Download Ligation mediated PCR performed at low denaturation temperatures

Nucleic Acids Research, 2003, Vol. 31, No. 18 e114
DOI: 10.1093/nar/gng116
Ligation mediated PCR performed at low
denaturation temperaturesÐPCR melting pro®les
Aleksander Masny* and Andrzej Pøucienniczak
Institute of Biotechnology and Antibiotics, Staroscinska 5, 02-516 Warsaw, Poland
Received June 18, 2003; Revised and Accepted August 2, 2003
ABSTRACT
We show that using low denaturation temperatures
(80±88oC) during ligation mediated PCR (LM PCR) of
bacterial DNA leads to the ampli®cation of limited
sets of the less stable DNA fragments. A set of
electrophoretic patterns of such fragments obtained
at different denaturation temperatures forms the
PCR melting pro®le (PCR MP). A single pattern
obtained for a given temperature and a set of patterns arising after application of several denaturation temperatures (PCR MP) are very speci®c for
the given bacterial genome and may be used
for strain characterisation and differentiation. The
method may also be used for ampli®cation and
isolation of the less stable DNA fragments in a
genome.
INTRODUCTION
Melting temperatures of genomic DNA fragments obtained by
digestion of restriction nuclease depends on their GC content
and length. It is known that intervals of temperatures
corresponding to the full transition from double- to singlestranded structure for a restriction DNA fragment may vary
from ~0.5°C to several degrees depending on GC content
distribution along the DNA fragment (1). From DNA
sequencing projects it is known that bacterial genomes show
substantial genomic heterogeneity in GC content (2,3). It
means that after digestion with restriction nuclease of bacterial
genomic DNA, fragments, which arise as a result of the
digestion, have different thermal stabilities. This feature may
be used to obtain sets of electrophoretic patterns of DNA
fragments ampli®ed during ligation mediated PCR (LM PCR)
(4) performed at various denaturation temperaturesÐPCR
melting pro®les (PCR MPs). PCR MPs are characteristic for a
given genome and restriction nuclease applied for digestion.
During PCR, denaturation temperatures (Td) around 94±95°C
are applied with the aim to achieve full separation of DNA
strands of all DNA fragments present in the ampli®ed sample.
Under such conditions, during LM PCR, all DNA fragments in
the sample should be ampli®ed. Lowering of Td applied during
LM PCR should decrease the number of ampli®ed fragments
because only single-stranded DNA molecules may serve as a
template during LM PCR. In this work, we show that using
low Td during LM PCR leads to limited and speci®c
ampli®cation of a small number of the less stable DNA
fragments. The electrophoretic patterns of DNA fragments
obtained after such ampli®cations are characteristic for the
bacterial strain taken for DNA isolation. The set of electrophoretic patterns (PCR MP) obtained at different Td values for
a given bacterial strain is a very speci®c ®ngerprint which may
be used for strain characterisation and differentiation because
the order of appearance of ampli®ed DNA fragments in
subsequent increasing denaturation temperatures (Td) is stable
for a given genomic DNA. The outline of the proposed method
is shown in Figure 1. The application of thermal stability of
the DNA restriction fragments as the selecting factor for the
creation of limited DNA representations seems to be a
completely new approach to the target template selection in
restriction site polymorphism based PCR ®ngerprinting
methods. Techniques such as AFLP (5) and other LM PCR
based methods (6,7) rely on the mutual distribution of two or
more relatively short DNA sequences within the distance
enabling ef®cient PCR ampli®cation. In contrast to the above
mentioned methods, PCR MP does not rely exclusively on the
distribution of two short DNA sequences within the PCR
ampli®cation range, because from the pool of DNA fragments
of similar length only the less stable ones are ampli®ed. For
this reason, PCR MP allows application of single, relatively
frequent cutter restriction nuclease without de®ning any
portion of the DNA sequence adjacent to a restriction site.
MATERIALS AND METHODS
Bacterial strains
The following bacterial strains were used: Escherichia coli:
K-12 MG 1655 (3), OM12, OM72, OM16, Klebsiella oxytoca
OM 36, 22 and Klebsiella pneumoniae OM 42 and OM 86.
