| Product Name |
Thermus aquaticus MutS DNA mismatch repair protein, N-6X His-MBP-Taq-Muts |
| Size |
100 µg |
| Description |
The Taq MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. This Muts protein binds in vitro to heteroduplex DNAs containing mispaired or unpaired bases over a wide temperature range from 4 to 70 °C and has a thermostable ATPase activity. This thermostable Taq MutS is active at temperature between 0 to 75°C. Since Taq MutS efficiently binds to 1-4 bases deletion (or insertion) and mismatch base pairs of GT, CT and AG, it is useful for detecting these mutations. Mutations can be detected in polyacrylamide gels or on a solid phase such as Ni agarose or amylose beads, or magnetic Ni-NTA particles.
|
| Applications |
- Remove mismatch DNA (error correction) from gene synthesis reaction
- Mutation detection and removal
- Rapid isothermal SNP detection
|
| Source |
E. coli |
| Fusion Tag |
6XHis tag and MBP tag at N-terminus |
| Purification Method |
FPLC |
| Concentration |
1 mg/ml |
| Purity |
~ 95% as determined by SDS-PAGE
|
| Accession # |
AAC43637, TAU3311, U33117 |
| Gene Name |
Thermus aquaticus MutS DNA mismatch repair protein |
| MW |
135.9 kDa |
| Protein Sequence |
1 MGSSHHHHHH GTKTEEGKLV IWINGDKGYN GLAEVGKKFE KDTGIKVTVE HPDKLEEKFP
61 QVAATGDGPD IIFWAHDRFG GYAQSGLLAE ITPDKAFQDK LYPFTWDAVR YNGKLIAYPI
121 AVEALSLIYN KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW PLIAADGGYA
181 FKYENGKYDI KDVGVDNAGA KAGLTFLVDL IKNKHMNADT DYSIAEAAFN KGETAMTING
241 PWAWSNIDTS KVNYGVTVLP TFKGQPSKPF VGVLSAGINA ASPNKELAKE FLENYLLTDE
301 GLEAVNKDKP LGAVALKSYE EELAKDPRIA ATMENAQKGE IMPNIPQMSA FWYAVRTAVI
361 NAASGRQTVD EALKDAQTGT DYDIPTTENL YFQGHMEGML KGEGPGPLPP LLQQYVELRD
421 QYPDYLLLFQ VGDFYECFGE DAERLARALG LVLTHKTSKD FTTPMAGIPL RAFEAYAERL
481 LKMGFRLAVA DQVEPAEEAE GLVRREVTQL LTPGTLLQES LLPREANYLA AIATGDGWGL
541 AFLDVSTGEF KGTVLKSKSA LYDELFRHRP AEVLLAPELL ENGAFLDEFR KRFPVMLSEA
601 PFEPEGEGPL ALRRARGALL AYAQRTQGGA LSLQPFRFYD PGAFMRLPEA TLRALEVFEP
661 LRGQDTLFSV LDETRTAPGR RLLQSWLRHP LLDRGPLEAR LDRVEGFVRE GALREGVRRL
721 LYRLADLERL ATRLELGRAS PKDLGALRRS LQILPELRAL LGEEVGLPDL SPLKEELEAA
781 LVEDPPLKVS EGGLIREGYD PDLDALRAAH REGVAYFLEL EERERERTGI PTLKVGYNAV
841 FGYYLEVTRP YYERVPKEYR PVQTLKDRQR YTLPEMKEKE REVYRLEALI RRREEEVFLE
901 VRERAKRQAE ALREAARILA ELDVYAALAE VAVRYGYVRP RFGDRLQIRA GRHPVVERRT
961 EFVPNDLEMA HELVLITGPN MAGKSTFLRQ TALIALLAQV GSFVPAEEAH LPLFDGIYTR
1021 IGASDDLAGG KSTFMVEMEE VALILKEATE NSLVLLDEVG RGTSSLDGVA IATAVAEALH
1081 ERRAYTLFAT HYFELTALGL PRLKNLHVAA REEAGGLVFY HQVLPGPASK SYGVEVAAMA
1141 GLPKEVVARA RALLQAMAAR REGALDAVLE RLLALDPDRL TPLEALRLLQ ELKALALGAP
1201 LDTMKG |
| Storage Buffer |
20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 0.1mM EDTA, 1 mM DTT, 50% Glycerol |
| Reaction Buffer |
100mM KCl, 50mM Tris-HCl, pH 8.5, 5~20mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 2% Glycerol, 65 °C |
| Storage |
-20 to -80 °C. |
| Shipping |
4°C or dry ice |
|
Protocol
(example)
|
1. After first round PCR, purify PCR fragments using Qiagen QIAquick PCR purification kit with elution in dH2O.
2. Dilute PCR product to 250 ng/µl in 10 mM Tris–HCl, pH 7.8, 50 mM NaCl and heat to 95 oC for 5 min followed by cooling at 0.1oC/s to 25 oC.
3. Add binding buffer (20 mM Tris–HCl, pH 7.8, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT and 5% glycerol) to annealed PCR product, adjust DNA concentration to 11.5 ng/µl, add 6XHis-MBP–MutS dimers to 950 nM.
4. Incubate the mixture at room temperature for 10 min, then add an equal volume of amylose resin (NEB) preequilibrated with 1X binding buffer, and incubate for 30 min at room temperature.
5. Gently spin down beads and save supernatant for subsequent processing (second round PCR, cloning etc).
|
| References |
1. Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. 2004 Nov 23;32(20):e162.
2. Correcting errors in synthetic DNA through consensus shuffling. Nucleic Acids Res. 2005 Mar 30;33(6):e55.
3. MutS as a tool for mutation detection. Acta Biochim Pol. 2005;52(3):575-83. Epub 2005 Aug 4.
4. One tube mutation detection using sensitive fluorescent dyeing of MutS protected DNA. Nucleic Acids Res. 2000 Apr 15;28(8):E36.
5. Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology. Nature Methods - 4, 257 - 262 (2007) doi:10.1038/nmeth1007
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