Báo cáo khoa học: Determination of the reopening temperature of a DNA hairpin structure in vitro pptx

Determination of the reopening temperature of a DNA hairpin
structure
in vitro
Xuefeng Pan
Institute of Microbiology, The Chinese Academy of Sciences, Beijing, China
A novel method, based upon primer extension, has been
developed for measuring the reopening temperature of a
single type of DNA hairpin structure. Two DNA oligo-
nucleotides have been utilized and designated as primers 1
and2.Primer1,withits5-and3¢-termini fully comple-
mentary to the hairpin flanking sequences, was used to
evaluate primer extension conditions, and primer 2, with its
3¢-end competing with the DNA hairpin stem, was used to
detect the DNA hairpin reopening temperature. A single
DNA hairpin structure was formed on the DNA template
by thermal denaturation and renaturation, and this hairpin
structure w as p redicted to prevent the annealing of the 3¢-end
of primer 2 with the template DNA, which leads to no pri-
mer extension. By incubating at different temperatures, the
DNA hairpin structure can be reopened at a particular
temperature where the primer extension can be carried out.
This resulted in the a ppearance of double-stranded DNA
that was detected on an agarose gel. This temperature is
defined here as the hairpin reopening temperature.
Keywords: DNA hairpin; non-B DNA secondary structure;
primer extension; reopening temperature; T
m
.
The significance of DN A folding into non-B secondary
structures (e.g. pseudohairpin, hairpin, palindromic, tri-
plex and G-tertraplex DNA molecules) is twofold.

Firstly, DNA non-B secondary structures play important
physiological roles. For example, some GC-rich DNA
sequences, that fold into G-tertraplex structures, partici-
pate in the regulation of gene expression [1] and the
maintenance of telomere structures [2–4], and some AT-
rich DNA sequences in the DNA replication origins of
bacterial plasmids that can adopt non-H bounded
conformations control DNA replication initiation [5].
Moreover, certain types of non-B DNA conformations
may also b e n eeded for the proper organization of
genetic material in chromosomes [6]. Secondly, non-B
DNA secondary structures can be aberrant DNA folds,
increasing the likelihood of genomic instability in DNA
replication, transcription, recombination or repair [7,8].
For example, non-B DNA secondary structures formed
on the lagging strand template of a DNA replication
fork have been found to increase the probability of
DNA replication impairment, and enhance DNA rear-
rangements through recombination and repair [8].
Recently, some trinucleotide repeats in the human
genome (e.g. CAG, CGG, GAA, and CGA) have been
found to generate repeat expansion and contraction
instabilities, responsible for the occurrence of more than
14 human genetic diseases a nd cancers [9–14]. Interest-
ingly, most of these disease-causing trinucleotide repeats
have been demonstrated to be capable of forming non-B
DNA secondary structures, such as hairpins, pseudohair-
pins, triplex and G-tertraplex DNA molecules in vitro,
which have been proposed to serve as intermediates for
producing expansion and contraction instabilities v ia

DNA replication, recombination or repair [10–12].
Moreover, in some DNA and RNA related molecular
experimental manipulations, such as DNA amplification by
PCR, primer extension on a DNA or RNA template, DNA
sequencing, and site-directed mutagenesis, various effects of
non-B DNA secondary structure formation have also been
reported [15–17]. These nucleic acid manipulations are
closely related to molecular hybridization of DNA or RNA
molecules, or DNA replication, reverse transcription and
RNA transcription, during which DNA or RNA molecules
should be unfolded [18]. Any folded DNA or RNA
conformation, if remaining unden atured o r reformed
through a reannealing step, may interfere with the recog-
nition of the molecules, o r affect the subsequent DNA
polymerization, RNA reverse transcription into DNA, or
RNA transcription [18].
All the replicative DNA polymerases so far character-
ized use single-stranded DNA as a template. However,
some DNA polymerases, such as the DNA polymerase of
bacteriophage U29 and thermostable Bacillus stearother-
mophilus DNA polymerase etc., have DNA double-strand
displacement activity, and enable the double-stranded
DNA segment in a non-B DNA structure (e.g. a DNA
hairpin) to open during DNA replication [19–21]. These
DNA polymerases can either remove a DNA hairpin
structure through their double-strand displacement activ-
ities or be stalled by the DNA hairpin structure [21,22].
In the latter case, the stalled DNA polymerase leaves a
3¢-end on the growing strand, which m ay subsequently
search out a short region of homology along the nearby