Strains designated OM were isolated from patients in one
hospital and all of them bear the INT37CZD integron
(accession number AF282595).
Converting entire genome to amplicons and melting
selective ampli®cation
From 3 ml overnight cultures of bacterial strains, 1.5 ml was
centrifuged at 1200 g for 2 min and DNA was isolated from
bacterial pellets using a protocol originally applied to DNA
isolation from blood, urine and cerebrospinal ¯uid (8), the
version with diatom suspension, only the last step of the
procedure was modi®ed: 150 ml of deionised water was added
to elute DNA after the isolation procedure; 1 ml of DNA
*To whom correspondence should be addressed. Tel: +48 22 849 60 51; Fax: +48 22 849 33 32; Email: [email protected]
Nucleic Acids Research, Vol. 31 No. 18 ã Oxford University Press 2003; all rights reserved
e114 Nucleic Acids Research, 2003, Vol. 31, No. 18
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Figure 1. Principle of the method. The thick black line represents a melting curve of a genomic DNA. Vertical and horizontal axes represent the percentage
of ssDNA and denaturation temperature (Td), respectively. Range a: sequences potentially ampli®able by PCR are not presentÐall DNA fragments remain
double-stranded and primers cannot bind. Range b: some DNA particles are single-stranded (grey shaded) and primers can bindÐPCR ampli®cation occurs.
Range c: majority of DNA particles are ssDNA thus are ampli®able, and at the end of the range all DNA fragments are ssDNAÐand are ampli®able. The
working range is from the right part of range a to the left part of range c and is limited by the lowest Td where a PCR occurs and the Td where the number of
PCR products reaches the border of the resolution capacity of polyacrylamide gels, respectively. PCR primers are of equal length as terminal complementary
sequences within every single strand and compared to ligated oligonucleotides are extended by a protruding end sequence and the remaining part of the
restriction site on their 5¢ ends. Terminal complementary sequences are created by ®lling in ssDNA termini obtained by ligation of oligonucleotides to
protruding 5¢ ends of restriction fragments.
solution was electrophoresed on 0.8% (w/v) agarose gels with
TAE buffer and subsequently stained in 0.5 mg/l ethidium
bromide solution for 10 min, DNA concentration was
estimated by comparison to reference DNA. The DNA
concentration was from ~100 ng/ml to several hundred ng/ml
in distinct samples. Genomic DNA was digested with HindIII
(10 U/ml). Digestion reactions were performed under uniform
conditions; 500 ng of DNA was dissolved in 30 ml buffer H
(50 mM Tris±HCl, pH 7.5, 10 mM MgCl2, 100 mM KAc,
1 mM DTE; Roche Molecular Biochemicals), 1 ml of the
endonuclease was added, after 2 h another 1 ml of the
endonuclease was added and the reaction was continued for 2 h
at 37°C. After digestion, the volume was adjusted to 100 ml
with TE, 100 ml of phenol pH 7.8 (9) was added, samples were
vortex mixed and centrifuged for 5 min at 12 000 g. After
centrifugation, 100 ml of aqueous phase was transferred into
new Eppendorf tubes and equal volumes of chloroform±
isoamyl alcohol (24:1) were added. Samples were vortex
mixed and centrifuged for 5 min at 12 000 g. Chloroform±
isoamyl alcohol extraction was repeated and the aqueous
phase was transferred into new tubes. Subsequently, 15 ml of
3 M sodium acetate and 3 ml of glycogene 20 mg/ml were
added, samples were mixed and 300 ml of 96% ethanol was
added. The samples were mixed again and incubated for
10 min on dry ice and centrifuged for 10 min at 12 000 g. The
pellets were washed with 200 ml of 75% ethanol for 5 min,
then centrifuged for 3 min at 12 000 g. Dry pellets of digested
DNA were dissolved in ligation mix comprising: two
oligonucleotides forming an adaptor, POWIE 5¢-CTCACTCTCACCAACGTCGAC-3¢ and HINLIG 5¢-AGCTGTCGACGTTGG-3¢, 20 pmol each, in total volume of 20 ml
ligation buffer (66 mM Tris±HCl, pH 8.5, 6.6 mM MgCl2,
10 mM DTT, 66 mM ATP; Amersham Pharmacia Biotech).
