Capillary Gel Electrophoresis for Ligase Detection Reaction Products
Masahiko HASHIMOTO*, Jun KAMIGORI** and Kazuhiko TSUKAGOSHI***
(Received October 7, 2009)
One technique that can distinguish low-abundant mutant DNA from wild-type DNA is the ligase detection reaction (LDR) coupled to a primary polymerase chain reaction (PCR). The LDR products obtained by the PCR/LDR assay can be analyzed in a variety of fashions such as microarray and slab gel electrophoresis. In the present work, we applied capillary gel electrophoresis (CGE) with fluorescence detection (FL) (CGE-FL) for analyzing the LDR products. Polyethylene oxide (PEO) polymer solution containing SYBR Gold, which could bind to a single strand DNA, was used as a separation matrix in the CGE-FL system. The LDR products could successfully be separated from other DNA fragments such as LDR primers and template, enabling the determination of the single base substitutions on codon 12 of K-ras gene.
-G[YQTFU: capillary gel electrophoresis, polymer solution, point mutation, ligase detection reaction
ࠠࡢ࠼: ࠠࡖࡇࠥ࡞㔚᳇ᵒേ㧘ࡐࡑṁᶧ㧘ὐ⓭ὼᄌ⇣㧘ࠟᬌᔕ
ࠠࡖࡇࠥ࡞㔚᳇ᵒേߦࠃࠆࠟᬌᔕ↢ᚑ‛ߩಽᨆ
ᯅᧄ 㓷ᒾ㧘 ⚐㧘Ⴆ ৻ᒾ
ߪߓߦ
TCU ㆮવሶߪߘߩ߹߹ߢߪ⊒≸ᵴᕈࠍᜬߚߥ
߇㧘ߎࠇࠄߩㆮવሶߦᄌ⇣߇↢ߓࠆߣ⊒≸ᵴᕈࠍ