downstream template and allow the DNA polymerase to
re-assemble at the 3¢-end and continue the DNA
replication (strand-slippage) [21–24].
Correspondence to X. Pan, Institute of Microbiology, The Chinese
Academy of Sciences, Beijing 100080, China.
E-mail: xpan@staffmail.ed.ac.uk or
Abbreviations: RF, replicative form; X-gal, 5-bromo-4-chloroindol-
3-yl b-
D
-galactoside.
(Received 5 March 2004, revised 5 July 2004, accepted 23 July 2004)
Eur. J. Biochem. 271, 3665–3670 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04301.x
The stability of a non-B DNA conformation (e.g. DNA
hairpin structure) can be characterized by its melting
temperature ( T
m
). Determination o f T
m
is essential f or
many nucleic acid-related experimental manipulations, such
as primer extensions with DNA or RNA, DNA or RNA
hybridization and oligonucleotide DNA primer design
[16,18,25,26]. So far, experimental methods, such as UV,
circular dichroism and NMR, and theoretical calculations,
such as nearest-neighbour analysis, have been widely used to
obtain thermodynamic parameters, including the T
m
,andto
detect nucleic acid structural transitions [27–35]. Amongst
these, UV spectroscopy is the most common experimental

method. H owever, under certain circumstances where the
critical transitions between DNA folding and unfolding are
invisible to UV [30], alternative m ethods, such as circular
dichroism, are required. On the other hand, the thermody-
namic parameters obtained by experimental methods reflect
only overall information on a DNA or RNA population, not
a local one (e.g. a partial sequence of a DNA or RNA
molecule [18,26]). Although theoretical calculations, such as
nearest-neighbour analysis, can be applied for a local
sequence analysis (e.g. of a single hairpin structure) it will
become inaccurate for certain DNA hairpin loops. For
example, a CG closing base pair enhances stability over o ther
closing base pairs and cannot be explained by the current
nearest-neighbour model [27–29,33,35]. A n experimental
method for m easuring a single type of DNA hairpin’s
reopening temperature embedded in a DNA molecule has
been developed in this work. The method utilizes the
knowledge of DNA hairpin structure formation in vitro
and the effects of DNA hairpin structure on a s ite-specific
oligonucleotide DNA-mediated primer extension reaction.
Materials and methods
Bacterial strains, media, DNA and biochemicals
E. coli strains TG1 [SupE hsdD5 thi D(lac-proAB)F¢]and
JM101 [F¢ traD36 lacIq D(lacZ)M15 proAB]wereusedin
this work [18,26]. Luria–Bertani broth was used for the
bacterial cultivation and M9 minimal medium was used
to maintain F¢ factors in E. coli strains [18]. All
manipulations were following standard methods [18],
unless indicated.
Plasmid pTac5 [37] and M13mp19 [18] were stocks of this

laboratory; the M13 single-stranded DNA construct was
the M13-E-E derivative with a 102 bp DNA deletion in an
inverted repeat region [38]. Oligonucleotide primer 1:
5¢-CCACCCTCG*T*C*GGCCAC-3¢ (where asterisks
refer to mismatched bases and G*T*C* is the marker);
primer 2: 5¢-ATGGCCTGAG*AGCCACCC-3¢ (G* as the
marker); and primer 3: 5¢-TCAG*AGGCCACAAACCA
CAC-3¢ (G* as the marke r) w ere s ynthesized using an
Applied Biosystems DNA synthesiser. The markers indica-
ted in each oligonucleotide primer were applied when DNA
sequencing to confirm the primer extension products.
Restriction enzymes, Eco RI, PstI, BamHI, T4 DNA ligase,
T4 DNA polymerase, and isopropyl thio- b-
D
-galactoside, 5-
bromo-4-chloroindol-3-yl b-
D
-galactoside ( X-gal) stock
solutions were purchased from Promega ( Beijing, China).
dNTPs, ATP and dithiothreitol were from Biochemicals
(San Diego, CA, USA).
Subcloning the template DNA and prepare ssDNA
Cloning of an 800 bp sequence from a prochymosin
expression plasmid p Tac5 onto M13mp19 was carried out
as the follows: pTac5 was digested by EcoRI restriction
enzyme, and the 800 bp EcoRI fragment was recovered by
using the low-melting agarose gel method [18]. This EcoRI
fragment was then subcloned into the EcoRI site in
M13mp19. The ligated DNA was used to transform TG1
competent cells as prepared by a CaCl