The mixture was heated in a water bath for 2 min at 56°C and
cooled for 10 min at room temperature. Subsequently, 1 ml of
T4 DNA ligase 1 U/ml was added and the samples were
incubated overnight at 16°C.
The PCR mixture contained: 50 pmol of POWAGCTT
primer 5¢-CTCACTCTCACCAACGTCGACAGCTT-3¢,
100 mmol each dNTPs and 1 ml of ligation reaction products
in a total volume of 50 ml of PCR buffer (50 mM KCl, 20 mM
Tris pH 8.9, 2.5 mM MgCl2, 0.01% gelatine). In an Eppendorf
thermal cycler (Mastercyclerâ 5330) PCRs were performed
as follows: 2 min at 72°C to release unligated oligonucleotides
HINLIG, subsequently 1 ml 2.5 U/ml of Taq DNA polymerase
(10) was added and mixed with a pipette tip. The reaction mix
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Nucleic Acids Research, 2003, Vol. 31, No. 18 e114
Figure 2. PCR MPs of E.coli K-12 MG1655 DNA. PCR products from two independent experiments (as de®ned in Materials and Methods) were loaded to
adjacent wells. Pairs of adjacent lanes are described with Td applied in PCR. Lane MÐDNA molecular weight marker; fragment lengths in bp are indicated.
(a) Eppendorf Mastercycler 5330 and (b) PTC-200 MJ Research cycler were used for ampli®cation. In the case of the Eppendorf cycler, 18 independent
experiments were carried out. In the case of (b), a Td gradient of 84±88°C was used in two independent experiments.
was incubated for an additional 5 min at 72°C to ®ll in the
single-stranded ends and create amplicons, followed by initial
denaturation at (80 + x)°C for 1 min and 21 cycles of
denaturation at (80 + x)°C for 40 s, annealing and elongation at
72°C for 2 min (where x is a digit between 0 and 8 and is
constant for a single PCR reaction). After the last cycle,
samples were incubated for 5 min at 72°C. PCRs were
performed in 500 ml tubes and the reaction mix was covered
with mineral oil. Primer annealing temperatures were calculated with the Premier Primer on-line applet and those
temperatures were used as the starting point for the annealing
temperature optimisation. Alternatively, PCR reactions were
performed in a gradient thermal cycler (MJ Research PTC200
DNA Engine Gradient) with a gradient range of 84±88°C in
200 ml thin walled PCR tubes with PCR mixtures as described
above. Cycler settings: sample type was set to tubes and
sample volume to 75 ml (50 ml reaction mixture and 25 ml oil)
and the heating lid was disabled. PCR reactions were
performed as follows: 2 min at 72°C, subsequently 1 ml
2.5 U/ml of Taq DNA polymerase was added and mixed with a
pipette tip. The reaction mix was incubated at 72°C for an
additional 5 min followed by initial denaturation at a gradient
across the thermal block of 84±88°C for 1 min 30 s and 21
cycles of denaturation at a gradient across the thermal block of
84±88°C for 1 min, annealing and elongation at 72°C for 2 min
15 s and 5 min at 72°C after the last cycle. Identical
experiments were performed at gradients of 85±87°C and
88±95°C. Reproducibility was tested in independent runs of
the cyclers. By independent runs, we mean two completely
independent experiments starting from DNA isolation. The
runs were performed in the second and fourth rows of
the gradient thermal cycler block. In the Eppendorf
Mastercyclerâ 5330, all the runs were performed in the
same position of one thermal block.
