Download Synthesis and isolation of a single-strand

[Manuscript in preparation]
Synthesis and isolation of a singlestranded DNA library with multiple LNAA modifications
Holger Doessing 1, Rakesh N. Veedu 2, Jesper Wengel 3, and Birte Vester 1,*
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1
Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense
M, Denmark; [email protected]
2
School of Chemistry & Molecular Biosciences, University of Queensland, St Lucia, Brisbane,
Queensland, Australia-4072; [email protected]
3
Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark;
[email protected]
*
Author to whom correspondence should be addressed; [email protected]; Tel.: +45-6550-2406;
Fax: +45-6550-2467.
Abstract
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We present new techniques for synthesis and isolation of LNA-containing DNA libraries. Locked
nucleic acids (LNA) are 2′-4′ bridged nucleic acids that show excellent base pairing stability and
resistance towards exoncucleases. This makes them attractive candidates for the preparation of nucleic
acid-based drugs, e.g. aptamers. Aptamers are discovered by in vitro selection, where ligand-binding
members of an oligonucleotide library are amplified, regenerated and subjected to additional selection
rounds under increasingly stringent conditions until the best candidates are obtained. The polymerases
normally used for in vitro selection are not compatible with LNA, meaning that LNA-containing libraries
have not been used for in vitro selection. Therefore, we have developed a strategy involving: (1)
Amplification of an LNA A-containing DNA library with Phusion High Fidelity DNA polymerase; (2) primer
extension on the amplified DNA with LNA ATP using KOD DNA polymerase; and (3) isolation of fulllength LNA extension products by an oligonucleotide capture approach. Together, these methods for
amplification and regeneration allow for in vitro selection using LNA-containing libraries.
Keywords
Locked nucleic acid (LNA), KOD DNA polymerase, in vitro selection, aptamer
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Introduction
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***fooo
Aptamers are oligonucleotides that assume a tertiary structure that allows them to bind a defined
target with high specificity and affinity.
*ref til syntese af LNA ATP og TTP: Veedu Vester Wengel ChemBioChem 2007 (8) 490-492
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*HVAD ER IN VITRO SELEKTION?*
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The low biostability of naked RNA and DNA limits the application in therapeutics. This can be
remedied by the introduction of various modifications to the oligonucleotide. These may either improve
nuclease resistance, chemical or physical stability, or a combination of these. However, modification of
aptamers ‘post selection’ comes at the risk of disrupting the aptamer’s overall structure or altering its
pharmacophoric features. It is therefore desirable to do the in vitro selection using libraries already
containing the modified nucleotide(s). Generally speaking, the in vitro selection procedure involves the
repeated partitioning, amplification, and regeneration of a sequence library. The procedure was
originally described for RNA [1, 2] and was later extended to single-stranded DNA [3]. Since then,
appropriate methods for in vitro selection using many different ribose-modified nucleoside analogues
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have been established, e.g. 2′-aminopyrimidines [4, 5], 2′-fluoropyrimidines [6-8], 2′-O-methyl
nucleotides [9, 10], and 4′-thioUTP and –CTP [11].
Locked nucleic acids (LNAs) are a class of nucleotide analogues that feature a 2′-4′ bridge that ‘locks’
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the ribose in the C3′-endo conformation [12-15]. Common features of LNA-modified nucleotides are
their very high affinity towards complementary nucleic acids *REFS* and resistance towards degradation
by exonucleases *REFS*. These are very attractive traits for improving aptamer stability and half-life.
LNA has been used for modification of aptamers post selection
Figure 1. Structure of locked nucleic acid adenosine triphosphate, LNA ATP.
* aptamerer med LNA-modifikation post-SELEX (Franks paper + referencerne deri) og andre
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In order for in vitro selection with LNA to work it is therefore essential to establish techniques that
are compatible with LNA chemistry. PCR amplification with LNA nucleotides has yet to be realized *BV:
men har været forsøgt (“despite attempts with”) og så noget med en Rakesh-ref? HBD: Har han da
skrevet noget om det?*. We have therefore sought a two-step solution to the matter of amplification
and regeneration: We present how Phusion High Fidelity DNA polymerase can use an LNA adenosinemodified DNA template for PCR under standard conditions. Moreover, we show that KOD DNA
polymerase can incorporate LNA monomers into a DNA library from both double- and single-stranded
templates. Finally, we also demonstrate how these full-length LNA-modified strands may be isolated by
way of an oligonucleotide capture probe. Together, these methods for amplification and regeneration
allow for in vitro selection using LNA-containing libraries.