2
method [18].
Replicative form (RF) DNA from the white plaques as
selected on Luria–Bertani agarose plates containing isopro-
pyl thio-b-
D
-galactoside and X-gal was isolated, and the
orientation of the insert in M13mp19 was determined by
PstI digestion. Single-stranded M13 DNA was prepared as
described by Sambrook et al. [18].
Annealing of the primer–DNA template and primer
extension analysis
Formation of the primer–single-strand DNA template and
the subsequent primer extension w ere performed based on
the method established by Kunkel [18,24], modified as
follows: primer was mixed with the dUTP-containing M13-
E-E ssDNA (extracted from RZ1032) at a ratio of 3 : 1 in
annealing buffer (10·) containing 200 m
M
Tris/Cl (pH 7.5),
20 m
M
MgCl
2
,500m
M
NaCl, respectively. These mixtures
were then kept at 70 °C for 5 mins and cooled to 12 °C,
22 °Cand30°C, respectively, in two different ways. One
way was to allow the annealing reaction to proceed at room

temperature, allowing slow annealing to 12 °C, 22 °Cand
30 °C, respectively. The other was to transfer the annealing
reaction into a waterbath held at 12 °C, 22 °Cor30°Cafter
the denaturing reaction at 70 °C (fast annealing), and then
store on ice. Primer extensions (second strand synthesis)
were carried out in a 20 lL reaction by adding buffer (10·)
containing 5 m
M
of each dNTP, 10 m
M
ATP, 100 m
M
Tris/
Cl (pH 8.0), 50 m
M
MgCl
2
,20m
M
dithiothreitol, one unit
of T4 DNA polymerase and three units of T4 DNA ligase,
and incubating at different temperatures for 90 mins. The
primer extension products (synthesized double-stranded RF
DNA) were analysed by running agarose gels.
Plating the M13 bacteriophage and DNA sequencing
To further confirm that the RF DNA was produced
through the DNA oligonucleotide primed primer extension
reaction, TG1 c ompetent cells prepared with the CaCl
2
method [18] were transformed with RF DNA from each

primer extension reaction. M13 bacteriophage carrying
primer 2: 5¢-ATGGCCTGAG*AGCCACCC-3¢ were pla-
ted on E. coli TG1 and the mutants were screened by DNA
sequencing of the DNA i n the plaques by t he method
described in [18]. The oligonu cleotide 5¢-GGTTGTC
GGCGTCGATAATCAAACT-3¢ was used as the sequen-
cing primer.
Determination of the hairpin reopening temperature
Oligonucleotide primer 2 was mixed with single-stranded
template DNA at a ratio of 3 : 1 in the annealing buffer.
This mixture was denatured at 70 °C for 5 mins, and then
3666 X. Pan (Eur. J. Biochem. 271) Ó FEBS 2004
slowly cooled to 12 °C (slow annealing). After these
treatments, one unit of T4 DNA polymerase was added
and the mixture was equally divided into a liquots that were
incubated at different temperatures for primer extension.
Following 90 mins of primer extension each aliquot was
analysed by agarose gel electrophoresis.
Results and Discussion
Experimental rationale
In order t o determine the r eopening temperature of an
individual secondary structure in a single DNA molecule
(Fig. 1 A,B), knowledge of DNA replication, DNA
folding and oligonucleotide-mediated primer extension
has been a pplied to establish a method. The molec ular
mechanism underlying the method is explained in
Fig. 1B,C. As can be seen in Fig. 1B,C, a DNA hairpin
structure can be adopted by a small region in the
template DNA sequence. Such a hairpin structure was
designed to abolish primer extension by interfering with

the annealing of the 3¢ bases of the DNA oligonucleotide
primer with the template DNA (Fig. 1B). However,
primer extension is possible as long as the hairpin
structure is melted and the DNA primer can anneal with
its complementary region in the DNA template. As can
be seen in Fig. 1A, two primers were d esigned as follows:
primer 1, with its 5 ¢-and3¢-ends fully complementary to
the hairpin flanking sequences, was used to monitor the
primer extension reaction conditions; and primer 2, with
its 3¢-terminus located in the hairpin stem, was used to
detect the DNA hairpin reo pening temperature (Fig. 1C).
It was expected that the primer–template annealing and
subsequent extension reactions from the primer 1–DNA
template hybrids could be used a s positive controls to
establish the conditions of the DNA polymerase-cata-
lysed primer extension reactions. Under the same condi-
tions, if the primer 1–DNA template hybrid appeared to
be extended by DNA polymerase while the primer
2–DNA template hybrid appeared not to be, i t could be
inferred that the failure of the primer 2–DNA template
hybrid primer extension reaction was due to the effects
of the hairpin structure on the process of primer 2
annealing to the DNA template.
Analysis of secondary structure formation by thermal
denaturation, renaturation and primer extension
To determine whether the expected secondary structure
can o r cannot f orm through denaturation and renatur-
ation manipulations, the fast annealing (fast renaturation)
and slow annealing (slow renaturation) procedures
(Materials and methods) were performed after the primer