ߒ㧘ᄌ⇣ࠍߔࠆ⚦⢩߇ᒻ⾰ォ឵ߐࠇࠆߎߣ߇⍮
ࠄࠇߡࠆ㧚TCU ㆮવሶ⟲*TCU㧘-TCU㧘0TCU㧘 4TCUࠍ᭴ᚑߔࠆ -TCU ㆮવሶߩᵴᕈൻဳᄌ⇣
-TCU ≸ㆮવሶߪ㧘ࡅ࠻ߩ≸ߦ߅ߡᦨ߽ᄙߊ
ߛߐࠇࠆ≸ㆮવሶߩ৻ߟߢࠅ㧘ߎࠇࠄߩ≸ߢ ߪ㧘-TCU ≸ㆮવሶ߆ࠄߩᄌ⇣ဳ -TCU ࠲ࡦࡄࠢ⾰
ߩᜬ⛯⊛ߥ⊒㧘↢ᚑ߇⚦⢩ߩ≸ൻ߅ࠃ߮≸⚦⢩ߣ ߒߡߩᒻ⾰⛽ᜬߦᔅⷐਇนᰳߢࠆߎߣ߇ࠄ߆ ߣߥߞߡࠆ㧚߃߫㧘-TCU ㆮવሶߩࠛࠠ࠰ࡦ 㧘
ࠦ࠼ࡦ ))6ߦ߅ߌࠆႮၮߪ㧘)#6㧘)%6㧘)66
߳ߣ৻Ⴎၮ⟎឵ߐࠇࠆ႐ว߇ࠅ㧘ߘࠇߙࠇࠕࡒࡁ
㉄ᱷၮ #UR㧘#NC㧘8CN ߳ߣ⠡⸶ߐࠇࠆ㧚ߎࠇࠄߩࠕ ࡒࡁ㉄ߪ㧘)62 ⚿วߦ߅ߡ㊀ⷐߥᓎഀࠍᨐߚߒߡ ߅ࠅ㧘ߎߩࠦ࠼ࡦߦ߅ߌࠆὐ⓭ὼᄌ⇣ߪ㧘)62 ߩട
᳓ಽ⸃ࠍᛥߒ㧘ᕡᏱ⊛ߦᵴᕈࠪࠣ࠽ࡦࠣᯏ⢻ࠍ
ߔࠆ≸ㆮવሶ RTCU ࠲ࡦࡄࠢ⾰ࠍ↢ᚑߔࠆ㧚ᄢ
⣺≸ߦ߅ߡߪ㧘ߔߴߡߩᄢ⣺≸ᖚ⠪ߩ⚂ ̆ ߦ߅ߡ≸⊒㆐ߩೋᦼᲑ㓏ߦ -TCU ㆮવሶߦὐ⓭ ὼᄌ⇣ࠍᜬߟߎߣ߇⍮ࠄࠇߡࠆ 㧚ߘࠇࠁ߃ -TCU ㆮવሶߦ߅ߌࠆ⓭ὼᄌ⇣ߩήߦ㑐ߔࠆᖱ ႎࠍᓧࠆߎߣߪ㧘≸∔∛ߩ▤ℂߦ↪ߢࠆ㧚
* Department of Chemical Engineering and Materials Science, Doshisha University, Kyoto Telephone:+81-774-65-6594, Fax:+81-774-65-6803, E-mail: mahashim@mail.doshisha.ac.jp
** Former graduate student of Doshisha University
*** Department of Chemical Engineering and Materials Science, Doshisha University, Kyoto Telephone:+81-774-65-6595, Fax:+81-774-65-6803, E-mail: ktsukago@mail.doshisha.ac.jp
⓭ὼᄌ⇣ߩᬌߦ߅ߡᦨᄢߩ㓚ოߪ㧘ᬌߩኻ
⽎ߣߥࠆᄌ⇣ &0# ࠦࡇ߇㧘ୟ⊛ᄙᢙࠍභࠆᱜ Ᏹဳ &0# ࠦࡇ⟲ߩਛߦ߆ߒ߆ሽߒߡߥ
ߎߣߢࠆ㧚ᄙߊߩ≸ߦ߅ߡ㧘ਥⷐߥ≸⚵❱ߦ߅
ߡߔࠄ㧘ᱜᏱ⚦⢩ߩ₸ߪ 㧑ߦ߽ߥࠆ 㧚 ߘࠇࠁ߃㧘⣲≌⚦⢩ߩ ࠥࡁࡓߩ৻ᣇߦߩߺᄌ
⇣߇ሽߔࠆߥࠄ߫㧘ᄌ⇣ &0# ߩ₸ߪ 㧑ߢ ߒ߆ߥ㧚ߎߩ୯ߪ㧘ࠨࡦࡊ࡞ណข߇ਥⷐߥ≸⚵❱
߆ࠄ㔌ࠇߡߒ߹ߞߚ႐ว㧘⪺ߒߊૐਅߔࠆ㧚ߒߚ߇ ߞߡ㧘ᄢᄙᢙߩᱜᏱဳ⚦⢩⟲ߦᭂዋ㊂߹ࠇࠆᄌ⇣
⚦⢩ࠍᱜ⏕߆ߟ㜞ᗵᐲߦ․ቯߔࠆᛛⴚߩ㐿⊒߇ᔅ ⷐߢࠆ㧚
ᱜᏱဳ &0# ߩਛ߆ࠄᓸ㊂ߩᄌ⇣ &0# ࠍ⼂ߔࠆᚻ ᴺߩ৻ߟߦ㧘ࡐࡔㅪ㎮ᔕࠟᬌ
