From the Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340
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ABSTRACT |
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Lengthy expansions of trinucleotide repeats are
found in DNA of patients suffering severe neurodegenerative age-related
diseases. Using a synthetic self-priming DNA, containing CAG and CTG
repeats implicated in Huntington's disease and several other
neurological disorders, we measure the equilibrium distribution of
hairpin folding and generate triplet repeat expansions by
polymerase-catalyzed extensions of the hairpin folds. Expansions occur
by slippage in steps of two CAG triplets when the self-priming sequence
(CTG)16(CAG)4 is extended with
proofreading-defective Klenow fragment (KF exo) from
Escherichia coli DNA polymerase I. Slippage by two triplets is 20 times more frequent than by one triplet, in accordance with our
finding that hairpin loops with even numbers of triplets are 1-2
kcal/mol more stable than their odd-numbered counterparts. By measuring
triplet repeat expansions as they evolve over time, individual rate
constants for expansion from 4 to 18 CAG repeats are obtained. An
empirical expression is derived from the data, enabling the prediction
of slippage rates from the ratio of hairpin CTG/CTG interactions to
CAG/CTG interactions. Slippage is initiated internally in the hairpin
folds in preference to melting inward from the 3' terminus. The same
triplet expansions are obtained using proofreading-proficient KF
exo+, provided 10-100-fold higher dNTP concentrations are
present to counteract the effect of 3'-exonucleolytic proofreading.
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INTRODUCTION |
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The human genome has an abundance of simple sequence repetitions that are unstable and tend to expand in large numbers in some genetic loci. A prime example is the CAG/CTG class of triplet repeats whose large expansions occur in genes associated with Huntington's disease and six other neurological disorders (1). Such expansions represent a novel form of mutation whose cause is unknown. An attractive possibility under investigation is primer/template slippage during DNA replication or repair of tandemly repeated sequences (2-8).
Strand slippage can occur when there is local strand separation in DNA regions containing tandem repetitions (9). For example, in a region of CAG/CTG repeats, local separation creates single-strand loops of CAGs and CTGs whose repetitive character may allow the two strands to be displaced (i.e. slipped) by an integral number of triplets. Such slippage in the presence of DNA polymerases can result in the addition of integral numbers of triplets to give triplet repeat expansions (2-8). Large loops that form stable hairpin structures are expected to increase the probability of slippage and expansion (8, 10-14).
We (14) and others (10, 12, 13, 15) have shown that single strands of CTG repeats form stable hairpin structures as a result of base pairing between antiparallel CTGs, yielding G·C and C·G base pairs alternating with T·T mispairs. Similar hairpins also form in strands of CAG repeats but are not as stable (14-16), probably because A·A mispairs are bulkier and more destabilizing than T·T (17). Our thermal denaturation studies of strand folding (14) indicated that hairpin stability increases more slowly than expected with increasing numbers of repeats. A plausible explanation is that strands with more repeating triplets have more degrees of freedom and tend to form several shorter hairpin loops instead of one long one. For example, a strand with 10 triplet repeats may fold into a single hairpin structure, whereas a longer strand with 30 repeats may form a more complex structure with two or three hairpin folds of similar size (14).
Evidence of strand slippage in CAG/CTG repeat regions during DNA polymerization has been obtained using various gel-based primer extension assays (5-8). Our assay differs from earlier ones mainly in the priming event. We engineered a DNA oligonucleotide of triplet repeats so that it is primed by intramolecular folding, rather than by intermolecular association. Being independent of concentration, the self-priming remains efficient at low DNA concentrations, unlike intermolecular priming that requires higher DNA concentrations. By using low concentrations, we avoid uncertainties arising from multimeric associations between DNA strands. Also, our starting primer 3'-end is fully complementary to template repeats, instead of partially complementary as in the only previous study of self-priming with triplet repeats (6).
