Short Technical Reports
`
`SHORT TECHNICAL
`REPORTS
`Manuscripts published in the Short
`Technical Reports section are shorter
`and less comprehensive in scope than
`full Research Reports.
`
`Elimination of Residual
`Natural Nucleotides from
`3¢¢ -O-Modified-dNTP
`Syntheses by Enzymatic
`Mop-Up
`
`BioTechniques 25:814-817 (November 1998)
`
`ABSTRACT
`
`Here, we describe a novel strategy
`called enzymatic “Mop-Up” that efficiently
`removes contaminating dNTPs from re-
`verse-phase, high-performance liquid chro-
`matography (RP-HPLC) purified 3¢-O-
`modified dNTP
`syntheses. Enzymatic
`mop-up takes advantage of the high selec-
`tivity of DNA polymerases for the former
`nucleoside triphosphates over the latter nu-
`cleotide analogs. We demonstrate the selec-
`tive removal of contaminating dATP and
`
`814 BioTechniques
`
`dTTP from RP-HPLC purified 3¢-O-methyl-
`dATP and 3¢-O-(2-nitrobenzyl)-dTTP syn-
`theses, respectively. These data highlight
`the importance of natural nucleotide conta-
`mination when interpreting enzymatic in-
`corporation data and provide an alternative
`hypothesis for the observed property of cat-
`alytic editing of DNA polymerases. More-
`over, the effective removal of natural nu-
`from 3¢-O-modified analogs
`cleotides
`addresses the important issue of nucleotide
`read-through for stop-start DNA sequenc-
`ing strategies, such as the base addition se-
`quencing scheme (BASS).
`
`INTRODUCTION
`
`Characterization of the behavior
`of deoxyribonucleoside triphosphate
`(dNTP) analogs as terminators of DNA
`syntheses has greatly increased our un-
`derstanding of DNA polymerases. In
`general, DNA polymerases show re-
`stricted selectivity and specificity for
`dNTP analogs that have been particu-
`larly useful for the development of an-
`tiviral compounds. While these analogs
`potentially have broad use for molecu-
`lar biology applications, in practice
`however, large screening assays must
`be used to identify specific DNA poly-
`merases that show the desired activity.
`Moreover, the study of 3¢ -O-modified-
`dNTPs has been particularly challeng-
`ing because the starting material for the
`chemical syntheses of these com-
`pounds is the natural 2¢ -deoxyribonu-
`cleoside, which can be carried-over
`into the chemical phosphorylation step.
`The subsequent use of chromatography
`is typically inadequate for the complete
`removal of the contaminating dNTPs
`from 3¢ -O-modified-dNTP synthesis.
`Previously, we demonstrated the
`feasibility of a stepwise DNA sequenc-
`ing strategy, called the base addition se-
`quencing scheme (BASS), by the syn-
`thesis and enzymatic incorporation of 7
`different 3¢ -O-modified-dATPs and 3¢ -
`O-methyl-dTTP (3). However, a major
`obstacle in BASS has been the presence
`of contaminating natural dNTPs carried
`over in the chemical synthesis of 3¢ -O-
`modified-dNTPs (2,3). Although re-
`verse-phase, high-performance liquid
`chromatography (RP-HPLC) can puri-
`fy 3¢ -O-modified-dNTPs ‡ 99.5%, the
`level of contamination remains suffi-
`
`ciently high that the DNA polymerase
`selectively incorporates the preferred
`natural dNTP over the 3¢ -O-modified-
`dNTP analog. Incorporation of the nat-
`ural dNTP at the desired termination
`base, referred to here as natural-nu-
`cleotide read-through, can significantly
`result in decreased signal intensities
`and higher background signals in sub-
`sequent base addition steps, thus mak-
`ing the interpretation of enzymatic in-
`corporation data more difficult. To
`overcome this problem, we have devel-
`oped an enzymatic “Mop-Up” strategy
`that specifically removes dNTP conta-
`mination from RP-HPLC-purified 3¢ -
`O-modified-dNTPs.