Gel electrophoresis of PCR products
PCR products, 8 ml out of 50 ml, were electrophoresed on
6% polyacrylamide gels (60:1 acrylamide/N,N-methylene
e114 Nucleic Acids Research, 2003, Vol. 31, No. 18
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Figure 3. Comparison of PCR MPs of two E.coli strains. (A) Clinical strain OM12 and (B) K-12 MG1655. Td gradients of 84±88°C were performed using
the PTC-200 MJ Research cycler. Lanes M, DNA molecular weight marker; fragment lengths in bp are indicated.
bis-acrylamide) with TAE buffer, stained in ethidium bromide
0.5 mg/l aqueous solution for 10±15 min. Images of the gels
were made using a White/Ultraviolet Transilluminator (UVP).
RESULTS AND DISCUSSION
PCR MP for E.coli strain K-12 MG1655 is shown in Figure 2.
The number of DNA fragments ampli®ed during LM PCR
depends on Td used in the denaturation step of the ampli®cation reaction. The comparison of the results of two
experiments shows that there are small differences between
the independent runs. The reason for this may be ¯uctuations
of temperature and salt concentration during independent
experiments. Analysis of many patterns like that shown in
Figure 3 leads to the conclusion that the most stable results for
E.coli strains are obtained when Td is in the range 87±88°C.
The resolution power of PCR MP patterns for bacterial strain
differentiation is illustrated in Figures 3 and 4. In Figure 3,
complete PCR MPs for two E.coli strains are compared.
Differences between the strains are visible for every Td applied
to obtain the pro®les. In Figure 4, it is seen that a very ef®cient
strain differentiation may also be carried out at a single Td
(88°C). In this case, different strains of E.coli and Klebsiella
were compared.
Optimal gradient range on a gradient cycler and
reproducibility of the results
The optimal gradient range should be established experimentally for the strains subjected to analysis and using a particular
thermal cycler. We consider the gradient range 84±88°C to be
optimal for differentiation of the analysed Enterobacteriaceae
strains, because it ensures good reproducibility and satisfactory differentiation ef®ciency. A steady increase in the number
of ampli®ed DNA fragments, which is dependent on Td
increase, is observed (Figs 2b and 5a). Surprisingly, a
Figure 4. Comparison of electrophoretic patterns obtained after LM PCR
for different E.coli and Klebsiella strains carried out at a single Td = 88°C.
Two independent experiments were performed using the PTC-200 MJ
Research cycler. Lanes M, DNA molecular weight marker; fragment lengths
in bp are indicated; the remaining lanes are described with OM series strain
numbers as follows: K.pneumoniae (42, 86), K.oxytoca (36, 22), E.coli
clinical strains (16, 72, 12) and E.coli K-12 MG1655 (MG).
relatively high rate of reproducibility is obtained for the Td
interval equal to 2°C between 85 and 87°C (Fig. 5b) despite
the fact that the differences between Td in adjacent tubes are
smaller than the accuracy of the cycler which is supposed to be
60.4°C. PCR MPs with a Td upper limit equal to 95°C are not
very useful for bacteria ®ngerprinting because of high
background and lower reproducibility. The upper temperature
gradient range limit, where reproducible results are obtained,
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Nucleic Acids Research, 2003, Vol. 31, No. 18 e114
Figure 5. Comparison of PCR MPs of E.coli MG 1655 performed at a temperature gradient of 84±88°C (a) and 85±87°C (b). PCR products from two independent experiments (as de®ned in Materials and Methods) were loaded to adjacent wells. Pairs of adjacent lanes are described with Td applied. Instability occurs only at 87.0°C at a temperature gradient of 84±88°C (a), and in the gradient range 85±87°C at temperatures: 85.6, 86.2, 87.0°C (b). Results were
obtained on another MJ Research PTC-200 gradient cycler unit than those presented in Figures 2b and 3B. Some DNA bands are missing on (a) lanes 88.0°C
compared to Figures 2b and 3B, lanes 88.0°C; however, they occur when higher Td values are applied (Fig. 6a, lane 88.6°C).
may depend on the particular cycler properties. The results
obtained at Td 88.0°C with the ®rst PTC-200 gradient thermal
cycler tested (Fig. 2b) differ by two DNA bands from the
results obtained at Td 88.0°C with the second cycler (Fig. 5a).
