Volume 17 Number 24 1989
`
`Nucleic Acids Research
`
`DNA sequencing with thermostable Tet DNA polymerase from Thermus thermophilus
`
`T.A.Bechtereva, Y.I.Pavlovl, V.I.Kramorov3, B.Migunova2 and O.I.Kiselev
`
`All-Union Research Institute of Influenza, 'Department of Genetics of Leningrad University, 2Al1-Union Institute
`of Vaccines, Leningrad and 3Institute of Applied Molecular Biology, Moscow, USSR
`Submitted November 14, 1989
`
`One of the modern exciting iLmprovements in Sanger dideoxynucleotide DNA sequen-
`cing method is in the replacement of the Klenow fragent of E.Coii DNA polymerase
`by a highly thermostable one frma Ttermus aquaticus (Taq poly-nerase) (1). Here we
`corunicate that another thermtable enzynw fm Txhermus ther:ropilus
`strain KM.
`(Tet polytnerase)
`can be succesfully used in
`DNA sequencing instead of
`expensive
`Taq polymerase. Tet polymerase share rn with Taq polymerase pr;perties:absence
`of 3-exonuclease activity, very high processivity dnd wide temperature range. Tet
`in DtNA sequencing both for
`fragnent
`polywerase is superior to Klero
`simplicity
`( the reactions take only 3 minues at 70°C ) and clear resolution of
`sequence
`ladder artefacts comn for E.coli enzyme ( fiy.A.,arrow ).
`Such high performance
`dideoKynucleotides
`reactions are achieved at a relatively increased amount of
`in
`mixes as cmpared with other polymerases including Taq polymerase. Generaly, this
`and we reproducibly obtain more seguen-
`not diminishes the nuaber of readable b
`ce data from a single reaction than with Klenw fragment.
`Fig.A. Autoradiograph of a polyacrylamide/urea gel demostrating comparison of
`by-Tet polemerase ( lane 1 to 8 ) and Klenow fragmt ( lane 9 to 12 ).
`sequencin
`Single-stranded p19 tlate
`ntaing 1,1 kb g13II-KpnX fragjamnt of the
`LYS2
`cerevisiae,
`P-labelled (GIPCH, teningrad) M13"forwerd"
`gene of S
`sequening primer wre prepared by sta
`methods. DA sequencing reaction
`d
`by
`Tet polymarase were perfom
`in the Tet buffer containing: 20M Tris(pH 8),lOnM
`.%gC
`, 0.05% Tween 20 and tionidet P-40. Fr annealirg reaction 10 mdl mixture
`cnting 5 ng of labelled prkowr.l,mk of t-plate Dim in s
`ing bufter was
`hate to 95 C for 2 min and incubated at 42C for 10 min,ooled to room tem-
`paratuceThen lmid (12 units) of Tet polyrase
`sd to this mxture and reslting aix was
`iudiatSly dinsed in 2 aild liquots into
`the 4 t
`aining the 2 sac temination
`Mmis. The ccof each deOKynucle-
`ttide
`in then we 4 smkM and idscleo-
`160 mk ddTP, in 'Amix and
`tides in G"mix
`'Tniix
`500 c*
`ddATP a ddrm, "C'mix 350 aWf
`C lan 1,3,5,7 ). Decreas
`the c ettion
`of ddTP do not lead to significance chge
`the data picture ( lanes
`2,4,6,8 ), but in
`seguence
`the bottom of
`ladder we can
`see
`additional bans due to nonspecific termina-
`naticn (fig.B."T"mix 500 akM a 250 akH ddrrP,
`lane
`a& 2). Reaction mixtures were incubated
`at 72 C for 3 main and reaction stoWed by
`the addition of 3 m5
`of formagide dye solu-
`tion. Samples were heated at 95 C for 3 min
`ing 1-2 mkl onto a wedge
`before l
`St acry-
`*lide gel arnd ran at constant pFer 40 W.
`
`B
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`/ 2
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`A
`f 2 3 4
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`66
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`8 910ff fZ
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`--:
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`REFERENCES
`1. M.A.Innes, K.B.MymAbo, D.H.Celfanrxi and
`M.A.D.Drcw.
`(1988) Proc.Natl.1.cad..Sci.USA
`85, 9436-9440.
`
`©IRL Press
`
`1 0507
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