`
`MATERIALS AND METHODS
`
`Reagents
`
`Nucleotides, Klenow fragment of
`DNA polymerase I and avian myelo-
`blastosis virus reverse transcriptase
`(AMV-RT) were purchased
`from
`Amersham Pharmacia Biotech (Piscat-
`away, NJ, USA). Bst DNA Polymerase
`was purchased from Bio-Rad (Her-
`cules, CA, USA). Streptavidin-coated
`magnetic Dynabeads(cid:210) M-280 were
`purchased from Dynal AS (Oslo, Nor-
`way). DNA synthesis reagents were
`purchased from PE Applied Biosys-
`tems (Foster City, CA, USA), except
`for the biotin phosphoramidite, which
`was purchased from Glen Research
`(Sterling, VA, USA). All oligonucleo-
`tides were synthesized trityl-on using a
`Model 394 DNA Synthesizer (PE Ap-
`plied Biosystems) and purified using
`Nensorb(cid:212)
`20 columns, according to
`the manufacturer’s protocol (NEN Life
`Science Products, Boston, MA, USA).
`
`Mop-Up Assay
`Biotinylated primer (500 pmol) (5¢ -
`CAGAGCCAAGGCTCTACCTAGCA-
`GTGTAAAACGACGGCCAGT-3¢ ) in
`100 m L of reaction buffer (10 mM Tris-
`HCl, pH 8.5, 10 mM MgCl2) was added
`to 100 m L of prewashed streptavidin-
`coated magnetic Dynabeads, incubated
`for 1 h in 10 mM Tris-HCl, pH 8.5, 10
`mM MgCl2, 1 M NaCl at ambient tem-
`perature and washed twice with reaction
`buffer by magnetic separation. Five
`
`Columbia Ex. 2002
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
`Vol. 25, No. 5 (1998)
`
`

`

`Short Technical Reports
`
`SHORT TECHNICAL
`REPORTS
`Manuscripts published in the Short
`Technical Reports section are shorter
`and less comprehensive in scope than
`full Research Reports.
`
`Elimination of Residual
`Natural Nucleotides from
`3¢¢ -O-Modified-dNTP
`Syntheses by Enzymatic
`Mop-Up
`
`BioTechniques 25:814-817 (November 1998)
`
`ABSTRACT
`
`Here, we describe a novel strategy
`called enzymatic “Mop-Up” that efficiently
`removes contaminating dNTPs from re-
`verse-phase, high-performance liquid chro-
`matography (RP-HPLC) purified 3¢-O-
`modified dNTP
`syntheses. Enzymatic
`mop-up takes advantage of the high selec-
`tivity of DNA polymerases for the former
`nucleoside triphosphates over the latter nu-
`cleotide analogs. We demonstrate the selec-
`tive removal of contaminating dATP and
`
`814 BioTechniques
`
`dTTP from RP-HPLC purified 3¢-O-methyl-
`dATP and 3¢-O-(2-nitrobenzyl)-dTTP syn-
`theses, respectively. These data highlight
`the importance of natural nucleotide conta-
`mination when interpreting enzymatic in-
`corporation data and provide an alternative
`hypothesis for the observed property of cat-
`alytic editing of DNA polymerases. More-
`over, the effective removal of natural nu-
`from 3¢-O-modified analogs
`cleotides
`addresses the important issue of nucleotide
`read-through for stop-start DNA sequenc-
`ing strategies, such as the base addition se-
`quencing scheme (BASS).
`
`INTRODUCTION
`
`Characterization of the behavior
`of deoxyribonucleoside triphosphate
`(dNTP) analogs as terminators of DNA
`syntheses has greatly increased our un-
`derstanding of DNA polymerases. In
`general, DNA polymerases show re-
`stricted selectivity and specificity for
`dNTP analogs that have been particu-
`larly useful for the development of an-
`tiviral compounds. While these analogs
`potentially have broad use for molecu-
`lar biology applications, in practice
`however, large screening assays must
`be used to identify specific DNA poly-
`merases that show the desired activity.