Results
Amplification of an LNA-containing library
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Classical in vitro selection of an aptamer starts with the preparation of an oligonucleotide library.
LNA phosphoramidites are readily available [16, 17] and the de novo synthesis of an LNA-containing
library is therefore reasonably straightforward. After partitioning of the library into ligand-binding and
non-binding members the pool of binding members must be amplified to obtain enough material for the
next selection round. We addressed the issue of amplification by synthesizing a DNA library with LNA
monomers. Our library contains a randomized core flanked by primer binding sites that allow for
amplification by PCR. We chose to distribute 7 LNA adenosines evenly within the 30-nucleotide core
(Figure 2A). The other 23 positions were randomized with deoxy-guanosine, -cytidine, and thymidine in
a 1:1:1 ratio. This design imitates the proportion of the four nucleobases in a fully randomized library.
Importantly, since the enzymes employed here may favor library members with little or no LNA content,
our library design also ensures that all library members will have the same level of LNA modification.
We amplified the LNA A-containing library to obtain the corresponding double-stranded DNA by
PCR. Remarkably, Phusion High Fidelity DNA polymerase is able to use the LNA library as template
without any changes to manufacturer’s protocol and still afford a single product of the correct size
(Figure 2B). This demonstrates that amplification of an LNA library is feasible.
Figure 2. DNA library encoding incorporation of 7 LNA adenosines.
(A) LNA adenosine incorporation sites are indicated in lowercase. ‘B’ denotes either of deoxyguanosine, -cytidine, and thymidine.
(B) Lane 1: Amplification of the library with Phusion High Fidelity DNA polymerase yields the desired
67 bp double-stranded DNA. Lane 2: Absence of template does not result in formation of any product.
A: LNA library
GGACAGGACCACACCCAG aBBBBaBBBaBBBaBBBaBBBaBBBaBBBB GGCCTTTTGTGTGTCGTTT
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B: PCR
Asymmetric PCR with LNA ATP
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We then set up primer extension reactions with KOD DNA polymerase supplemented with dGTP,
dCTP, dTTP, and either LNA ATP or dATP, as well as a fluorophore-labeled forward primer. The PCRamplified LNA library was used as template. Amplification with fluorophore-labeled primers enabled us
to discern both the forward and reverse DNA strands as well as the primer extension product on
polyacrylamide gels by each strand’s specific label (Figure 3A). After extension at 72 °C for 5 minutes the
reaction product was heat-denatured and then re-extended again at 72 °C. This process was repeated
for 30 cycles, effectively rendering our setup an asymmetric PCR.
The progress of the asymmetric PCR could be monitored by drawing samples from various cycles and
resolving them by polyacrylamide gel electrophoresis (PAGE). Gel scans are shown in Figure 3B and C.
With LNA ATP we clearly saw the displacement of the forward strand from the template duplex to a
single-stranded state during the first cycle (left panel in Figure 3B, lane 1-2; turquoise and green,
respectively). We also saw annealing of the primer to form the primer extension duplex (lane 1-2; red
and purple). In subsequent rounds we saw the formation of free extension products (Figure 3B, lanes 15; red bands). The full-length nature of these products was verified by analyzing the samples by PAGE
under denaturing conditions, where strands resolve according to size (Figure 3C). We clearly observed
an increase in primer size consistent with extension to full-length. Moreover, we were able to achieve
successive extensions, as the yield of the full-length product increased until 15 cycles (Figure 3C, left
panel). However, when dATP was used in the extension reaction (Figure 3B, center panel), we saw initial
primer extension, but both the product and template strands were completely degraded in subsequent
cycles (Figure 3B, lanes 6-8). This indicates that the polymerase’s strong 3′-5′ exonuclease activity *REF*
readily digests DNA strands but not LNA A-modified strands. We have previously observed similar
behavior *REF TIL RAKESH*. Eventually, after a number of cycles various run-away products appeared
(Figure 3B, lanes 9-10). This was also the case when no ATP source was present at all (Figure 3B, lanes
11-15). The nature of the run-away products is unknown, but denaturing PAGE suggests some form of
multi-mer formation involving the forward strands (exemplified in Figure 3 C, right panel; lanes 9-10).