ᔕ2%4.&4ࠕ࠶ࠗ߇⍮ࠄࠇߡࠆ 㧚 2%4.&4 ࠕ࠶ࠗߢߪ㧘ᔕ⚳ੌᓟ㧘ᔕ↢ᚑ‛
.&4 ↢ᚑ‛ࠍᧂᔕߩᔕ‛߆ࠄಽ㔌ߒᬌߔ ࠆᔅⷐ߇ࠆ㧚ᧄ⎇ⓥߢߪ㧘2%4.&4 ࠕ࠶ࠗߦ߅ ߌࠆಽᨆല₸ߩะࠍ⋡⊛ߣߒ㧘↢㜞ಽሶߦኻߒ 㜞ㅦ߆ߟ㜞ಽ㔌⢻ࠍߔࠆࠠࡖࡇࠥ࡞㔚᳇
ᵒേCGEߦࠃࠅ .&4 ↢ᚑ‛ߩಽᨆࠍⴕߞߚ㧚
2%4.&4 ࠕ࠶ࠗߩේℂ
PCR/LDR
ࠕ࠶ࠗߩ⇛࿑ࠍFig. 1ߦ␜ߔ㧚߹ߕ㧘PCRߦࠃࠅࠥࡁࡓ DNAߩὐ⓭ὼᄌ⇣ࠍ
㗔ၞࠍPCRߦࠃࠅჇߔࠆ㧚ߎߎߢᓧࠄࠇߚPCR
↥‛ࠍ࠹ࡦࡊ࠻DNAߣߒߡ↪LDRᔕࠍ ⴕ߁㧚
LDR
ߢߪ㧘3ƍ
ᧃ┵ߦߘࠇߙࠇ⇣ߥࠆࠢࠝ࠴࠼
(A, G, T, C)
ࠍᜬߟ4
⒳ߩ⼂ࡊࠗࡑ㧘5ƍ
ᧃ┵߇ࡦ㉄ൻߐࠇ3ƍ
ᧃ┵߇Ⱟశࡌ࡞ൻߐࠇߚㅢࡊࠗࡑߩ㧞⒳ߩ
LDR
ࡊࠗࡑ߅ࠃ߮DNA
ࠟࠍ࠹ࡦࡊ࠻DNA
ߦട߃ࠆ㧚ߎ ߩLDR
ᔕࠞࠢ࠹࡞ࠍടᾲߔࠆߎߣߦࠃߞߡ㧞ᧄ㎮ߩ࠹ࡦࡊ࠻
DNA
ࠍ1
ᧄ㎮DNA
ߦᾲᄌᕈߐ ߖ㧘ߘߩᓟLDR
ࡊࠗࡑߩTm୯ઃㄭ߹ߢ಄ළߒ㧘LDR
ࡊࠗࡑࠍ࠹ࡦࡊ࠻DNA
ߩ⋧㎮ߦ ࠕ࠾ࡦࠣߐߖࠆ㧚⼂ࡊࠗࡑߩ3ƍ
ᧃ┵ߩႮ ၮ߇ᄌ⇣ㇱߩႮၮߣ⋧⊛ߥ႐วߦߩߺ㧘⼂ࡊࠗࡑߩ
3ƍ
ᧃ┵ߣㅢࡊࠗࡑߩ5ƍ
ᧃ┵ߪDNA
ࠟߦࠃࠅㅪ⚿ߐࠇ㧘3ƍ
ᧃ┵߇Ⱟశࡌ࡞ൻߐࠇߚ৻ᧄߩࠝࠧࠢࠝ࠴࠼
(LDR
↢ᚑ‛
)
߇ᓧࠄࠇࠆ㧚LDR
ᔕߪ㧘⠴ᾲᕈDNA
ࠟࠍ↪ࠆߎߣߦࠃࠅ㧘⸥᷷ᐲࠨࠗࠢ࡞ࠍ➅ࠅ
ߔߎߣ߇น⢻ߣߥࠅ㧘
LDR
↢ᚑ‛ࠍჇߐߖࠆߎ ߣ߇ߢ߈ࠆ㧚LDR⚳ੌᓟ㧘
LDR
↢ᚑ‛ߩήࠍ⏕ߔࠆߚߦ㧘
LDR
↢ᚑ‛ࠍᧂᔕߩㅢࡊࠗࡑ߆ࠄಽ 㔌ߒⰯశᬌߔࠆᔅⷐ߇ࠆ㧚ᧄ⎇ⓥߢߪ㧘LDR⚳ੌᓟߩᔕṁᶧਛߩ
LDR
↢ᚑ‛ࠍᧂᔕߩㅢ ࡊࠗࡑ߆ࠄCGE
ߦࠃࠅಽ㔌ߒ㧘ࠝࡦࠗࡦߢⰯశ (FL) ᬌߒߚ㧚
Fig. 1. Conceptual schematic of the PCR/LDR assay.
⹜⮎߅ࠃ߮ታ㛎ᣇᴺ ⹜⮎
ᧄ⎇ⓥߦ↪ߚ⹜⮎ߪ㧘ోߡᏒ⽼ߩ․⚖ຠࠍ↪
ߚ㧚ࠞ࡞ࡏࠠࠪࡔ࠴࡞࡞ࡠࠬ
(CMC)
㧘ࠣࠪࡦߪ㧘శ⚐⮎Ꮏᬺࠃࠅ⾼ߒߚ㧚࠻ࠬ
(
ࡅ࠼ࡠࠠࠪࡔ࠴࡞
)
ࠕࡒࡁࡔ࠲ࡦ(Tris)
㧘ࠛ࠴ࡦࠫࠕࡒࡦ྾㈶㉄
(EDTA)
ߪ㧘࠽ࠞࠗ࠹ࠬࠢࠃࠅ⾼ߒߚ㧚10x Tris-
ࡎ ࠙ ㉄-EDTA
✭ ⴣ ṁ ᶧ ߪ 㧘Bio-Rad Laboratories
ࠃࠅ⾼ߒߚ㧚Taq DNAࡐࡔ㧘dNTP mix
߅ࠃ߮ᱜᏱဳ(wild-type)
ࡅ࠻ࠥࡁࡓDNA (G12G; GGT)
ߪ㧘Promega
ࠃࠅ⾼ߒߚ㧚TaqDNA
ࠟߪ㧘New England BioLabs
ࠃࠅ⾼ߒ ߚ㧚SYBR Gold
ߪ㧘Invitrogen
ࠃࠅ㧘ࡐࠛ࠴ࡦࠝࠠࠪ࠼
(PEO; M
r8,000,000)
