ᚑくᄢቇℂᎿቇ⎇ⓥႎ๔
J. Fac. Sci. Tech., Seikei Univ.
Vol.47 No.2 (2010) pp.31-32㧔⎇ⓥㅦႎ㧕ญ ⣧ ☼ ⤑ ߆ ࠄ ߩ 㪪㪥㪧 ᬌ ಴ 㧙ன㓁ᕈࠍ㒐ᱛߔࠆߦߪ㧙ᷰㄝ ዷሶ㧖㧘⩲⼱ 㤗Ꮧ㧖㧘ਭን ኼ㧖

SNP of aldehyde dehydrogenase 2 in oral mucosa samples
Nobuko Watanabe㧖, Maki Sugaya㧖, Hisashi Hisatomi㧖(Received September 21, 2010)㧝⢛ ᥊ SNP ᬌ಴ߦઍ⴫ߐࠇࠆㆮવሶ⸻ᢿߢߪ, DNA ណขߩߚ߼ណⴊ߇৻⥸⊛ߢ޽ࠆ߇, ቬᢎ਄ߩℂ↱߿ᗵᨴ㒐ᱛߥ৻Ⴎၮᄙဳ (SNP, Single Nucleotide Polymorphism)ߤߩⷰὐ߆ࠄ, ណⴊߩઍࠊࠅߦญ⣧☼⤑ࠍࠬࡢࡉߢណข

ߪ⚂ 30 ం୘ߩႮၮኻ߆ࠄ᭴ᚑߐࠇࠆࡅ࠻ߩ DNA ߩ߁ߔࠆᣇᴺ߽ណ↪ߐࠇߡ޿ࠆ‫ߩߎޕ‬ᣇᴺߪ, ࠝ࡝ࡦࡇ࠶ࠢߜ, 1000 Ⴎၮߦ 1 ߟ⒟ᐲሽ࿷ߒ, ᄙ᭽ᕈߦ㑐ਈߒߡ޿ࠆ‫ޕ‬SNP ߩᬌ಴ߦࠃࠅ, ⮎೷ߦኻߔࠆല⢻ࠍ੍߼ផ᷹ߒ, ᴦߢߩㆮવሶ⸻ᢿߢ߽ណ↪ߐࠇߡ߅ࠅ, ⺕ߦߢ߽◲නߦDNA ࠍណขߢ߈ࠆ೑ὐ߇޽ࠆ‫ᧄޕ‬ታ㛎ߢߪ DNA ♖⵾ߩᚻ㑆ࠍ⋭߈, ญ⣧☼⤑߆ࠄ⋥ធ PCR ෻ᔕࠍⴕ߁ߚ߼,≮ᴺߩㆬᛯ߿⮎೷ᛩਈ㊂ߩ᳿ቯߥߤ, ᦨㆡߥᴦ≮ᴺࠍ࠺