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Figure 3. Validation of the generation of an LNA A-modified DNA library by asymmetric PCR using KOD
DNA polymerase.
(A) Color legend for panel B and C indicating the three different fluorophores used to 5′-label the
primer and the two strands in the experiment. Colors are additive, i.e. Cy3 (green) and Cy5 (red) comigration produces a yellow band.
(B,C) 0, 5, 15 and 30 rounds of asymmetric PCR using dGTP, dCTP, and dTTP supplemented with
either LNA ATP (‘LATP’), dATP, or water (‘no ATP’).
(B) Non-denaturing polyacrylamide gel electrophoresis (PAGE) of samples. Red: free primer or
extension product. Turquoise: Template duplex. Green: Displaced forward strand. Purple:
Primer:template duplex.
(C) Denaturing polyacrylamide gel electrophoresis of the LATP and ‘no ATP’ samples from panel B.
LATP samples: The primer (red) is extended to full length. No ATP samples: Only diminutive primer
extension. Strand degradation and run-away synthesis commences (lanes 8-10). Note that full-length
strands do not co-migrate due to their fluorophore labels.
B: Non-denaturing PAGE
A: Color legend
C: Denaturing PAGE
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The next question we wanted answered was whether our asymmetric PCR setup would work on a
single-stranded template as well. To eliminate the forward strand of the template duplex a 5′ phosphate
was introduced by PCR using a phosphorylated forward primer. The PCR product could then be treated
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with lambda exonuclease, which preferentially digests a 5′-phosphorylated strand of a DNA duplex [18],
leaving only the reverse strand (i.e. the template). This procedure is successfully demonstrated in Figure
4A. We then tested our incorporation of LNA A by asymmetric PCR using either the template duplex or
the single-stranded template generated by lambda degradation. We found no substantial differences in
either reaction progress or yield when comparing the reactions by PAGE after 10 cycles (Figure 4B, C).
Thus, KOD DNA polymerase can perform primer extension with LNA ATP from both single- and doublestranded DNA templates, thereby allowing the regeneration of an LNA A-containing pool of in vitroselected oligonucleotides.
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Figure 4. Comparison of LNA A-incorporation by asymmetric
PCR on single- and double-stranded DNA using KOD DNA
polymerase.
(A) Agarose gel showing lambda exonuclease digestion of the
5′-phosphorylated strand in a 67 bp DNA duplex. The
complementary stand is labeled with FAM (green). When doublestranded the DNA binds ethidium bromide (red) and retains its
FAM fluorescence (producing yellow; lane 1). After digestion the
sample is single-stranded and no longer binds ethidium bromide
(lane 2).
(B, C) Color coding as in Figure 3. ‘λ exo-digest remnants’
indicates by-products of over-digestion with lambda exonuclease.
Extension with dGTP, dCTP, dTTP, and LNA ATP yields full-length
product.
(B) Non-denaturing polyacrylamide gel electrophoresis.
(C) Denaturing polyacrylamide gel electrophoresis.
A: Digestion of forward
DNA strand
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B: Non-denaturing PAGE
C: Denaturing PAGE
Purification of full-length LNA strands
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Before the LNA A-containing library obtained by the asymmetric PCR can be used for selection it
must be purified. The reaction mixture contains both template strands and a range of partially extended
products as seen in Figure 3C, lanes 2-5. In order to isolate only the fully extended product strands we
used an oligonucleotide capture approach, where an immobilized oligonucleotide with complementarity
to the 3′ end of the fully extended product strands is used for hybridization to extract the desired
product as illustrated in Figure 5A. After isolation and washing of the hybridized strands on the beads
the LNA A-containing strands are eluted off the beads by heating. Only DNA:DNA hybridization takes
place, and therefore the interaction is readily disrupted by heating. The purity of the eluate was verified
by PAGE, which showed a clean product (Figure 5B).