`Moreover, the study of 3¢ -O-modified-
`dNTPs has been particularly challeng-
`ing because the starting material for the
`chemical syntheses of these com-
`pounds is the natural 2¢ -deoxyribonu-
`cleoside, which can be carried-over
`into the chemical phosphorylation step.
`The subsequent use of chromatography
`is typically inadequate for the complete
`removal of the contaminating dNTPs
`from 3¢ -O-modified-dNTP synthesis.
`Previously, we demonstrated the
`feasibility of a stepwise DNA sequenc-
`ing strategy, called the base addition se-
`quencing scheme (BASS), by the syn-
`thesis and enzymatic incorporation of 7
`different 3¢ -O-modified-dATPs and 3¢ -
`O-methyl-dTTP (3). However, a major
`obstacle in BASS has been the presence
`of contaminating natural dNTPs carried
`over in the chemical synthesis of 3¢ -O-
`modified-dNTPs (2,3). Although re-
`verse-phase, high-performance liquid
`chromatography (RP-HPLC) can puri-
`fy 3¢ -O-modified-dNTPs ‡ 99.5%, the
`level of contamination remains suffi-
`
`ciently high that the DNA polymerase
`selectively incorporates the preferred
`natural dNTP over the 3¢ -O-modified-
`dNTP analog. Incorporation of the nat-
`ural dNTP at the desired termination
`base, referred to here as natural-nu-
`cleotide read-through, can significantly
`result in decreased signal intensities
`and higher background signals in sub-
`sequent base addition steps, thus mak-
`ing the interpretation of enzymatic in-
`corporation data more difficult. To
`overcome this problem, we have devel-
`oped an enzymatic “Mop-Up” strategy
`that specifically removes dNTP conta-
`mination from RP-HPLC-purified 3¢ -
`O-modified-dNTPs.
`
`MATERIALS AND METHODS
`
`Reagents
`
`Nucleotides, Klenow fragment of
`DNA polymerase I and avian myelo-
`blastosis virus reverse transcriptase
`(AMV-RT) were purchased
`from
`Amersham Pharmacia Biotech (Piscat-
`away, NJ, USA). Bst DNA Polymerase
`was purchased from Bio-Rad (Her-
`cules, CA, USA). Streptavidin-coated
`magnetic Dynabeads(cid:210) M-280 were
`purchased from Dynal AS (Oslo, Nor-
`way). DNA synthesis reagents were
`purchased from PE Applied Biosys-
`tems (Foster City, CA, USA), except
`for the biotin phosphoramidite, which
`was purchased from Glen Research
`(Sterling, VA, USA). All oligonucleo-
`tides were synthesized trityl-on using a
`Model 394 DNA Synthesizer (PE Ap-
`plied Biosystems) and purified using
`Nensorb(cid:212)
`20 columns, according to
`the manufacturer’s protocol (NEN Life
`Science Products, Boston, MA, USA).
`
`Mop-Up Assay
`Biotinylated primer (500 pmol) (5¢ -
`CAGAGCCAAGGCTCTACCTAGCA-
`GTGTAAAACGACGGCCAGT-3¢ ) in
`100 m L of reaction buffer (10 mM Tris-
`HCl, pH 8.5, 10 mM MgCl2) was added
`to 100 m L of prewashed streptavidin-
`coated magnetic Dynabeads, incubated
`for 1 h in 10 mM Tris-HCl, pH 8.5, 10
`mM MgCl2, 1 M NaCl at ambient tem-
`perature and washed twice with reaction
`buffer by magnetic separation. Five
`
`Vol. 25, No. 5 (1998)
`
`

`

`hundred picomoles of the mop-up tem-
`[5¢ -(T)26ACTGGCCGTCG-
`plate
`TTTTACA-3¢ ) in 100 m L of reaction
`buffer were added to the primer-cap-
`tured beads and annealed by heating to
`80(cid:176) C for 5 min. The reaction was slow-
`ly cooled to ambient temperature and
`washed twice with reaction buffer. In-
`corporation of the natural nucleotide
`was performed according to the enzy-
`matic conditions described for Bst DNA
`polymerase (4 U) or Klenow fragment
`(10 U) (3) at 65(cid:176) or 37(cid:176) C for 1 h, respec-
`tively. The mop-up solution containing
`
`the 3¢ -O-modified-dNTP was isolated
`by magnetic separation and directly
`used in the incorporation assay. For en-
`zymatic mop-up of dTTP analogs, the
`template was used: [5¢ -
`following
`(A)26CTGGCCGTCGTTTTACA-3¢ ].