However, those two missing bands occurred at a Td gradient of
88±95°C at Td 88.6°C in the experiment performed with the
second cycler (Fig. 6a). The shortest DNA fragment ampli®ed
in PCR conducted with the second MJ Research PTC-200
gradient cycler at Td 85.8°C (Fig. 5a), occurs at Td 85.2°C in
PCR MP performed at the same Td gradient (84±88°C) on the
®rst of the tested cyclers (Fig. 2b). Variability of the results
obtained with two separate PTC-200 cyclers probably resulted
from a shift in calibration by at least 0.6°C.
On the other hand, for some DNA fragments, the transition
temperature from dsDNA to ssDNA may be as low as 0.5°C
(1). The temperature of the thermal block wells may differ by
0.4°C from the nominal temperature. For this reason, some
DNA fragments do not melt in every cycle of PCR. For
example in PCR MP performed at a gradient of 84±88°C, with
the ®rst PTC-200 cycler, at Td 87.8°C one unstable band
occurred which did not occur at Td 87.4°C but was ampli®ed
at 88.0°C (Fig. 2B). The most probable causes of this
phenomenon were temperature ¯uctuations within the cycler
accuracy range. For the reasons presented above, some
instability of the results should be expected at particular
Td values. Td values where high rates of reproducibility
are obtained must be determined experimentally and it is
improbable to establish a gradient where the reproducibility
of the results would be always complete at all Td values.
The second and fourth rows of the thermal block were
tested and the results obtained at de®ned Td values in
independent runs did not depend on the thermal block
row used. It must be emphasized that the order of appearance
of DNA fragments along with an increase in Td values is
always the same and does not depend on the thermal cycler
used (Figs 2b and 5a). Thus the order of appearance of
DNA bands in PCR performed in subsequent increasing
temperatures is invariable for a given genomic DNA and
should be treated as the most important criteria for the
interpretation of results. Reproducibility de®ned as complete
repetitiveness of the results at any Td is not achieved, as
described above, but reproducibility of the results de®ned as
the order of appearance of DNA bands is complete for both
cyclers tested.
We have not tested how polymerases from different sources
in¯uence the results.
e114 Nucleic Acids Research, 2003, Vol. 31, No. 18
PAGE 6 OF 6
interpretation of the results obtained even on an old type
cycler with relatively low accuracy.
CONCLUSIONS
The proposed method allows speci®c gradual ampli®cation of
the genomic DNA differing in the thermal stability starting
from the less stable DNA fragments ampli®ed at lower Td
values to more stable ones ampli®ed at higher Td values.
The PCR MPs are very speci®c ®ngerprints for bacterial
strains and may be used for strain differentiation.
PCR MPs may be obtained very conveniently with PCR
gradient cyclers with the possibility of Td programming across
a heating block.
The order of appearance of DNA fragments at subsequent,
increasing Td values obviously depends on the investigated
DNA properties and is characteristic of a given genomic DNA;
furthermore, the order does not depend on the cycler used.
PCR primers should be longer (extended on their 5¢ ends by
a protruding end sequence and the remaining part of the
restriction site) compared to ligated oligonucleotides, in order
to overcome the PCR suppression phenomenon (11).
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Figure 6. Comparison of PCR MPs of E.coli MG1655 performed at a
temperature gradient of 88±95°C in two independent experiments.
Stability of the results for an old type cycler
We obtained the best reproducibility in particular Td values
applied when independent experiments were performed in the
same position of the thermal block (Fig. 2a) and more variable
results were obtained at individual Td values when distant
wells were used (results not shown). Measurements performed
in Eppendorf instruments revealed that temperatures in distant
wells within the same thermal block can differ by about 1°C at
72 and 96°C. It should be emphasised that the Eppendorf
Mastercyclerâ 5330 is an old type cycler, with an accuracy
range of +0.8/±1.2 at 94°C and 60.6 at 72°C. However, DNA
fragments which were not ampli®ed repetitively at a particular
Td, were ampli®ed in the Td higher by 1°C (Fig. 2a). Thus, the
accuracy of the thermal cycler (Td ¯uctuations) was the most
probable source of variable results in some Td values.
Nevertheless, the order of appearance of the DNA bands
along with the increase in Td is stable, which enables
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