߅ࠃ߮ࡐࡆ࠾࡞ࡇ ࡠ࠼ࡦ(PVP; M
r130,000)
ߪ㧘Sigma-Aldrich
ࠃࠅ⾼ߒߚ㧚
PCR
߅ࠃ߮LDR
ߦ↪ߚࠝࠧDNA (Table 1)
ߩวᚑߪ㧘Invitrogen
߹ߚߪSigma Genosys
ߦଐ㗬ߒߚ㧚 ᔕ ṁ ᶧ ߩ ⺞ ߦ ߪ 㧘
nuclease-free water (Promega)
ࠍ↪ߚ㧚CE
ಽᨆ↪ߩṁᶧߩ⺞ߦߪ㧘⚐᳓ㅧⵝ⟎
(MILLIPORE, Elix 3 UV)
ߢ♖ߐ ࠇߚRO
᳓ࠍ↪ߚ㧚K-ras
ㆮવሶߩࠛࠠ࠰ࡦ1
㧘ࠦ࠼ࡦ
12.2
ߦᄌ⇣(G12V; GTT)
ࠍᜬߟࡅ࠻ᄢ⣺≸↱᧪⚦⢩ᩣ
(SW480)
ߪ㧘Gene Tex
ࠃࠅ⾼ߒ㧘Wizard SV Genomic DNA Purification System (Promega)
ࠍ↪ߡ♖ߒߚ㧚ታ㛎ᣇᴺ 2%4
200
μLߩPCR
࠴ࡘࡉߦ㧘1.5 mM
ߩMgCl
2ࠍ
1x PCR
ࡃ࠶ࡈࠔࠍട߃㧘ߘߩᓟ200 μM dNTPs
㧘500 nM K-ras forward
߅ࠃ߮reverse
ࡊࠗࡑ(Table 1)
㧘4.2 ng/ȝL
࠹ࡦࡊ࠻DNA
ࠍ㗅ߦട߃ ߚᓟ㧘Master cycler (Eppendorf)
ߦPCR
࠴ࡘࡉࠍ࠶࠻ߒߚ㧚
95
͠ߢ2 min
ߞߚᓟ㧘2.5 U DNA
ࡐࡔࠍട߃ߚ
(
ᔕᶧో㊂50 μL)
㧚ᒁ߈⛯߈㧘95
͠ߢ30 s
㧘60
͠ߢ1 min
㧘72
͠ߢ1 min 30 s
㑆ᜬߒ㧘ߎߩࠨࠗࠢ࡞ࠍ
40
࿁ⴕߥߞߚ㧚ࠨࠗࠢ࡞⚳ੌᓟ㧘ቢోߥ
2
ᧄ㎮DNA
ࠍᓧࠆߚߦ72
͠ߢ3 min Table 1. Sequences of oligonucleotide used in the PCR/LDR assays
Primer Sequences (5’ ĺ 3’) Size (mer)
K-ras forward TTAAAAGGTACTGGTGGAGTATTTGATA 28
K-ras reverse AAAATGGTCAGAGAAACCTTTATCTGT 27
K-ras Com-2 pTGGCGTAGGCAAGAGTGCCT-Fluorescein 20
K-rasc12.2 WtG AAACTTGTGGTAGTTGGAGCTGG 23
K-ras c12.2V AAACTTGTGGTAGTTGGAGCTGT 23
K-ras c12.2D AAACTTGTGGTAGTTGGAGCTGA 23
K-ras c12.2A AAACTTGTGGTAGTTGGAGCTGC 23
Fig. 2. Size confirmation for the PCR products derived from (a) wild-type and (b) mutant (SW480).