`
`RESULTS AND DISCUSSION
`
`Here, the mop-up assay is described
`by which the contaminating natural nu-
`cleotide is enzymatically removed from
`a 3¢ -O-modified-dNTP solution by
`
`Common biotlnyfale<'I ptlmer
`
`'-O-modified-dNTP
`
`dATI'
`
`dA'fP
`
`dA'l"P
`
`dATP
`
`•
`l' Bind to trep1avidin-ooaced mugae~ beads
`~c ................ + A~~eal "Jl~ifk Jl01Y(N) le.rnpl;i,1e
`
`~C•-..,m ......... _...,_,,...._J'l'l"i'l"l''l'l'I'I'l"l'l,TlLT'l'l'T'l'r"l'l'TI
`+ Add DNA pofym.er·
`<§ @f
`~~
`@sdATI'
`
`SeJ~tive inoorporlil.io
`.fdNTPooly
`
`.dlATP
`
`OCH3
`
`'l"I'I'TI'Tl'
`
`Di card
`
`Mo Up purified
`'-0-modil'iei:l-d)','TP solutiqn
`
`+
`
`Figure 1. Enzymatic mop-up assay to remove contaminating dNTPs from a 3¢¢ -O-modified-dNTP
`solution. A common biotinylated primer is attached to streptavidin-coated magnetic beads, washed and
`annealed to a specific poly(dN) template. The mop-up polymerase, buffer and 3¢ -O-modified-dNTP are
`incubated to allow the mop-up enzyme selectively to remove the contaminating dNTP by DNA synthesis
`of the solid-phase bound primer-template complex. Once completed, the mop-up purified 3¢ -O-modified-
`dNTP is isolated from the primer-template complex by magnetic separation and directly used in the in-
`corporation assay.
`
`Vol. 25, No. 5 (1998)
`
`

`

`Short Technical Reports
`
`c -'"4---- Readtltrough
`o
`(presence of dA 'fP)
`G
`A
`G
`G
`T
`G
`
`Pseudo-termination
`(ab ence of dA TP)
`
`0 0.2S 0.S0 125 2.50
`µ M
`
`fL25
`1-1M
`dATP
`
`dATP after
`Mop-Up
`
`Figure 2. Capacity of the mop-up assay. Enzymatic mop-up was performed on a range of dATP con-
`centrations (0, 5, 10, 25 and 50 m M) using Bst DNA polymerase and assayed (final concentrations: 0,
`0.25, 0.50, 1.25 and 2.5 m M, respectively) using AMV-RT. Conditions for the AMV-RT incorporation as-
`say have been described previously (3) and are 32P-labeled universal primer-annealed to the oligo-tem-
`plate in AMV-RT buffer. All reactions contained dCTP, dTTP, ddGTP and the compound listed below
`each lane.