2 and th e single-stranded DNA template mixture were
Fig. 1. Organization and the working mech-
anism. (A) DNA template and potential hair-
pin structu res (free energy va lues labelled were
computed by a program [25]), and the loca-
tions of the primer 1, 2 and 3 pairing. (B)
Paring of primer 2 with the template when a
hairpin structure has been formed through
denaturation and renaturation (free energy
value labelled was computed by a program
[25]). (C) Illustration of the working mechan-
ism for measuring the reopening temperature
of a small DNA hairpin structure.
Ó FEBS 2004 Reopening temperature of single DNA hairpin (Eur. J. Biochem. 271) 3667
denatured at 70 °C for 5 mins. Primer exte nsion r eactions
were then started by adding one unit of T4 DNA
polymerase to these renaturation mixtures. The primer
extension products were compared by agarose gel
electrophoresis. As can be seen in Fig. 2A, the slow
renaturation reaction produced smeared DNA products
(Fig. 2 A, lane 1), while the f ast annealing reaction
showed two dominant bands (Fig. 2A, lane 3), indicating
that the p rimer extensions with primer 2 produce
different products as a function of the different annealing
manipulations. This suggested that the conformations of
the template DNA formed after t he two different
annealing reactions were different. Heteroduplex DNA
formed by p rimer 2 and the template DNA generated
through fast annealing to 22 °C seemed to allow the T4
DNA polymerase to synthesize double-stranded DNA

strands more completely, while the heteroduplex DNA
generated through slow annealing to 22 °Cpreventedthe
T4 DNA polymerase from carrying out primer extension
at 22 °C. Fast annealing may decrease the likelihood of
the self-folding of the template DNA, while it may
increase the probability of primer 2 binding to the
template DNA. By contrast, slow annealing with primer
2 may form incompletely paired heteroduplex DNA as
shown in F ig. 1B. Under this condition, the two ÔGGÕ
bases i n t he template may b e unavailable due to
formation o f a hairpin structure, w hich leaves the two
ÔCCÕ bases at the 3¢-end of primer 2 unpaired.
To further confirm that the problem encountered for
primer 2 mediated primer extension was due to the effects of
template DNA folding (forming a hairpin structure),
annealing reactions between primer 1 and single-stranded
DNA were also carried out using the slow annealing
manipulation. As expected, heteroduplex molecules formed
by primer 1 and the template DNA allowed T4 DNA
polymerase to produce double-stranded DNA, irrespective
of the a nnealing temperatures ( data not shown). More
impressively, this DNA re plication was detected when the
reacting temperature was as low a s 12 °C (Fig. 3, lane 3),
while in the same situation, the primer 2–single-strand DNA
template cannot be used by T4 DNA polymerase to
synthesise any double-stranded DNA products (Fig. 3, lane
5). In addition to primer 1 extension, primer 3 [designed to
prevent formation of the h airpin structure (Fig. 1A)] can
also enable primer extension to proceed under either slow
(Fig. 2 B, lane 3) or fast (Fig. 2B, lanes 2 and 4) annealing.

These data taken together indicate that the hairpin-forming
region (as indicated in Fig. 1A) can indeed affect primer 2
DNA primer extension, most likely due to hairpin structure
formation (Fig. 1B).
Measuring the reopening temperature for a single type
of DNA hairpin
As can be seen in Fig. 3, primer extension with primer 1 can
produce e xtended double-stranded DNA products (Fig. 3,
lane 3), but primer 2 cannot (Fig. 3 , lane 5). When
considering that these two reactions used the same conditions
and only differed in the primer DNA, it is reasonable to
believe that the f ailure of primer 2-mediated primer extension
was simply due to hairpin structure formation on the
template DNA, which left the ÔCCÕ bases of primer 2
Ôflapping Õ (Fig. 1B). The DNA conformation depicted in
Fig. 1B cannot be used as template by the DNA polymerase
to synthesize the second strand DNA due to the unpaired
3¢-end of the primer, until the ÔGGÕ bases in the hairpin stem
are freed by elevating the reaction temperature and paired
with the ÔCCÕ bases at the 3¢-end of primer 2. In this work, the
reopening of the hairpin structure and the subsequent primer
extensions were attempted by dividing the reaction into
aliquots, and incubating at different temperatures. The
results of these manipulations are presented in Fig. 4. As can
be seen in Fig. 4, primer extension products were detected
when the reaction temperature (reopening and extension
temperature) was above 19 °C. The appearance of the
12345
1234 5
6