PCR 㒖ኂ‛⾰ࠍਛ๺ߔࠆ Ampdirect (Shimadzu) ࠍ↪

ࠩࠗࡦߔࠆ࠹࡯࡜࡯ࡔࠗ࠼ක≮߇น⢻ߣߥࠅ, ੹ᓟߩ⥃ᐥᔕ↪߇ᦼᓙߐࠇࠆ‫⃻ޕ‬࿷㧘SNP ߩ⒳‫ߩޘ‬ᬌ಴ᴺ߇㐿⊒޿ߡ, AS-PCR ߦࠃࠆ SNP ᬌ಴ࠍⴕߞߚ‫ޕ‬ᣇᴺ߇ SNP ᬌ಴ᴺߣߒߡఝࠇߡ޿ࠆ߆್ᢿߒߦߊ޿‫ޕ‬㧞㧚᧚ᢱߣᣇᴺߦ߅ߌࠆன㓁ᕈߩ㒐ᱛᣇᴺߦߟ޿ߡ⚫੺ߔࠆ‫ޕ‬AS-PCR 2TKOGT ߩ⸳ቯߐࠇߡ޿ࠆ߇, ᠲ૞ᕈ߿ା㗬ᕈߥߤߩ໧㗴߇޽ࠅ, ߤߩߘߎߢᧄႎ๔ߢߪ allele-specific PCR (AS-PCR) ᴺ 1㧕ߪᄌ⇣ㇱ૏߇ 3’ ᧃ┵ߦ૏⟎ߔࠆࠃ߁ߦ primer ࠍ⸳ቯߒ,ᄌ⇣߇޽ࠆ႐วߪ PCR ߦࠃࠆჇ᏷߇⿠ߎࠄߥ޿ߎߣࠍForward primer ࠍ 5’-GTCCTGGGAGTGTAACCCATAACCC-3’ߣߒ, Reverse primer ߪㅢᏱߩ AS-PCRᴺߢ૶↪ߔࠆ 3’ᧃ┵ߦᄌ⇣ㇱ૏ࠍ౉ࠇߚ R1, 3’ᧃ┵ߩ 1೑↪ߒߚᄌ⇣ᬌ಴ᴺߢ޽ࠆ‫ߒ߆ߒޕ‬, ℂ⺰ㅢࠅߦ primerߟᚻ೨ߦછᗧߩᄌ⇣ࠍ౉ࠇߚ R2, 3’ᧃ┵ߩ 2 ߟᚻ೨ߦછ

ࠍ⸳ቯߒߡ߽ primer ߩႮၮ㈩೉߿㉂⚛ߥߤߩ෻ᔕ᧦ઙᗧߩᄌ⇣ࠍ౉ࠇߚ R3 ࠍᱜᏱဳ (wild type) ߅ࠃ߮ᄌ⇣ဳ (mutant type) ߘࠇߙࠇߦߟ޿ߡ⸳ቯߒߚ (Table 1)‫ޕ‬ߦࠃࠅℂ⺰ㅢࠅߩ⚿ᨐ߇ᓧࠄࠇߥ޿ߎߣ߇ᄙ‫ᧄޕࠆ޽ޘ‬Ⓜߢߪ, aldehyde dehydrogenase 2 (ALDH2) ߩ SNP ࠍ଀ߦ AS-PCR ᴺߦ߅ߌࠆன㓁ᕈߩ㒐ᱛᣇᴺࠍㅀߴࠆ‫ޕ‬

6CDNG4GXGTUGRTKOGT ߩႮၮ㈩೉

ALDH2 ߪ㘶㈬ᓟ, ៨ขߒߚࠛ࠲ࡁ࡯࡞ߩઍ⻢ߦࠃߞ

ฬ⒓         ㈩೉         㩷 wild R1 5’-GGTCCCACACTCACAGTTTTCACTTC-3’
mut R1 5’-GGTCCCACACTCACAGTTTTCACTTT -3’

ߡ↢ߓࠆࠕ࡞࠺ࡅ࠼ࠍ㈶㉄ߦಽ⸃ߔࠆઍ⻢㉂⚛ߢ޽ࠅ,ࡅ࠻ߢߪ 12q24.2 ߦ૏⟎ߒ, 44239 bp ߩ DNA ߆ࠄ 13

wild R2 5’-GGTCCCACACTCACAGTTTTCACTVC-3’
mut R2 5’-GGTCCCACACTCACAGTTTTCACTVT -3’

୘ߩ exon ߢ᭴ᚑߐࠇࠆ mRNA ߦォ౮ߐࠇ, 517 ୘ߩࠕࡒࡁ㉄ߦ⠡⸶ߐࠇࠆ‫ޕ‬ALDH2 ߦߪ 504 ⇟⋡ߩࠣ࡞࠲ࡒࡦ㉄߇࡝ࠪࡦߦᄌ⇣ߔࠆ SNP (rs671 : G/A) ߇ႎ๔ߐࠇ

wild R3 5’-GGTCCCACACTCACAGTTTTCACVTC-3’
mut R3 5’-GGTCCCACACTCACAGTTTTCACVTT -3’