500 bp PCR product was used as a size standard.
Conditions: gel matrix, 0.5%(w/v) PEO in 1xTBE buffer containing 1x SYBR Gold; applied voltage, 12 kV.
ᔕߐߖߚ㧚ᓧࠄࠇߚ
PCR
ࡊࡠ࠳ࠢ࠻ߩࠨࠗ࠭㧔290 bp㧕ߪ㧘PEO ࠍಽ㔌ࡑ࠻࠶ࠢࠬߦ↪ߚ
CGE
ߦࠃࠅ⏕ߒߚ (Fig. 2)㧚.&4
200
μl ߩࡑࠗࠢࡠ࠴ࡘࡉߦ㧘20 mM Tris-HCl(pH 8.3)㧘25 mM
㈶㉄ࠞ࠙ࡓ㧘10 mM ㈶㉄ࡑࠣࡀࠪ࠙ࡓ㧘10 mM DTT㧘1 mM NAD+߅ࠃ߮
0.1 % Triton X-100
ࠍLDR Reaction buffer㧘30 nM
⼂ࡊࠗࡑ㧘
30 nM
ࡈ࡞ࠝࡠࠗࡦᮡ⼂ㅢࡊࠗࡑ (Table 1)㧘PCRࡊࡠ࠳ࠢ࠻߅ࠃ߮
2 U Taq DNA Ligase
ࠍᷙวߒߚ (ో㊂50 μL)㧚Master cycler
ࠍ↪ߡᔕṁᶧࠍ94͠ߢ 2 min
ടᾲߒߚᓟ㧘94͠
ߢ
30 s㧘ߘߩᓟ 65͠ߢ 2 min
ᜬߔࠆ᷷ᐲࠨࠗࠢ࡞ࠍ
40
࿁➅ࠅߒߚ㧚ᧄ⎇ⓥߦ߅ߌࠆ
LDR
ߢߪ㧘4
⒳ߩ⇣ߥࠆႮၮࠍ3ƍ
ᧃ┵ߦᜬߟ⼂ࡊࠗࡑࠍߘࠇߙࠇട߃ߚ4
ߟ ߩᔕṁᶧࠍ⺞ߒߚ㧚ߘߩਛߢ㧘࠹ࡦࡊ࠻DNA (G12V) ߩᄌ⇣ߒߚႮၮߦኻߒ⋧⊛ߥႮၮ
ࠍ3ƍᧃ┵ߦᜬߟ⼂ࡊࠗࡑ (K-ras c12.2V in
Table 1)
ࠍട߃ߚᔕṁᶧߦ߅ߡߩߺ㧘Ⱟశࡌ࡞ൻߐࠇߚ
43-mer
ߩLDR
↢ᚑ‛߇ᓧࠄࠇ㧘ὐ⓭ὼ ᄌ⇣߇ᬌߐࠇࠆ㧚ಽ㔌ࡑ࠻࠶ࠢࠬ
ᧄ⎇ⓥߢߪ㧘1x TBE✭ⴣᶧ (89 mM Tris-ࡎ࠙㉄
-2 mM EDTA (pH 8.3)) ߢ⺞ߒߚ 0.5 % (w/v)
ߩPEO
ࡐࡑṁᶧ߅ࠃ߮25 mM TGE
✭ⴣᶧ (25mM Tris-ࠣࠪࡦ-5 mM EDTA (pH 8.3)) ߢ⺞ߒ
ߚ0.3 % (w/v) CMC
ࡐࡑṁᶧࠍಽ㔌ࡑ࠻࠶ࠢࠬߣߒߡ↪
CGE
ಽᨆࠍⴕߞߚ㧚ࡐࡑṁᶧ⺞ᤨߦߪ㧘ࠄ߆ߓࠬ࠲ߢ✭ⴣṁᶧࠍ
900 rpm
ߢᠣᜈߒߡࠆ⁁ᘒߢ㧘⒊㊂ߒߚࡐࡑࠍߘ ߩਛᔃ߳ṛࠅߥߊട߃ࠆߎߣߢ㧘ߢ߈ࠆ㒢ࠅဋ৻ߦ ṁ⸃ߐߖߚ㧚߹ߚ㧘60 minᠣᜈߒߚᓟ㧘15 min 㖸ᵄᮏߦࠇࡐࡑࠍቢోߦṁ⸃ߒߚ㧚%'(. ಽᨆ
ᧄ⎇ⓥߦ↪ߚ
CE
ࠪࠬ࠹ࡓߪ㧘ࡈࡘ࠭࠼ࠪࠞࠠࡖࡇ࠴ࡘࡉ (GL Sciences)㧘㜞㔚Ḯ
(Model HZCE-30PNO.25㧘Matsuda Precision Devices
Co.)㧘FL
ᬌེ (RF-550㧘Shimadzu)㧘ࠗࡦ࠹ࠣ࠲ (CR-6A㧘Shimadzu)ߢ᭴ᚑߐࠇߡࠆ㧚ࠠ