Verifying LNA content
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Finally, we verified the presence of LNA moieties in our eluate by an unconventional enzymatic
degradation approach. We utilized the fact that in the absence of triphosphates the 3′-5′ exonuclease
activity of the KOD DNA polymerase readily degrades DNA strands (cf. Figure 3), whereas LNA-modified
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strands are resistant to degradation (our unpublished data). The Cy5-labeled eluate was mixed with
equimolar amounts of the LNA A-modified crude library and the all-DNA FAM-labeled template library
generated by lambda exonuclease digestion. KOD DNA polymerase was added and the mixture was left
for reaction. While the DNA strand was completely degraded over the course of 80 minutes the LNAmodified crude library could only be chopped down until the 3′-most LNA moiety as is clearly shown by
PAGE analysis in Figure 5C (blue and green bands, respectively). The eluate (red) showed a degradation
pattern identical to that of the crude LNA library, thereby demonstrating the incorporation of LNA A in
the purified extension products.
Figure 5. Extraction and quality control of full-length extension products.
(A) Strategy for purification of single-stranded extension products: Extension products (red) can
anneal onto a biotinylated capture oligonucleotide, which is bound to streptavidin-coated magnetic
nanoparticles. Unbound reaction components can be washed away. The capture oligomer binds the 3′
end of the library, ensuring that only full-length extension products are bound. Only DNA:DNA
hybridization takes place, and therefore the interaction is readily disrupted by heating.
(B) Denaturing PAGE of primer extension mixture before and after purification by oligonucleotide
capture. Color coding as in Figure 3.
(C) Digestion assay. Equimolar amounts of purified primer extension product (5′-Cy5; red), crude
LNA A library (5′-32P; green), and template DNA generated by lambda exonuclease (5′-FAM; blue) were
combined and subjected to the 3′-5′ exonucleolytic activity of KOD DNA polymerase. Differences in
electrophoretic mobility of the three species involved are mainly due to the nature of their different 5′
labels. Below: Color legend. LNA moieties are indicated with circles. Observed extent of degradation is
indicated with dashes.
A: Oligonucleotide capture strategy
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B: Oligonucleotide capture results
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C: Digestion assay
Discussion
*BV: Man kunne måske godt starte med at pointere vanskelighederne ved modifcierede biblioteket.
LNA svært / ribosemethylRNA relativt let. Og relativt til DNA og RNA bibl.! og understrege at disse
vanskeligheder må løses med alternative eller i hvert fald kraftigt modificerede metoder.
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The library that we chose for testing the approaches described above has a core of 30 nucleotides,
of which 7 are fixed LNA adenosines and 23 are randomized between guanosine, cytidine, and
thymidine. The total number of unique members is therefore (323 ≈) 94×109. A fully randomized library
would encode (430 ≈) 1.2×1018 members, a billion-fold more. It is likely that the positioning of the LNA
moieties will have a negative effect on the diversity of the structures that may be selected, if our pilot
design was employed in an in vitro selection experiment. However, we chose this design over a fully
randomized pool to ensure that we would not amplify and regenerate library members with no or very
few LNA moieties. The fixed positioning of the LNA adenosines also allowed us to verify the presence of
the 3′-most LNA in the purified product by exonucleolytic degradation with KOD DNA polymerase
(Figure 5C). We expect, however, that our protocol can be extended to fully randomized templates as
well.
We have previously shown that Phusion High Fidelity DNA polymerase is able to read an LNAmodified DNA template [19-21]. However, the enzyme had to be supplemented with Mn2+ and betaine
to afford the desired products. This is the first report of how Phusion High Fidelity DNA polymerase can
amplify a 67 nucleotide LNA A-modified DNA template under standard conditions. Imanishi et al.