In the present study we use a self-priming strand containing a large number of CTGs relative to CAGs, to measure the influence of CTG/CTG interactions on strand slippage in polymerase-catalyzed expansions of correctly paired (CAG/CTG) repetitions. This approach provides for the first time an opportunity to measure slippage and expansion rates as a function of the number of interacting triplets, for DNA polymerases with and without proofreading 3'-exonuclease activity.
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EXPERIMENTAL PROCEDURES |
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Materials
DNA Polymerases--
Escherichia coli DNA polymerase
I Klenow fragment (KF)1
mutant, exo (D355A,E357A), devoid of proofreading
3'-exonuclease activity, was purified from overproducing strains (18).
Normal proofreading-proficient KF polymerase (exo+) was
obtained commercially (U. S. Biochemical Corp./Amersham Corp.).
Oligonucleotide Synthesis--
The self-priming DNA 60-mer,
(CTG)16(CAG)4, and 30-mer
(CTG)6(CAG)4 were synthesized by an Applied
Biosystems DNA/RNA synthesizer, using -cyanoethyl phosphoramidites,
and purified by denaturing polyacrylamide gel electrophoresis. The
90-mer DNA marker (CTG)16(CAG)14 was purchased
from Operon Technologies Inc. and obtained lyophilized after high
pressure liquid chromatography purification. Purified DNA samples were
dialyzed extensively against a low ionic strength buffer (5 mM NaH2PO4, 5 mM
Na2HPO4, 1 mM Na4EDTA,
pH 7.0) and stored at
70 °C.
Methods
Melting Analysis-- Thermal denaturation profiles were obtained for the 60- and 30-mer DNAs at the same strand concentration (1.9 µM) in low ionic strength buffer, by measuring UV absorbance A260 versus temperature, while raising temperature from 25 to 85 °C at a constant rate of 2 °C/min.
End Labeling and Equilibration--
Samples of the self-priming
60-mer used in polymerase reactions were 5'-end-labeled with
32P, using [-32P]ATP and T4 polynucleotide
kinase (U. S. Biochemical Corp./Amersham Corp.) in kinase buffer
(50 mM Tris-HCl, pH 7.6, 10 mM
MgCl2, and 10 mM 2-mercaptoethanol). Labeled
samples at a strand concentration of 100 nM were
heat-denatured at 100 °C for 5 min and allowed to renature by
cooling at room temperature. The results obtained in polymerase
reactions showed no dependence on the rate of cooling, as expected for
intramolecular folding. The labeled strands were stored at 4 °C to
avoid any potential intermolecular associations promoted by freezing
(14).
Extension Reactions-- Radiolabeled DNA at 10 nM strand concentration was incubated 5 min at 37 °C in reaction buffer (50 mM Tris-HCl,, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, and 0.05 mg/ml bovine serum albumin) to allow equilibration of DNA structure. Typically, 60 µl of DNA solution was then micropipeted into 15 µl of enzyme + dNTP solution (same buffer) held in a microcentrifuge tube at 37 °C, at which point running time (t) for reaction was started. Final enzyme concentration was approximately 60 nM in each case; the dNTPs used (N = C, A, G) were in equimolar amounts ranging from 0.1 to 10 µM. At times indicated (t = 0.5 min, etc.) a 5-µl aliquot of reaction mixture was removed and quenched with 25 µl of 20 mM EDTA + 20 M formamide.
Denaturing Gel Electrophoresis-- Reaction products were separated into bands according to product size, by denaturing gel electrophoresis at 2000 V on 12% polyacrylamide (40 cm × 40 cm × 0.2 mm) with 16 M formamide as denaturant, in TBE buffer (89 mM Tris borate, pH 8.3, 2 mM Na2EDTA). Gels dried on paper were scanned with Molecular Dynamics Storm 860 PhosphorImager, and band intensities were integrated by FragmeNT Analysis software. Each band intensity was evaluated as percent of total integrated band intensities in the corresponding lane.