࠺ࡅ࠼ߩಽ⸃⢻ജ߇ᒙ߹ࠅ, Ქᕈߩᓇ㗀ࠍฃߌ߿ߔ޿૕

ߡ޿ࠆ‫ޕ‬ᄌ⇣ဳߢߪ ALDH2 ᵴᕈ߇ૐਅߔࠆߚ߼, ࠕ࡞Ⴎၮ㈩೉V : A, C or G⾰ߣߥࠆ 2)‫ ߼ߚߩߘޕ‬ALDH2 ߩ SNP ᬌ಴ߪ㘶㈬ߦኻߔ

㩷&0#RQN[OGTCUG ߩㆬᛯ 12 ⒳㘃ߩ DNA polymerase ߣߒߡ AmpliTaq Goldࠆ⠴ᕈߩ್ቯߦ↪޿ࠄࠇߡ޿ࠆ‫ޕ‬(Applied biosystems) , Tfi, Pfx, Pfx50 (Invitrogen) ,*1

FastStart Taq, Expand High Fidelityplus, Pwo Super

㧦ℂᎿቇ⎇ⓥ⑼ℂᎿቇኾ᡹ඳ჻೨ᦼ⺖⒟*2Yield (Roche Diagnostics) , Paq (Stratagene), Hot Start㧦‛⾰↢๮ℂᎿቇ⑼ ഥᢎ*3㧦‛⾰↢๮ℂᎿቇ⑼ ᢎ᝼(hisatomi@st.seikei.ac.jp)

㧙㧙


Taq (Greiner bio-one) , PrimeSTAR GXL (TaKaRa) ,Wild Type Reverse primer

6CDNG*QV5VCTV6CS&0#RQN[OGTCUG ߦࠃࠆㆬ೎KOD-plus-Neo, KOD FX (TOYOBO) ࠍ↪޿ߡญ⣧☼⤑߆ࠄ⋥ធ PCR ࠍⴕߥ޿, Ⴧ᏷ല₸ࠍ⺞ߴߚ‫߅ߥޕ‬, Ⴧ᏷ߦߪ Reverse primer ߣߒߡ wild R1 ߣ mut R1 ߩᷙวㆮવሶဳ

ℂᗐ

wild R1wild R2wild R3ᱜᏱဳࡎࡕჇ᏷޽ࠅჇ᏷޽ࠅჇ᏷޽ࠅჇ᏷޽ࠅᄌ⇣ဳࡎࡕჇ᏷ߥߒჇ᏷޽ࠅჇ᏷ߥߒ

Ⴧ᏷ߥߒ

Mutant Type Reverse primer‛ࠍ↪޿ߚ‫ޕ‬ㆮવሶဳℂᗐ

mut R1mut R2mut R3ᱜᏱဳࡎࡕჇ᏷ߥߒ

Ⴧ᏷޽ࠅ

Ⴧ᏷ߥߒ

Ⴧ᏷޽ࠅ

ᄌ⇣ဳࡎࡕ

Ⴧ᏷޽ࠅ

Ⴧ᏷޽ࠅ

Ⴧ᏷޽ࠅჇ᏷޽ࠅ㩷 ญ⣧☼⤑߆ࠄߩ 2%4 ᴺ

PCR ෻ᔕṁᶧ (Ampdirect 5 µL, Hot Start Taq DNAߒ, ᄌ⇣ဳߩ DNA ߪ Reverse primer mut R2 ߢߩჇ᏷bio-one), 200 µM dNTP, 1 µM Forward primer, 1 µM