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attempted to do primer extension on LNA T-modified DNA templates with Phusion High Fidelity DNA
polymerase under standard conditions but saw little or no full-length extension [22]. It is possible that
the nature of the LNA nucleoside may impact on the enzyme’s ability to function properly.
**Triphosphates of LNA guanosine, thymidine, 5-methylcytidine, and uridine are also available [21,
23]*REFS FOR GTP/m5CTP*, and their usage by KOD DNA polymerase *OG PHUSION?* has been
demonstrated [24, 25]. *NOGET MED AT MAN OGSÅ KU KIGGE PÅ DE ANDRE TRIFOSFATER IFT.
BIBLIOTEKER*
* Evt også UNA aptamerer som modpol (Anna Pasternaks paper), og hvor enzymatisk
polymerisering nok er endnu sværere? Men måske lidt lang ude?!
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We [23-25] and others [22] have also established how KOD DNA polymerase or exonucleasedeficient variants hereof are able to incorporate LNA triphosphates under conditions normally used for
polymerase-mediated primer extension. Here, we show how this can be used to regenerate a pool of
LNA-containing DNA oligonucleotides. The 3′-5′ exonuclease domain of KOD DNA polymerase is
supposedly active against single-stranded species only [26]. It is curious, though, that we do not observe
degradation of the 3′ terminus of the fully extended products during asymmetric PCR, as this 23
nucleotide region contains no LNA moieties. Likewise, we also do not see degradation of either the
forward or reverse strand of the template duplex in the presence of LNA ATP (cf. Figure 3C, left panel).
One explanation for the lack of degradation under asymmetric PCR with LNA ATP could be that the LNAmodified strands hybridize more readily to the template. These LNA:DNA duplexes then act as decoy
substrates for KOD DNA polymerase, leaving little or no free polymerase to act on the single-stranded
species. In the absence of LNA only DNA:DNA duplexes will form and their thermal stability will lower,
leading to free inactive KOD polymerase, whose exonucleolytic activity may then dominate and cause
complete degradation. Further experimentation is needed to clarify this particular aspect.
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The library design employed here does not contain LNA in the flanking regions. We opted not to
introduce LNA moieties here, as the increased base-pairing potential of LNA could lead to selection of
aptamers with dependence on the flanking regions. Moreover, the oligonucleotide capture approach
relies on the sequence-specific pairing between the capture oligomer and full-length extension products
only. The subsequent elution by heating might be impeded if LNA:DNA hybridization is involved.
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Alternatively, the 3′ terminus of the extension products could be designed to contain 1-2 LNA moieties.
These could offer protection against exonucleases, meaning that the reaction mixture could be cleared
of DNA-only strands by adding appropriate enzymes, e.g. snake venom phosphodiesterase [27-29]. In
this case, enzymes, buffers and salts, and partially extended LNA-containing products would still have to
be removed by other means.
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Conclusion
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In summary, we have established that a DNA library with an LNA content and distribution that
mimics that of a fully randomized library can acts as a suitable substrate for Phusion High Fidelity DNA
polymerase under standard PCR conditions. The PCR product can then be used as template by KOD DNA
polymerase in successive primer extensions (i.e. asymmetric PCR) with LNA ATP leading to the
production of a single-stranded LNA-containing pool. The PCR product may either be used directly in its
double-stranded form, or it may be treated by lambda exonuclease to afford the single-stranded
species, both of which are suitable as templates for partial LNA incorporation by KOD DNA polymerase.
Use of the single-stranded species enabled us to isolate the full-length LNA strands by oligonucleotide
capture on magnetic nanoparticles. Finally, we verified the presence of LNA in the purified products
using an unconventional exonuclease assay with the KOD DNA polymerase.
By addressing the issues of amplification, re-generation, and purification of an LNA-containing pool
we have provided the tools for use of LNA-containing libraries for in vitro selection experiments. The
future goal is thus the discovery of e.g. aptamers that fully employ the unique properties of LNA.
Acknowledgements
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H.D. and L.H.L were funded by a stipend from the Danish Agency for Science, Technology and
Innovation (FTP). This work was carried out at the Nucleic Acid Center, which is funded by the Danish
National Research Foundation.