ࡖ ࡇ ࠴ ࡘ ࡉ ᄖ ო ߩ ࡐ ࠗ ࡒ ࠼ ⵍ ⷒ ࠍ
Window Maker
TM(Micro Solv) ࠍ↪ߡ㐳ߐ 2 mm
㒰߈ᬌ⓹ߣߒ㧘ബశ߇ᬌ⓹ࠍᾖߔࠆࠃ߁ ߦࠠࡖࡇ࠴ࡘࡉࠍ⸳⟎ߒߚ㧚ࠠࡖࡇ
࠴ࡘࡉߪ㧘೨ಣℂߣߒߡ
1 M NaOH
᳓ṁᶧߢ10 min㧘0.2 M NaOH
᳓ṁᶧߢ5 min㧘ߐࠄߦ RO
᳓ߢ2 min
ᵞᵺߒߚ㧚߹ߚ㧘㔚᳇ᶐㅘᵹࠍዊߐߊߒߚ႐วߦߪ㧘↪೨ߦ
2.0 % (w/v) PVP
ࡐࡑṁᶧ ࠍࠠࡖࡇౝߦㅢߒߚ㧚೨ㅀߒߚPEO
ࠆߪ
CMC
ࡐࡑṁᶧࠍలႯߒ㧘㜞㔚Ḯࠍ↪ߡࠠࡖࡇ࠴ࡘࡉ (ౝᓘ
75
μm㧘ᄖᓘ150
μm,70 cm (ታല㐳 50 cm))
ߩਔ┵ߦ㜞㔚ࠍශടߒ㧘ⵍ ᬌࠍᵒേߐߖFL
ᬌེࠍ↪ߡᬌߒߚ㧚߹ߚ㧘FL
ᬌེߩബ߅ࠃ߮ᬌᵄ㐳ࠍ㧘ⵍᬌߩᦨᄢ ബᵄ㐳߅ࠃ߮ᦨᄢⰯశᵄ㐳ߦวࠊߖߡታ㛎ࠍⴕ ߞߚ㧚⚿ᨐ߅ࠃ߮⠨ኤ
%/% ࡐࡑṁᶧࠍ↪ߚ %'(. ಽᨆ
ᧄ⎇ⓥߦ↪ߚࡅ࠻ᄢ⣺≸⚦⢩↱᧪ᩣSW480 (G12V)
ߪK-ras
ㆮવሶߩࠛࠠ࠰ࡦ1㧘ࠦ࠼ࡦ 12.2
ߦ߅ߡ㧘Ⴎၮ߇C
߆ࠄA
ߦᄌ⇣ߒߡࠆ6)㧚ࠃ ߞ ߡ4
⒳ ߩ ⼂ ࡊ ࠗ ࡑ (K-ras c12.2WtG, c12.2V, c12.2D, c12.2A) (Table 1) ߩ ਛ ߢ K-ras c12.2V
ࠍ↪ߡLDR
ࠍⴕߞߚ႐วߦߩߺLDR
↢ ᚑ‛߇ᓧࠄࠇࠆߎߣ߇੍ᗐߐࠇࠆ㧚LDR
ᓟߦᓧࠄࠇߚ4
ߟߩLDR
ᔕṁᶧࠍFL
ᬌߦࠃࠆ
CMC
ࡐࡑᵒേᶧࠍ↪ߚCGE
ߦࠃ ߞߡߘࠇߙࠇಽᨆߒߚ㧚ᔕᓟߩṁᶧߦLDR
↢ᚑ‛߇↢ᚑߒߡࠇ߫㧘ߘߩ↢ᚑ‛ߩࡇࠢ߅ࠃ߮ᱷ
ᷲߩⰯశᮡ⼂ㅢࡊࠗࡑߩࡇࠢ߇ᓧࠄࠇࠆ ߣ⠨߃ࠄࠇࠆ㧚ߒ߆ߒ㧘Fig. 3(a) ߦ␜ߔࠃ߁ߦ
4
ߟߩᔕᶧߣ߽ߦࡈ࡞ࠝࠗࡦᮡ⼂ㅢࡊࠗࡑߦࠃࠆ㕖ᏱߦዊߐߥⰯశࠪࠣ࠽࡞ߩߺ߇ᬌ
ߐࠇ㧘⋡⊛ߩ
LDR
↢ᚑ‛ߩࡇࠢࠍᬌߔࠆߎߣ ߪߢ߈ߥ߆ߞߚ㧚ߎࠇߪ㧘ᧄಽᨆࠪࠬ࠹ࡓ߇㧘ᓸ㊂ߩ
LDR
ᔕ↢ᚑ‛ࠍᬌߔࠆߩߦචಽߥᬌᗵᐲ ߦ⥋ߞߡߥߚߢࠆߣ⠨߃ࠄࠇࠆ㧚FL
ᬌߦࠃࠆࡈ࡞ࠝࠗࡦᮡ⼂ㅢࡊࠗࡑߩᬌ
㒢⇇
(2 x 10
-7M)
߆ࠄ㧘LDR
ߩᔕ₸ߪ30 %
એ ਅߢࠆߎߣ߇ផ᷹ߐࠇࠆ㧚ᰴߦ㧘
LDR
ᓟߩᔕṁᶧࠍỚ❗ߒߡಽᨆࠍⴕߞ ߚ㧚Ớ❗ߩ㓙ߦߪ4
࿁ಽߩLDR
ᔕṁᶧ(
ฦ50
μL) ࠍ1
ߟߩ࠴ࡘࡉߦട߃(
ో㊂200
μL)㧘ߎࠇ ࠍᷫਅߢੇ῎ߐߖౣ߮ṁᇦࠍ10 μl
ട߃ࠆߎߣߢ20
ߦỚ❗ߒߚ㧚ห᭽ߦ㧘CMC
ࡐࡑࠍಽ㔌ࡑ࠻࠶ࠢࠬߦ↪ߚ
CGE-FL
ᬌߦࠃߞߡߎߩỚ❗ߒߚᔕṁᶧࠍಽᨆߒߚ㧚ߎߩߣ߈㧘
Fig. 3 (b)
ߦ␜ߔࠃ߁ߦ㧘Ớ❗೨ߣห᭽ߦࡈ࡞ࠝࠗࡦᮡ⼂
ㅢࡊࠗࡑߦၮߠߊࡇࠢߩߺ߇ᓧࠄࠇ㧘
LDR