Expansion Rate Analysis-- The 60-mer sequence (A) forms hairpin loops (An) with even and odd numbers of bases in the hairpin bend (Fig. 1). In the presence of DNA polymerase and dNTPs, the loops are rapidly extended (in seconds) to blunt-ended forms, An + (CAG)n, seen as gel band intensities In, where n is the number of CAG triplets added (Fig. 3). The band intensities change gradually (in minutes) because of slippage and further expansion. Since bands with n = 0, 2, 4, etc. are much more intense than those with n = 1, 3, 5, etc., even-numbered loops are evidently more stable then odd-numbered, as suggested earlier (10). Thus slippage by two triplets and expansion by (CAG)2 is favored over slippage by one triplet and expansion by (CAG)1. To verify that added triplets have the sequence CAG, chain termination with dideoxy-NTPs is used. In separate reactions, a single dideoxy-NTP is added, at 100 × the corresponding dNTP concentration, to obtain termination at N = C, A, or G. In this way (data not shown) we confirm the positions of bands corresponding to addition of (CAG)n for both even and odd values of n.
Assuming band intensities change successively by 2-triplet slippage and expansion, so that In converts to In+2 with rate constant kn, we evaluated rate constants k0 for I0 ![]() |
RESULTS |
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The self-priming sequence, A = (CTG)16(CAG)4, forms a series of hairpin loops (Fig. 1), to which integral numbers of CAG triplets can be added by polymerase-catalyzed reaction with appropriate dNTP substrates (N = C, A, G). In Fig. 1, A0 represents the most stable loop in the form of a blunt-ended hairpin; A1 corresponds to slippage of A0 by a single triplet; A2, slippage by two triplets; etc. Initially, there is an equilibrium distribution of loops reflecting their thermodynamic stability in the absence of polymerase. We verify that stable folds are formed by examining thermal denaturation profiles obtained by plotting UV absorbance (A260) versus temperature (Fig. 2).
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When DNA polymerase and dNTPs are added, the original loops are
extended to blunt ends in a matter of seconds, so that the rates at
which blunt-end hairpins rearrange by slippage and expand by triplet
additions can be accurately measured over a period of minutes. To
establish the equilibrium amounts of individual hairpins and their
extension rates, we analyzed polymerase-catalyzed reaction products
separated into discrete bands by electrophoresis on a denaturing,
formamide-polyacrylamide gel. The products are first analyzed as a
function of reaction time (t) at physiologically low (0.1 to
1 µM) dNTP concentration, using a proofreading-deficient DNA polymerase, Klenow fragment exo. The results shown
(Fig. 3) are obtained with 0.2 µM each of the three dNTP substrates (N = C, A, G). This concentration appears high enough to obtain near-maximum
velocity of extension with a minimum of artifacts such as terminal
transferase activity, seen at higher [dNTP] as an increase in
background "pause" bands (data not shown).
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Thermal Denaturation Profiles-- Upon heat denaturation (Fig. 2), the 60-mer (CTG)16(CAG)4 shows two sigmoidal transitions, with melting temperature Tm = 56 and 80 °C at low ionic strength. By comparison, the 30-mer (CTG)6(CAG)4, having 10 fewer CTG repeats, shows only the higher transition with Tm near 80 °C. The biphasic melting curve of the 60-mer is consistent with the kind of hairpin folding shown in Fig. 1, in which the 4 CAG triplets are correctly base-paired with 4 CTG repeats to give the more stable component melting at 80 °C, whereas the remaining 12 CTG triplets are more weakly paired to give the less stable component melting at 56 °C. The latter Tm value is close to that observed for hairpin folding of CTG triplets alone, e.g. (CTG)10 and (CTG)30, each with Tm = 51 °C (14). However, the melting curve is unable to resolve the kinds of hairpin folds present. The various possible folds (Fig. 1) initially have the same strand length, but when extended with polymerase they acquire increasing lengths, which can be separated by electrophoresis on formamide-polyacrylamide gel.