Experimental
Oligonucleotides
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LNA A library: GGACAGGACCACACCCAGaBBBBaBBBaBBBaBBBaBBBaBBBaBBBBGGCCTTTTGTGTGTCGTTT (lowercase indicates LNA adenosine); forward primer: GGACAGGACCACACCCAG; reverse primer:
AAACGACACACAAAAGGCC. Whenever appropriate, chemically synthesized primers with 5′modifications – phosphate, Cy3, Cy5, or 6-carboxyfluorescein (FAM) – were used. Radiolabeling was with
T4 polynucleotide kinase and 32P-γ-ATP [30]. DNA synthesis was by Sigma; LNA synthesis was in-house at
the Nucleic Acid Center.
Polymerase chain reaction
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0.5 nM template (all-DNA or LNA-containing single-stranded DNA) and 0.5 µM of each primer were
combined in 1× Phusion HF buffer with 200 µM of each deoxyribonucleotide triphosphate and 0.02
units/µl Phusion High Fidelity DNA polymerase (Finnzymes). PCR conditions were: 98 °C/5 min., 24 cycles
(98 °C/5 s, 53 °C/10 s, 72 °C/5 min.), 4 °C/hold (with minor deviations). Products were resolved by
agarose gel electrophoresis and visualized by fluorescence scanning on a Typhoon Trio system (GE
Healthcare).
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Asymmetric PCR
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Reaction conditions were: 1×KOD buffer #2, 3 mM MgSO4, 0.2 mg/ml BSA, 0.2 u/µl KOD DNA
polymerase (Novagen), 2.5 µM primer, 250 ng double-stranded DNA template (67 bp), and 0.25 mM of
each nucleotide triphosphate as required. Thermocycling was set to: 95°/1 min., 30 cycles (95°/15 s,
60°/35 s, 72°/5 min), 4°/hold. Reactions were quenched with 8.3 mM (final) EDTA and stored at −20 °C.
Products were resolved on 10% non-denaturing polyacrylamide gels at 4 °C or on 13% denaturing (7 M
urea) polyacrylamide gels and visualized by fluorescence scanning on a Typhoon Trio system (GE
Healthcare) to assess molecular interactions or sizes, respectively.
Lambda exonuclease reaction
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Double-stranded DNA was prepared with a 5′-phosphorylated primer and a 5′ fluorophore-labeled
primer. Digestion with lambda exonuclease (New England Biolabs) was with 6.7 units/µg doublestranded DNA (50 ng/µl final DNA concentration) at 37 °C for 25 min. The reaction was stopped by
quenching with addition of 1 vol. 50 mM EDTA or by heating to 75 °C for 15 min. Full digestion of the 5′phosphorylated strand was verified by agarose gel electrophoresis and ethidium bromide staining.
Fluorescence scanning was on a Typhoon Trio system (GE Healthcare).
Oligonucleotide capture on magnetic beads
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30 µl asymmetric PCR reaction was quenched with EDTA and combined with at least 4-fold molar
excess of biotinylated oligomer complementary to the library’s 3′ end. The mix was heated to 65 °C for 5
min. and then slow-annealed over 1 hour. The solutions were adjusted to 1×B&W (according to
manufacturer’s instructions) and combined with 1.5 mg Dynabeads M-280 Streptavidin (Invitrogen)
washed twice in 1×B&W buffer. The number of beads corresponded to an excess of biotin binding sites.
After 20 min. with gentle agitation the beads were washed twice in 1×B&W before heat-elution of the
non-biotinylated species into 50 µl phosphate-buffered saline with 0.2% Tween-20 (75 °C, 5 min.).
KOD exonuclease assay
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Reaction conditions for exonuclease activity by KOD DNA polymerase were similar to those of
asymmetric PCR, except that no primer and triphosphates were used, and KOD DNA polymerase was
added to 0.02 u/µl. Incubation was at 72 °C. Samples were quenched with EDTA and resolved by 13%
denaturing PAGE. Bands were visualized by fluorescence scans of Cy5 and FAM and autoradiography of
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P and combined in Photoshop (Adobe) using gel wells as reference markers.
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