A
B
Fig. 2. Primer extension reactions with primer 2 and primer 3 under fast
and slow annealing conditions. (A) Primer extension with primer 2
under slow and fast annealing conditions: lanes 1 and 2, primer
extension product and primer-free control under slow anne aling; lanes
3 and 4, primer exte nsion product and primer-free control under fast
annealing; lane 5, ssDNA control. (B) Primer extension product with
primer 3 unde r slow and fast annealing: lane 1, RF DNA con trol; lanes
2 and 4, primer extension products with p rimer 3 under fast annealing;
lane 3, the primer extension product under slow annealing with ATP;
lane 5, primer-free control and lane 6, the ssDNA template control.
1234567
RFDNA
Products
RNA
Fig. 3. Primer extension reactions with primer 1 and primer 2. Primer
extension products using primer 1 and primer 2 have been indicated.
Lanes1and2,RFDNAcontrols;lane3,primerextensionwithprimer
1at12 °C u nder slow annealing; lane 4, the primer 1-free cont rol; lan e
5, primer extension with primer 2 at 12 °C under slow annealing; lane
6, primer 2-free cont rol; lane 7, single-stranded DNA template control.
3668 X. Pan (Eur. J. Biochem. 271) Ó FEBS 2004
double-stranded DNA products on the agarose gel was taken
to indicate the temperature at which the hairpin structure had
been reopened and where the 3 ¢ ÔCCÕ of p rimer 2 had formed
a fully hydrogen bonded heteroduplex with the single-
stranded DNA template. Further confirmation th at the
double-stranded DNA molecules seen in Figs 2B, 3 and 4
weremadethroughprimer1,2and3-mediatedprimer

extensions was obtained by transformation. E. coli TG1
competent cells were transformed with these double-stran-
ded DNA products and single-stranded DNA molecules
isolated from the M13 plaques were sequenced (data not
shown) to check the DNA markers carried by primers 1, 2
and 3.
Comparison between the experimentally obtained
reopening temperature and the
T
m
values with the
nearest-neighbour thermodynamic calculation
As indicated in Fig. 4, the reopening temperature of the
hairpin (Fig. 1B) was  19 °C. This temperature has been
compared with the T
m
values calculated b y using a nearest-
neighbour thermodynamics based software [25,39]. The
theoretical T
m
, when calculated based on the folding
temperatures of 12 °C, 19 °C, 22 °Cand37°C, and a
DNA folding condition of 1.0
M
Na
+
,are27.5°C, 27.9 °C,
28.7 °C and 28.1 °C, respectively. H owever, as the actual
experimental DNA folding concentration of Na
+

is 50 m
M
,
the correspo nding T
m
calculated at those folding temper-
atures are 20.4 °C, 21 °C, 19.9 °Cand20.2°C, respectively,
which are fairly close to the experimentally obtained
reopening temperatures.
Conclusion
Based on the effects of a hairpin structure formed in
template DNA on DNA primer annealing and DNA
polymerase-catalysed primer extension, a novel method for
measuring the reopening temperature for a single type of
hairpin structure in DNA has been established. The
reopening temperature obtained e xperimentally was fairly
close to the T
m
values obtained by a nearest-neighbour
thermodynamics calculation, suggesting that it can be useful
for evaluating the reopening temperature for a DNA
hairpin in a local region of DNA, and for comparing the
reopening temperatures for a group of hairpin structures
when the reaction system is defined.
Acknowledgements
This work was supported in part by a grant from The National
High Tech Program of China. The author is very much grateful to
Professor David Leach, the University of Edinburgh for constructive
suggestions, comments and the correction for the manuscript, and
those people at Institute of Microbiology, The Chinese Academy of

Sciences for help.
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Fig. 4. Measuring the re ope ning tempera ture. Annealed primer 2 and
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