`
`s ~
`
`Readthrough
`presence o dA TP)
`
`ddATPor
`3' ~ O~methy 1~dA TP
`termination
`
`G
`A
`G
`G
`T ~
`G
`G
`
`I) 1 1ABAIIAlfl
`dd .TI' y y y
`
`'
`d . f f>
`
`-~
`
`2!i
`fJl},,t}
`
`5j
`
`1¥)
`
`3" -0-rru:thyl-dA TP
`A: o Mop-Up
`B; &JMop-- p
`
`Figure 3. Complete termination of 3¢¢ -O-methyl-dATP by AMV-RT. As shown, various concentrations
`of 3¢ -O-methyl-dATP are assayed in the absence (lanes A) or after enzymatic mop-up (lanes B). All re-
`actions contained 32P-labeled universal primer-annealed to the oligo-template and AMV-RT in reaction
`buffer (lane 1). In addition, lane 2 contained dCTP and ddGTP. All other reactions contained dCTP,
`dTTP, ddGTP and compounds listed below each lane.
`
`816 BioTechniques
`
`DNA synthesis and the extension of a
`solid-phase bound primer-template
`complex containing a complement oli-
`go(dN) 5¢ -end overhang (Figure 1). The
`mop-up strategy takes advantage of
`DNA polymerases that show high affin-
`ity for natural nucleotides and minimal
`specificity for 3¢ -O-modified-dNTPs
`by standard-template incorporation as-
`says. Once enzymatic removal of the
`natural nucleotide is complete, the puri-
`fied 3¢ -O-modified-dNTP solution is
`isolated by magnetic separation and
`can be directly used for the incorpora-
`tion assay. Heat inactivation or chemi-
`cal extraction of the mop-up poly-
`merase can be performed before the
`template assays, although experiments
`omitting this step have resulted in simi-
`lar incorporation data.
`To test the capacity of enzymatic
`mop-up, a range of dATP concentra-
`tions (0, 5, 10, 25 and 50 m M) were
`tested using Bst DNA polymerase as
`the mop-up enzyme and assayed (final
`concentrations: 0, 0.25, 0.50, 1.25 and
`2.5 m M, respectively) using AMV-RT
`for the presence of nucleotide read-
`through (Figure 2). Under these condi-
`tions, Bst DNA polymerase efficiently
`mopped-up dATP concentrations be-
`tween 25 and 50 m M. Similar results
`have been obtained for the enzymatic
`mop-up of dTTP (data not shown).
`Typically, 3¢ -O-modified-dNTPs have
`been evaluated at a final concentration
`range of 12.5–50 m M in incorporation
`assays (References 2 and 3; data not
`shown), corresponding to a capacity to
`efficiently mop-up a 2.5%–20% level
`of natural-nucleotide contamination.
`Previously, the incorporation of RP-
`HPLC purified 3¢ -O-methyl-dATP by
`AMV-RT was shown to cause both ter-
`mination and natural nucleotide read-
`through by the oligo-template assay (3)
`(Figure 3, lanes A). Bst DNA poly-
`merase that was previously shown not to
`incorporate 3¢ -O-methyl-dATP (3) was
`used to selectively remove dATP conta-
`mination from an RP-HPLC purified 3¢ -
`O-methyl-dATP solution. Following en-
`zymatic mop-up, the incorporation of
`3¢ -O-methyl-dATP by AMV-RT shows
`complete termination without natural
`nucleotide read-through (Figure 3, lanes
`B). Klenow fragment has also been used
`to efficiently mop-up both contaminat-
`ing dATP from a 3¢ -O-methyl-dATP so-
`
`Vol. 25, No. 5 (1998)
`
`

`

`incorporation data.
`Here, we have demonstrated that en-
`zymatic mop-up is a viable strategy that
`uses conventional reagents for the pu-
`rification of novel RP-HPLC-purified
`3¢ -O-modified-dNTP analogs. The de-
`velopment of enzymatic mop-up also
`addresses the important issue of nu-
`cleotide read-through in stop-start DNA
`sequencing strategies such as BASS.
`
`REFERENCES
`
`1.Canard, B., B. Cardona and R.S. Sarfati.
`1995. Catalytic editing properties of DNA
`polymerases. Proc. Natl. Acad. Sci. USA
`92:10859-10863.
