| Product Name |
Thermus aquaticus MutS DNA mismatch repair protein, C-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 |
MBP tag at N-terminus and 6XHis tag at C-terminus |
| Purification Method |
FPLC |
| 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 MGKIEEGKLV IWINGDKGYN GLAEVGKKFE KDTGIKVTVE HPDKLEEKFP QVAATGDGPD
61 IIFWAHDRFG GYAQSGLLAE ITPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN
121 KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW PLIAADGGYA FKYENGKYDI
181 KDVGVDNAGA KAGLTFLVDL IKNKHMNADT DYSIAEAAFN KGETAMTING PWAWSNIDTS
241 KVNYGVTVLP TFKGQPSKPF VGVLSAGINA ASPNKELAKE FLENYLLTDE GLEAVNKDKP
301 LGAVALKSYE EELVKDPRIA ATMENAQKGE IMPNIPQMSA FWYAVRTAVI NAASGRQTVD
361 EALKDAQTNS SSNNNNNNNN NNLGDDDDKL EVLFQGPHME GMLKGEGPGP LPPLLQQYVE
421 LRDQYPDYLL LFQVGDFYEC FGEDAERLAR ALGLVLTHKT SKDFTTPMAG IPLRAFEAYA
481 ERLLKMGFRL AVADQVEPAE EAEGLVRREV TQLLTPGTLL QESLLPREAN YLAAIATGDG
541 WGLAFLDVST GEFKGTVLKS KSALYDELFR HRPAEVLLAP ELLENGAFLD EFRKRFPVML
601 SEAPFEPEGE GPLALRRARG ALLAYAQRTQ GGALSLQPFR FYDPGAFMRL PEATLRALEV
661 FEPLRGQDTL FSVLDETRTA PGRRLLQSWL RHPLLDRGPL EARLDRVEGF VREGALREGV
721 RRLLYRLADL ERLATRLELG RASPKDLGAL RRSLQILPEL RALLGEEVGL PDLSPLKEEL
781 EAALVEDPPL KVSEGGLIRE GYDPDLDALR AAHREGVAYF LELEERERER TGIPTLKVGY
841 NAVFGYYLEV TRPYYERVPK EYRPVQTLKD RQRYTLPEMK EKEREVYRLE ALIRRREEEV
901 FLEVRERAKR QAEALREAAR ILAELDVYAA LAEVAVRYGY VRPRFGDRLQ IRAGRHPVVE
961 RRTEFVPNDL EMAHELVLIT GPNMAGKSTF LRQTALIALL AQVGSFVPAE EAHLPLFDGI
1021 YTRIGASDDL AGGKSTFMVE MEEVALILKE ATENSLVLLD EVGRGTSSLD GVAIATAVAE
1081 ALHERRAYTL FATHYFELTA LGLPRLKNLH VAAREEAGGL VFYHQVLPGP ASKSYGVEVA
1141 AMAGLPKEVV ARARALLQAM AARREGALDA VLERLLALDP DRLTPLEALR LLQELKALAL
1201 GAPLDTMKGK LAAALEHHHH HH |
| Storage Buffer |
20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 0.1mM EDTA, 1 mM DTT, 50% Glycerol |
| Reaction Buffer |
100 mM KCl, 50 mM Tris-HCl, pH 8.5, 5~20 mM 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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