C ߩႮၮ㈩೉ࠍ೎‫ࠈߎߣߚߒ⹜ߦޘ‬, A ߦᄌ⇣ߐߖߚߎpolymerase 0.2 units, GC-rich Ⴧ᏷ buffer 1 µL (Greinerല₸߇߿߿ᖡ߆ߞߚߚ߼, ᷙ๺ߒߚ V Ⴎၮߢߪߥߊ A, G,Reverse primer) ߦ, ࠬࡢࡉࠃࠅណขߒߚࡅ࠻ߩญ⣧☼ߣߢ, ࠃࠅㆬ೎ߩ♖ᐲ߅ࠃ߮Ⴧ᏷ല₸߇਄߇ߞߚ‫ߩߎޕ‬⤑ࠍ⫳⇐᳓ (200 µL) ߦᷙ๺ߒߚṁᶧ 1 µL ࠍ PCR ṁᶧprimer set (mut R4) ࠍ↪޿ߡ 9 ੱߩญ⣧☼⤑߆ࠄ PCR10 µL ߦ ᷝടߒ, 95͠/5min ߩᓟ, 95͠/20sec, 62͠ࠍⴕߥߞߚߣߎࠈ, ㆮવሶဳ߇್ቯ಴᧪ߚ (Figure 1)‫ޕ‬/25sec, 72͠/20sec ࠍ 1 ࠨࠗࠢ࡞ߣߒߚ PCR ෻ᔕࠍ45cycles ⴕߞߚ‫ޕ‬ญ⣧☼⤑ 1 ࠨࡦࡊ࡞ߦߟ߈ᱜᏱဳ, ᄌᱜᏱ ᄌ⇣

ᱜᏱ ᄌ⇣

ᱜᏱ ᄌ⇣

ࡋ࠹ࡠဳ, 1 ߟߩߺჇ᏷ߒߚ႐วߪߘࠇߙࠇᱜᏱဳߩࡎᱜᏱဳࡎ

ࡋ࠹ࡠဳ

ᄌ⇣ဳࡎ

⇣ဳߘࠇߙࠇߩ෻ᔕṁᶧࠍ↪ᗧߒ, 2 ߟߣ߽Ⴧ᏷ߔࠇ߫

(KIWTG#.&*&0# ߩ 502 ㆬ೎
Reverse primer ߦߪ mut R4 ࠍ↪޿ߚ‫ޕ‬ࡕဳ, ᄌ⇣ဳߩࡎࡕဳߣ್ቯߒߚ‫ޕ‬㧠⠨ ኤ㧟⚿ ᨐᧄ᧪, AS-PCR ߪ 3’ᧃ┵ߦᄌ⇣ㇱ૏߇૏⟎ߔࠆࠃ߁ߦ ALDH2 ߦ߅ߌࠆ 12 ⒳㘃ߩ DNA polymerase ߩჇ᏷primer ࠍ⸳⸘ߔࠆ‫ߒ߆ߒޕ‬, ታ㓙ߦ෻ᔕࠍⴕ߁ߣ᳿ߒߡല₸ࠍ Table 2 ߦ␜ߔ‫ޕ‬ญ⣧☼⤑ࠍ⋥ធ PCR ෻ᔕߦ↪

allele specific ߥ⚿ᨐߦߥࠆࠊߌߢߥ޿‫ߢߎߘޕ‬, ੍߼ 3’޿ࠆߎߣߢ෻ᔕߦ㒖ኂ߇߆߆ࠅ, Ⴧ᏷ല₸߇ᖡ޿㉂⚛߇ᧃ┵ߩ SNP ߣߘߩౝ஥ (5’஥) ߦછᗧߩᄌ⇣ࠍ౉ࠇࠆᄙ߆ߞߚ‫ߩߘޕ‬ਛߢ, FastStart Taq, Pwo Super Yield,Paq, Hot Start Taq ߪ⦟ᅢߥჇ᏷߇⏕⹺ߐࠇߚ‫ߦࠄߐޕ‬,ߎߣߢㆬᛯᕈࠍჇߐߖࠆߎߣߦᚑഞߒߚ‫⎇ᧄޕ‬ⓥߩઁߦ,Un-coupling 1 ㆮવሶߢ߽ห᭽ߥ⚿ᨐ߇ᓧࠄࠇߡ߅ࠅ,6CDNGฦ &0#RQN[OGTCUG ߦ߅ߌࠆჇ᏷ല₸DNA polymerase㩷 Ⴧ᏷ല₸DNA polymerase㩷 Ⴧ᏷ല₸AmpliTaq Gold㩷 㩷 㩷 㩷 Pwo Super Yield٤Tfi㩷 㩷 㩷 㩷 Paq
٤Pfx㩷 㩷 㩷 㩷 Hot Start Taq
٤Pfx50ٌ㩷 㩷 㩷 㩷 PrimeSTAR GXL FastStart Taq٤㩷 㩷 㩷 㩷 KOD-plus-Neoٌ
Expand High Fidelityplus KOD FX㩷ٌprimer ߩ 1 Ⴎၮౝ஥ߦછᗧߩᄌ⇣ࠍ౉ࠇࠆᣇᴺߪઁߩSNP ߩᬌ಴ߦ߽ᔕ↪ߢ߈ࠆߣ⠨߃ࠄࠇࠆ‫ޕ‬ AS-PCR ߪ㔚᳇ᵒേߦࠃߞߡ PCR ↥‛ߩࠛࠕࡠ࠱࡞