`2.Canard, B. and R.S. Sarfati. 1994. DNA
`polymerase fluorescent substrates with re-
`versible 3¢ -tags. Gene 148:1-6.
`3.Metzker, M.L., R. Raghavachari, S.
`Richards, S.E. Jacutin, A. Civitello, K.
`Burgess and R.A. Gibbs. 1994. Termination
`of DNA synthesis by novel 3¢ -modified-de-
`oxyribonucleoside 5¢ -triphosphates. Nucleic
`Acid Res. 22:4259-4267.
`
`This work was supported in part by
`Grants 1 R01 HG01459 and 1 R01 AI33334
`and grants to K.B. from the Texas Advanced
`Technology Program and National Insti-
`tutes of health HG/GM01745. Address cor-
`respondence to Dr. Michael Metzker, De-
`partment of Human Genetics, Merck
`Research Laboratories, West Point, PA
`19486, USA. Internet: michael_metzker
`@merck.com
`
`Received 23 March 1998; accepted 15
`July 1998.
`
`Michael L. Metzker, Ramesh
`Raghavachari1, Kevin
`Burgess2 and Richard A.
`Gibbs
`Baylor College of Medicine
`Houston, TX
`1LI-COR
`Lincoln, NE
`2Texas A&M University
`College Station, TX, USA
`
`lution before incorporation by human
`immunodeficiency virus type 1 (HIV-
`1)-RT (data not shown) and contaminat-
`ing dTTP from a 3¢ -O-(2-nitrobenzyl)-
`dTTP solution before incorporation by
`Bst DNA polymerase (data not shown).
`Thus, these data show that dNTP conta-
`mination of RP-HPLC 3¢ -O-modified-
`dNTPs can be effectively removed by
`the enzymatic mop-up assay.
`Canard et al. (1) have provided evi-
`dence for the hydrolysis of 3¢ -O-ester
`linkages by a mechanism of catalytic
`editing of DNA polymerases. Both 3¢ -
`O-methyl and 3¢ -O-(2-nitrobenzyl) pro-
`tecting groups described in the current
`study are attached to the sugar ring by
`ether linkages. The oligo-template as-
`say used here and elsewhere (3) has
`been designed to differentiate between
`the incorporation of the natural dNTP
`(read-through) and the 3¢ -O-modified-
`dNTP (true termination) (Figures 2 and
`3). Using this assay, we have observed
`that the majority of RP-HPLC purified
`3¢ -O-modified-dNTPs contains mini-
`mal, but significant levels of dNTP
`contamination. Previously, we evaluat-
`ed 7 different 3¢ -O-modified-dNTPs, 3
`of which contained ester linkages that
`were not incorporated by 8 different
`DNA polymerases (3). One of these 3¢ -
`linked analogs, 3¢ -O-acyl-
`O-ester
`dATP, can terminate terminal deoxynu-
`cleotidyl transferase (TdT) synthesis in
`a template-independent manner (data
`not shown). The dATP contamination
`for 3¢ -O-acyl-dATP was estimated at
`0.4% by TdT nucleotide-extension as-
`says. Following chain termination of
`3¢ -O-acyl-dATP by TdT, DNA synthe-
`sis could not be reinitiated after a dATP
`chase suggesting that the presence of
`contaminating natural nucleotides, not
`catalytic editing of DNA polymerases,
`is a major cause for nucleotide read-
`through past the desired termination
`base. Moreover, the levels of natural
`nucleotide contamination can vary sig-
`nificantly for different base substituents
`containing the same 3¢ -O-protecting
`group resulting in no read-through [3¢ -
`O-(2-nitrobenzyl)-dATP] (3) or read-
`through [3¢ -O-(2-nitrobenzyl)-dTTP]
`(data not shown). Taken together, our
`data highlight the importance of natur-
`al nucleotide contamination of the
`3¢ -O-modified-dNTPs in the characteri-
`zation and interpretation of enzymatic-
`
`Vol. 25, No. 5 (1998)
`
`