Initial Hairpin Loops-- As seen in Fig. 1, starting at the 5'-end marked by *, the 60-mer sequence A has a potential template of 16 CTG repeats followed by a primer of 4 CAG repeats. Upon hairpin folding, so that the CAGs align with CTGs in antiparallel fashion, the (CAG)4 primer can be correctly base-paired with any 4 successive antiparallel CTGs, shown as (GTC)4 in Fig. 1. The first loop, A0, is a blunt-ended structure with 3'- and 5'-ends juxtaposed. This is the most stable fold because it allows remaining CTGs to form the maximum number of base pairs with each other.
The even-numbered loops A2, A4, ... , A10 belong in the same group as A0, because they each have the same kind of 4-base hairpin bend and an even number of overhanging triplets. The odd-numbered loops A1, A3, ... , A11 belong in a separate group, because they each have a 3-base bend and an odd number of overhanging triplets. The even-numbered loops are expected to be somewhat more stable than the odd-numbered (10), because they each have one more base pair stabilizing the hairpin bend. For completeness, much less stable loops, A12 to A15, having fewer than 4 correctly paired CAG/CTG combinations, are also shown (Fig. 1). Being blunt-ended, A0 is not suitable for extension by DNA polymerases until it rearranges by strand slippage into one of the other possible forms with an overhanging 5'-template, (GTC)n* (Fig. 1). These forms, An (n = 1, 2, etc.), are easily extended to blunt-end products, An + (CAG)n, by the addition of (CAG)n opposite (GTC)n*. The products are resolved as a series of bands on a denaturing gel, with band intensity In proportional to the amount of product, (An + (CAG)n). The 5'-end (*) is radiolabeled with 32P to obtain quantitative band intensity measurements with a PhosphorImager.Loop Resolution by Electrophoretic Analysis of Polymerase-catalyzed
Extension Products--
Using proofreading-deficient DNA polymerase KF
exo, we observed complete extension of initial loops
within 0.5 min of reaction (Fig. 3). A 6-fold excess of polymerase is
used to saturate all the self-primed loops originally present so that
they can be completely extended to blunt ends in seconds, before
appreciable primer/template slippage can occur. Also, we use a dNTP
concentration low enough (0.2 µM) to avoid the formation
of anomalous pause bands arising by base misinsertion or by blunt end
addition of a single nucleotide (19).
Measurement of Expansion Rates for Primer/Template Slippage in
Polymerase KF exo Reactions--
By examining the band
patterns in Fig. 3, we find that as the original 60-mer band (0 triplets added) decays with t, each subsequent even-numbered
band, representing addition of 2, 4, etc. CAG triplets, intensifies to
a maximum before decaying. This is better illustrated in Fig.
4, where the band intensity
In, expressed as percent of total intensity in each
lane, is shown plotted versus t for the first three
even-numbered bands, n = 0, 2, and 4. Since
odd-numbered bands (additions of 1, 3, etc. triplets) remain faint at
all times, we see that slippage by two triplets is strongly favored
over slippage by one triplet.
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Relationship between Slippage Rate and Number of Interacting
Triplets--
In Table I (last column) we see that the slippage rate
constant kn decreases with increasing number,
n, of added CAGs. The rate constant is highest (0.8 min1) for n = 0, the initial blunt-end
hairpin A0 (Fig. 1), which has the highest
number of CTG/CTG interactions relative to CAG/CTG interactions. As
Fig. 1 shows, A0 has 4 strong CAG/CTG base
pairing interactions and 5 weaker CTG/CTG interactions, so that
(CTG/CTG)/(CAG/CTG) = 5/4. The longer blunt-end hairpins, An + (CAG)n, formed by adding n
CAG triplets complementary to the template (GTC)n* (Fig. 1),
gain n more CAG/CTG interactions while losing n/2
CTG/CTG interactions. Thus, while (CAG/CTG) = 4 + n
increases with n, both (CTG/CTG) = 5
(n/2) and the ratio, (CTG/CTG)/(CAG/CTG) = (10
n)/(8 + 2n), decrease with n.
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Effect of Proofreading Activity on Expansion Rate--
When KF
exo is replaced by normal proofreading-proficient Klenow
fragment, the 3'-exonuclease activity of proofreading markedly inhibits
expansion at low dNTP concentrations (Fig.