߇⊒↢ߒ, ࠦࡦ࠲ࡒࡀ࡯࡚ࠪࡦߩෂ㒾ᕈ߇Ⴧߔ‫ޕ‬૗ᐲߢ߽߿ࠅ⋥ߒ߇಴᧪ࠆࠨࡦࡊ࡞ߥࠄߣ߽߆ߊ, ⥃ᐥᬌ૕ߢߪ 1 ᐲ㒢ࠅߩᬌ಴ߦߥࠅ, ᄬᢌߪ⸵ߐࠇߥ޿‫࠱ࡠࠕࠛޕ‬࡞ ࠍ 㒐 ᱛ ߔ ࠆ ߚ ߼ , SYBR Green I ߩ ᷝ ട ߦ ࠃ ࠆ

٤:Ⴧ᏷⦟ᅢ, ٌ:Ⴧ᏷ᓸᒙ, :Ⴧ᏷⏕⹺ߖߕ

real-time PCR ߢߩᬌ಴߇ᦸ߹ߒ޿ߣ⠨߃ࠄࠇࠆ‫ޕ‬ߎ ߩ 㧠 ⒳ 㘃 ߩ DNA polymerase ߦ ߅ ޿ ߡ , Forward

primer ߦኻߒ 6 ⒳㘃ߩ Reverse primer ࠍߘࠇߙࠇ↪޿ߡᄌ⇣ߩㆬ೎ࠍ⏕⹺ߒߚ‫ޕ‬Table 3 ߦᦨ߽ㆬ೎♖ᐲ߇㜞ෳ⠨ᢥ₂߆ߞߚ Hot Start Taq ߩ⚿ᨐࠍ␜ߔ‫߽ᦨޕ‬ㆬ೎♖ᐲ߇㜞޿

Hot Start Taq ߢ߽, ℂ⺰਄ㆬ೎ߢ߈ࠆߪߕߩ Reverse1) Takeda S, Ichii S, Nakamura Y. : Detection of K-rasߥ޿ߎߣ߇್᣿ߒߚ‫ߚ߹ޕ‬, 3’ᧃ┵ߩ 2 ߟᚻ೨ߦછᗧߩ

amplification (MASA). Hum. Mutat. 2:112-117, 1993.

mutationprimer wild R1 ޽ࠆ޿ߪ mut R1 ࠍ↪޿ࠆߣ, ㆬ೎ߢ߈ᄌ⇣ࠍ౉ࠇߚ wild R3 ߢ߽ห᭽ߦㆬ೎ߪߢ߈ߥ߆ߞߚ‫ޕ‬ߒ߆ߒ, Reverse primer ߩ 3’ᧃ┵ߩ 1 ߟౝ஥ߩႮၮࠍ, T߆ࠄ A, G, C ࠍᷙ๺ߒߚ V ߦᄌᦝߒߚ wild R2, mut R2ࠍ↪޿ࠆߎߣߢ SNP ㆬ೎߇น⢻ߣߥߞߚ (Table 3)‫ߛߚޕ‬㧙㧙

insputumbymutant-allele-specific2) Yoshida A. : Molecular genetics of human aldehyde
dehydrogenase. Pharmacogenetics 2:139-147, 1992.