↢ᚑ‛ࠍᬌߔࠆ߹ߢߦᬌᗵᐲࠍᡷༀߔࠆߎߣ ߪߢ߈ߥ߆ߞߚ㧚ࠩశḮࠍ↪ߚ
LDR
↢ᚑ‛ߩᬌ߇ႎ๔ߐࠇߡࠆ߇㧘ࠕࠢశḮࠍ↪
ߚ႐วߪ㧘ㅢࡊࠗࡑߦ࠲ࠣߐࠇߚⰯశߦၮ ߠߊᬌߪᗵᐲߩὐߢ࿎㔍ߢࠆߣ⠨߃ࠄࠇࠆ㧚
2'1 ࡐࡑṁᶧࠍ↪ߚ %'(. ಽᨆ
ࠗࡦ࠲ࠞ࠲ߪ
2
ᧄ㎮DNA
ߩࠄߖࠎ᭴ㅧ ߦࠗࡦ࠲ࠞ࠻ߒ㧘DNA
ߩ⋧⊛Ⴎၮኻߩਛ ߦࠅㄟߎߣߢᒝⰯశࠍ␜ߔ‛⾰ߢࠆ㧚ࠗࡦ࠲ࠞ࠲ߩ৻⒳ߢࠆ
SYBR Gold
ߪ㧘2
ᧄ㎮ߛߌߢߥߊ
1
ᧄ㎮ߩDNA
ߣ߽⚿วߔࠆߎߣ߇⍮ࠄࠇߡ߅ࠅ㧘ㄭᐕ㧘
SYBR Gold
߅ࠃ߮ዩ⚛ࠍᵒേᶧߦട߃ߚ
CGE
ߦ߅ߡ㧘1
ᧄ㎮DNA
ߩቯ㊂ࠍⴕߞߚ߇ႎ๔ߐࠇߡࠆ9)㧚
ㅢࡊࠗࡑߩ
3ƍ
ᧃ┵ߦᮡ⼂ߐࠇߚⰯశߦၮ ߠߊᬌ߇࿎㔍ߢߞߚ೨ㅀߩ⚿ᨐࠍฃߌ㧘SYBR Gold
ࠍᵒേᶧߦട߃ߚLDR
↢ᚑ‛ߩᬌߦߟߡ ᬌ⸛ߒߚ㧚SYBR Gold
ࠍᧄࠪࠬ࠹ࡓߦㆡ↪ߔࠆ႐ว㧘
SYBR Gold
ߦࠃߞߡᨴ⦡ߐߖࠆߎߣ߇น⢻ߥLDR
↢ᚑ‛ߣᔕᶧߦ߹ࠇࠆߘߩઁߩDNA
ࡈࠣࡔࡦ࠻
(
࠹ࡦࡊ࠻DNA
㧘⼂ࡊࠗࡑ㧘ㅢࡊࠗࡑ
)
ࠍಽ㔌ߔࠆᔅⷐ߇ࠆ㧚ߎߩಽ㔌 ࠍ㆐ᚑߢ߈ࠇ߫㧘LDR
↢ᚑ‛ߩㆬᛯ⊛ᬌ߇น⢻ߣߥࠅ㧘ߘߩᄌ⇣ߩ⒳㘃ࠍ․ቯߢ߈ࠆ㧚
LDR
ᓟߩᔕṁᶧࠍ20
Ớ❗ߒ㧘SYBR Gold
ࠍ ṁ⸃ߐߖߚPEO
ࡐࡑᵒേᶧࠍࠠࡖࡇߦ లႯߒCGE
ಽᨆࠍⴕߞߚ㧚ᔕṁᶧߦK-ras c12.2 WtG
㧘c12.2V
㧘c12.2D
߅ࠃ߮c12.2A
ࡊࠗࡑࠍߘࠇߙࠇട߃ߡ
LDR
ᔕࠍⴕ㧘ߎࠇࠄࠍCGE-FL
ࠪࠬ࠹ࡓߢಽᨆߒߚ⚿ᨐࠍFig. 4 (a)
̆(d)
ߦ␜ߔ㧚Fig. 4 (a)
㧘(c)
߅ࠃ߮(d)
ߦ߅ߡ㧘ห᭽ߩ
3
ᧄߩࡇࠢ߇ࠄࠇߚ㧚ߎࠇࠄߪ⼂ࡊࠗࡑ㧘ㅢࡊࠗࡑ߅ࠃ߮࠹ࡦࡊ࠻
DNA
ߦࠃ ࠆࡇࠢߢࠆߣ⠨߃ࠄࠇࠆ㧚ߎߩಽ㔌♽ߦ߅ߡ ߪ㧘ࠨࠗ࠭߇ዊߐߥ߽ߩ߶ߤṁᤨ㑆߇ᣧߚ㧘⚂
11 min
ઃㄭߦࠄࠇࠆ㧞ᧄߩࡇࠢ߇ߘࠇߙࠇ⼂ࡊࠗࡑ߅ࠃ߮ㅢࡊࠗࡑߦၮߠ߈㧘⚂
13 min
ઃㄭߦࠄࠇࠆࡇࠢ߇࠹ࡦࡊ࠻ߦࠃࠆ ߽ ߩ ߢ ࠆ ߣ ⠨ ߃ ࠄ ࠇ ࠆ 㧚 ㅢ ࡊ ࠗ ࡑ
(20-mer)
ࠃࠅ߆ߦࠨࠗ࠭ߩᄢ߈⼂ࡊࠗࡑ
(23-mer)
߇వߦᬌߐࠇߚߩߪ㧘ㅢࡊࠗࡑFig. 3. Electropherograms of the LDR products without (a) and with 20-fold
preconcentration (b).
Conditions: gel matrix, 0.3%(w/v) CMC in 25 mM TGE buffer; applied voltage, 12 kV.
ߦ࠲ࠣߐࠇߡࠆⰯశ⦡⚛ࡈ࡞ࠝࠗࡦߩᓇ 㗀ߢࠆߣ⠨߃ࠄࠇࠆ㧚Fig. 4(b) ߦ߅ߡߪ㧘Fig.