7). At 0.2 µM dTNP, band
intensities similar to those found initially with KF exo
are observed in the 1st min of reaction, but expansion by slippage is
greatly reduced because of 3'-end degradation with increasing time
(Fig. 7). As dNTP concentration is increased to counteract the
degradation, the band patterns for expansion by slippage are recovered
for longer times. At 1.0 µM dNTP, the same patterns of
band intensities as in Fig. 3 are observed for the first 10 min, but
some decreases in long expansions (notably I14
and I12) are evident at longer times. At 10 µM, no significant decreases are evident for at least 60 min. Thus, to obtain the same degree of expansion in the presence of
proofreading as in its absence, apparently 10 to 100 times as much dNTP
is required.
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DISCUSSION |
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Single DNA strands containing tandemly repeated CTG or CAG
triplets form hairpin structures which, although not as stable as
normal DNA duplexes, are nonetheless remarkably stable (10-15, 20). To
investigate the influence that such secondary structures have on strand
slippage in polymerase-catalyzed repeat expansions, we have constructed
a strand sequence, (CTG)16(CAG)4, forming a
series of self-primed hairpins (Fig. 1) to which repeats of CAG
triplets can be added by DNA polymerases. This sequence provides an
opportunity to measure the influence of hairpin CTG/CTG interactions on
primer/template slippage in repeating CAG/CTG regions. By using a DNA
polymerase devoid of exonuclease activity (KF exo), we
find that initial hairpin loops (formed at equilibrium) are fully
extended to blunt-ended forms in a matter of seconds, so that the rates
at which blunt-end loops rearrange by slippage and add more repeats can
be measured over a period of minutes to about 1 h (Fig. 3). With
this construct, we observe triplet repeat expansions in a series of
discrete slippage steps for which rate constants can be evaluated.
Of the various hairpins formed initially (Fig. 1), those with an even number of bases in the bend appear most stable since they are found in the largest amounts (Table I). The series of even-numbered loops from A0 to A10 account for over 90% of all folded structures. As expected, the most stable loop is the blunt-ended structure A0, whose initial amount is 61% of total structures at 37 °C. The next most stable loops are initially present in amounts of 5-7%, these being the five even-numbered loops, A2 to A10. Surprisingly, each of the latter is as abundant or more abundant than all the odd-numbered loops collectively. The abundance of even-numbered loops does not decrease from A2 to A10 as might be expected but instead shows a minimum at A4 (5%). Possibly the longer overhanging templates, shown simply as (GTC)n* in Fig. 1, form a second hairpin fold providing some additional stability.
The initial amounts of loops formed at equilibrium, in the absence of
polymerase, can be used to calculate relative loop stabilities in terms
of standard free energy, Go =
RTlnKeq. From the ratio of
equilibrium amounts, Keq = 7/61, we find
A2 is less stable than A0
by RTln(61/7) = 1.3 kcal/mol at T = 37 °C
or 310 K. Similarly, from Keq = 2/61, A1 is found to be 2.1 kcal/mol less stable than
A0 and thus 0.8 kcal/mol less stable than
A2. Finding A1 about 1 kcal/mol less stable than A2 is consistent with our observation (Table I, footnote) that slippage by 1 triplet, converting A0 to A1, is
an order of magnitude slower than the rate for 2-triplet slippage
converting A0 to A2.
As shown in Fig. 6B, the rate of slippage by 2 triplets may be simply related to the ratio of CTG/CTG interactions to CAG/CTG interactions involved in slippage. The logarithm of the slippage rate constant, when plotted against the ratio (CTG/CTG)/(CAG/CTG) in the blunt-ended hairpin undergoing slippage, shows a linear relationship with a positive slope. The slope indicates that slippage rate increases exponentially with the ratio. The implication is that hairpin CTG/CTG interactions promote slippage by 2 triplets and thereby provide a favorable way of propagating waves of expansion in CAG/CTG repeat regions.
