 Á—º °œ˜µ¤
ª·›¸„µ¦ °Š Áž¨ºÊ °Š (2549) ¨³Äo total RNA ‹µ„ÁœºÊ °Á¥ºÉ°˜nµŠÇ ª·Á‡¦µ³®r„µ¦Â—Š°°„ °Š¥¸œ HbCCH —oª¥Áš‡œ·‡
semi-quantitative RT-PCR (sqRT-PCR) ץčo 18S rRNA Áž}œ internal control
Ÿ¨„µ¦ª·‹´¥Â¨³°£· ž¦µ¥Ÿ¨
¨Îµ—´Á °Š¥¸œ HbCCH
‹µ„„µ¦Á¡·É¤ž¦·¤µ– cDNA °Š HbCCH —oª¥Áš‡œ·‡ RACE ¡ªnµµ¤µ¦™Á¡·É¤ž¦·¤µ–¥¸œÁžjµ®¤µ¥Å—o
™ cDNA °Š 5c-RACE œµ—ž¦³¤µ– 200 ‡¼nÁÂ¨³ 3c-RACE œµ—ž¦³¤µ– 500 ‡¼nÁ Á¤ºÉ °œÎµÅžª·Á‡¦µ³®r
¨Îµ—´Á ¡ªnµ¤¸¨Îµ—´Áš¸ÉÁ®¤º°œ„´œÂ¨³š´Žo°œ„´¨Îµ—´Á °Š TDFRMA69 š¸ÉčoÁž}œÂÄœ„µ¦ design
GSP °Š 5c-RACE ¨³ 3c-RACE —ŠªnµÁž}œ¨Î µ—´Á °Š¥¸œÁ—¸¥ª„´œ ¨Îµ—´ Á¦ª¤ °Š¥¸œš´ÊŠ®¤—
Ášn µ„´  567 ‡¼nÁÁ¤ºÉ °Âž¨¦®´ ¨Îµ—´ÁÁž} œ¦®´ „¦—°³¤· Üץčoަ„¦¤ ExPaSy-Translate tool
(http://www.expasy.ch/tools/dna.html) ¡ªnµž¦³„°—oª¥ 5c-noncoding region 92 ‡¼nÁ 3c-noncoding region
217 ‡¼n ÁÂ¨³ open reading frame š¸Éž¦³„°—oª¥ ¦®´ Á¦·É¤˜oœ (start codon) ¨³¦®´ °Š„¦—°³¤·Ãœ˜nµŠÇ
˜n°ÁœºÉ °Š„´œ‹Îµœªœš´ÊŠ®¤— 258 ‡¼nÁ (Áž}œ¦®´ °Š„¦—°³¤·Ãœ‹Îµœªœ 86 ˜´ª) ‹¹ Š˜µ¤—oª¥¦®´ ®¥»— (stop codon)