4(a), (c), (d)ߢߪⷰࠄࠇߥ․⇣⊛ߥࡇࠢ߇ᬌ
ߐࠇ㧘ߘߩࠨࠗ࠭ࠃࠅ್ᢿߔࠆߣ㧘LDR ↢ᚑ‛ߩ ࡇࠢߢࠆߣ⠨߃ࠄࠇࠆ㧚ߎߩߣ߈↪ߚ⼂ࡊ
ࠗࡑߪ
K-ras c12.2V
ࡊࠗࡑߢࠆߎߣ߆ ࠄ㧘G ψ TߩDNA
ὐ⓭ὼᄌ⇣ࠍᬌߔࠆߎߣ߇ ߢ߈ߚ㧚ߎࠇࠄߩߎߣࠃࠅ㧘ࠗࡦ࠲ࠞ࠲ߣ ߒߡSYBR Gold
ࠍ↪ߚCGE
ߦࠃߞߡLDR
↢ᚑ‛ࠍಽᨆߔࠆߎߣߢ
DNA
ὐ⓭ὼᄌ⇣ߩ․ቯ߇น⢻ߢࠆߣ⠨߃ࠄࠇࠆ㧚
⚿⺰
ᧄ⎇ⓥߢߪ㧘PCR/LDRࠕ࠶ࠗߦ߅ߌࠆᦨ⚳ࡊ ࡠ࠳ࠢ࠻ߢࠆLDR
↢ᚑ‛ࠍCGE
ߦࠃࠅಽ㔌ߒ㧘ࠩశḮࠃࠅଔߥࠕࠢశḮࠍ↪Ⱟశᬌ
ߔࠆߎߣࠍ⹜ߺߚ㧚ᧄታ㛎♽ߦ߅ߌࠆ
LDR
ߩᔕ₸ߪ
30 %એਅߢࠅ㧘ᓸ㊂ߩ LDR
↢ᚑ‛ࠍᬌߔࠆߦߪ㧘ㅢࡊࠗࡑߦ࠲ࠣߐࠇߚ⦡⚛ߩⰯ
శߦၮߠߊᬌᴺߢߪᬌᗵᐲ߇ਇචಽߢߞߚ㧚
ࠗࡦ࠲ࠞ࠲ߢࠆ
SYBR Gold
ࠍಽ㔌ࡐࡑṁᶧߦᷝടߒߚ
LDR
↢ᚑ‛ߩCGE
ಽᨆᴺߦ ߟߡᬌ⸛ߒ㧘⚦⢩♽SW480 (G12V)ߩ K-ras
ㆮવ ሶ㧘ࠛࠠ࠰ࡦ1㧘ࠦ࠼ࡦ 12.2
ߦ߅ߡႮၮ߇C
߆ ࠄA
߳ᄌ⇣ߒߡࠆߎߣ߇⏕ߢ߈㧘PCR/LDRࠕ࠶ࠗߦ
SYBR Gold
ࠍಽ㔌ࡑ࠻࠶ࠢࠬߦᷝടߒߚ
CGE-FL
ᬌᴺ߇DNA
ὐ⓭ὼᄌ⇣ߩ․ቯߦ↪ߢࠆߎߣࠍߒߚ㧚
ᧄ⎇ⓥߩ৻ㇱߪ㧘ޟℂᎿቇ⎇ⓥᚲ 2008 ᐕᐲหᔒ
␠ᄢቇℂᎿቇ⎇ⓥᚲ⎇ⓥഥᚑ㊄㧔ੱ㧕ޠߩᡰេࠍ ฃߌߚ㧚ߎߎߦ⸥ߒߡ㧘⻢ᗧࠍߔࠆ㧚
ෳ⠨ᢥ₂
1) M. Hashimoto, F. Barany and S. A. Soper, “Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations”, Biosens. Bioelectron., 21, 1915-1923 (2006).
2) N. P. Gerry, N. E. Witowski, J. Day, R. P. Hammer, G.
Barany and F. Barany, “Universal DNA microarray method for multiplex detection of low abundance point mutations”, J. Mol. Bio., 292, 251-262 (1999).
3) M. Khanna, P. Park, M. Zirvi, W. Cao, A. Picon, J. Day, P. Paty and F. Barany, “Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors”, Oncogene, 18, 27-38 (1999).
4) K. Li, B. Chen, Y. Zhou, R. Huang, Y. Liang, Q. Wang, Z. Xiao and J. Xiao, “Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction-ligase detection reaction (PCR-LDR) ”, J Microbiol Methods., 76, 289-294 (2009).
5) D. K. Toubanaki, T. K. Christopoulos, P. C. Ioannou and C. S. Flordellis, “Identification of single-nucleotide polymorphisms by the oligonucleotide ligation reaction:
Fig. 4. Electropherograms of the LDR products obtained using the different discriminating primers of (a) K-ras c12.2WtG, (b) K-ras c12.2V, (c) K-ras c12.2D and (d) K-ras c12.2A.
Conditions: gel matrix, 0.5%(w/v) PEO in 1xTBE buffer containing 1x SYBR Gold; applied voltage, 12 kV.
a DNA biosensor for simultaneous visual detection of both alleles”, Anal. Chem., 81, 218-224 (2009).
6) M. Hashimoto, M. L. Hupert, M. C. Murphy and S. A.
Soper, “Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations”, Anal.
Chem., 77, 3243-3255 (2005).
7) R. Sinville, J. Coyne, R. J. Meagher, Y.-W. Cheng, F.
Barany, A. Barron and S. A. Soper, “Ligase detection reaction for the analysis of point mutations using free-solution conjugate electrophoresis in a polymer microfluidic device”, Electrophoresis, 29, 4751-4760 (2008).
8) P. Yi, Z. Chen, Y. Zhao, J. Guo, H. Hu, Y. Zhou L. Yu and L. Li, “PCR/LDR/capillary electrophoresis for detection of single-nucleotide differences between fetal and maternal DNA in maternal plasma”, Prenat. Diagn., 29, 217-22 (2009).
9) R. Oba, Y. Kudo, N. Sato, R. Noda and Y. Otsuka, “A new method of competitive reverse transcription polymerase chain reaction with SYBR Gold staining for quantitative analysis of mRNA”, Electrophoresis, 27, 2865-2868 (2008).