In the expansion of the blunt-ended loops in our system, there are
several reasons to think that slippage begins internally with CTG/CTG
interactions rather than at the primer 3' terminus with CAG/CTG
interactions. We observe (Fig. 2) that the CTG/CTG region has a melting
temperature 24 °C lower than the CAG/CTG region, so that local
melting needed to initiate slippage is much more likely to occur in the
CTG/CTG region. To initiate slippage by 2 triplets, three CAG/CTG
interactions need to be disrupted at the primer 3'-end, but only one
next to the CTG/CTG region where two weaker CTG/CTG interactions are
more easily disrupted. Slippage by 2 triplets is favored over slippage
by 1 triplet, in accordance with even-numbered loops of CTGs being more
stable than odd-numbered (Table I). The rate constant for 2-triplet slippage increases with CTG/CTG interactions but decreases with CAG/CTG
interactions (Fig. 6A), so that the rate constant increases exponentially with the ratio of CTG/CTG to CAG/CTG interactions, as
seen by the linear plot of log(rate constant) versus this
ratio (Fig. 6B). Furthermore, in the presence of
3'-exonucleolytic proofreading (Fig. 7), as long as there are CTG/CTG
interactions present (n = 0-8) and slippage rates are
0.28 min1 or higher (Table I), less than 1.0 µM dNTP concentrations are needed to restore normal
slippage patterns, whereas 10 µM concentrations are
needed when CTG/CTG interactions are absent (n = 12 and
14) and slippage rates fall to 0.08 min
1 or lower (Table
I).
Since our system is single-stranded and relatively short, it is not certain how many of the points above may apply to long double-stranded DNA molecules in vivo. Nevertheless, with these points in mind, we can propose a simple model (Fig. 8) indicating how triplet repeat expansion may occur when there is local strand separation in a region of triplet repeats. Such separation could arise, for example, by negative supercoiling (21) of the kind needed to activate transcription or replication. Once separated in a region of triplet repeats, the strands may fold on themselves to form hairpin loops that are initially opposite each other but because of repetition are able to migrate apart by strand slippage and remain separated for longer periods. While apart, if a nick (single-strand cleavage) occurs opposite one of the loops, that loop can stretch and become linear so that a DNA polymerase, perhaps a proofreading-deficient polymerase involved in DNA repair, may fill in the gap to create an expansion on one strand. After the gap is filled and sealed by ligase, a nick opposite the other loop can lead to expansion on the other strand. According to this model, single-strand loops that become separated and nicked in the presence of polymerase may behave like the simple hairpin system we have studied.
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Our simple system enables one to evaluate the contributions that individual single-strand interactions make to strand slippage in duplex repeat regions. By using the sequence (CTG)16(CAG)4, we have made an evaluation of CTG/CTG interactions contributing to slippage and expansion of CAG/CTG repeats. Similarly, by using the sequence (CAG)16(CTG)4, a corresponding evaluation can be made for CAG/CAG interactions. Since CAG/CAG interactions are known to be weaker, forming less stable hairpin folds (14-16, 20), it is of interest to see how much difference this makes in the slippage rate constants. Previously it has been generally considered (2, 3, 8, 11, 12, 20) that the greater the stability of single-strand folding, the greater the probability of strand slippage and expansion in repeat regions. However, it is also possible that a difference in folding stability on opposite strands may be important in determining strand slippage and expansion rates. This may be a reason why repeats of triplets such as GAA/TTC, having only purines on one side and pyrimidines on the other, also expand to give diseases such as Friedrich's ataxia (22).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AG 11398 and GM 21422.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The abbreviations used are: KF, Klenow fragment; A, self-priming DNA sequence, 5'-(CTG)16(CAG)4-3'; An, initial hairpin fold of sequence A, having n CTG triplets at the 5'-end available as template for extending the primer 3'-end by n CAG triplets; An + (CAG)n, blunt-end hairpin formed by extending An by n CAG triplets with DNA polymerase.
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REFERENCES |
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