4736-4737 Nucleic Acids Research, 1995, Vol. 23, No. 22
. 1995 Oxford University Press
Prevention of product carry-over by single tube
two-round (ST-2R) PCR: application to BCR-ABL
analysis in chronic myelogenous leukemia
J. Trka+, V. Divoky and T. Lion*
Children's Cancer Research Institute (CCRI), St Anna Kinderspital, Kinderspitalgasse 6, A-1 090 Vienna, Austria
Received August 8, 1995; Revised and Accepted September 14, 1995
False positive results in two-round nested PCR reactions most
frequently originate from carry-over of amplified products. It is
desirable, therefore, to avoid handling of the first round PCR
product prior to amplification with internal primers. Ideally, both
amplifications steps should be performed in a single tube without
having to open it between first and second PCR round. Earlier
approaches to this task have not entirely eliminated problems
such as interference of external primers during the second round
of amplification (1,2). This may result in the formation of
spurious products requiring subsequent hybridization to ensure
specific detection of target sequences. Here we present a simple
two-round PCR protocol which permits highly sensitive and
specific amplification in a single tube, and demonstrate its
application to the detection of leukemic cells carrying a
BCR-ABL rearrangement in patients with chronic myelogenous
leukemia (CML).
Total RNA was extracted from peripheral blood leukocytes of
patients with CML, of a healthy volunteer donor, and from the
CML cell line K562 as described (3). A minimum of 1 gg RNA
was converted to cDNA using M-MLV reverse transcriptase
(Gibco-BRL) in a total volume of 20 ul (3), and 10-100 ng cDNA
were used as template in the PCR reactions. The PCR mixtures
were prepared in 200 pl MicroAmp reaction tubes (Cetus). The
lower phase PCR reaction mix (MIX-1) contained 10 mM
Tris-HCl (pH 9.0 at 250C), 50 mM KCI, 0.1% Triton-X 100, 1.5
mM MgCl2, 200 ,uM of each dNTP, 0.6 U Taq polymerase
(Promega), 10 pmol of the primers 1 and 2 in a total volume of
25 pl. The reaction MIX-I was overlaid with 150 gl silicone oil
displaying a viscosity of 350 mm2/s at 25°C (AK 350; Wacker
Chemie, Munchen, Germany), and the reaction mix with internal
primers (MIX-2) was pipetted onto the oil layer. MIX-2 contained
10 mM Tris-HCl (pH 9.0 at 25°C), 50 mM KCl, 0.1% Triton-X
100, 2.25 mM MgCl2, 300 jM of each dNTP, 2 U Taq
polymerase, 20 pmol of the primers 3 and 4 in a total volume of
50 pl. The primer sequences and their characteristics are shown
in Figure 1.
The aqueous reagent mix submerges spontaneously, and forms
a layer within the silicone oil. PCR cycling was carried out in
Perkin-Elmer 9600 and/or 2400 thermocyclers. The cycling
conditions for the first amplification round were: initial denaturation at 95 0C for 3 min, and in the subsequent cycles at 94.5 °C for
*
3
1.
327 bp
4
Figure 1. Schematic representation of the relative primer positions along the
327 bp BCR-ABL fragment amplified during ST-2R-PCR in patients with the
b3/a2 variant of the rearrangement (4). The melting temperatures (Tm) indicated
were calculated for 50 pM NA (nucleic acid) and I M salt concentrations.
Primer l-Bcr-Ex-S (Tm = 63°C): 5'-TTC AgA AgC TTC TCC CTg-3'
Primer 3-Bcr-Ex-S-long (Tm = 81 C): 5'-TTC AgA AgC ITTC TCC CTg ACA TCC g-3'
Primer 2-Abl-Ex-AS (Tm = 66°C): 5'-CTC CAC Tgg CCA CAA AAT-3'
Primer 4-Abl-Ex-AS-long (Tm = 78°C): 5'-CTC CAC Tgg CCA CAA AAT CAT ACA g-3'
30 s, annealing at 56°C for 30 s and extension at 72°C for 30 s
during a total of 12 cycles. Subsequently, the temperature in the
thermocycler was held at 85°C while individual reaction tubes
were removed, briefly centrifuged at 5000 r.p.m. to merge MIX-I
and -2 to a total volume of 75 pl, and placed back into the heating
block. In the second round of PCR, 37 cycles were performed
with denaturation at 94°C for 30 s, annealing at 69°C for 40 s and
extension at 72°C for 40 s, with a final extension step for 7 min.
The products were electrophoresed in 3% Agarose gels (1.5%
NuSieve-Agarose FMC; 1.5% NA-Agarose, Pharmacia) and
visualized by ethidium bromide staining.
To assess the sensitivity of our protocol, the CML cell line
K562 carrying a BCR-ABL rearrangement was serially diluted
into leukocytes derived from a healthy volunteer donor at ratios
ranging from 1:101 to 1:106. A total of 2 x 107 cells/sample were
processed as described above. In repeated experiments, we were
able to detect cells carrying the BCR-ABL fusion gene at a
sensitivity of 105 (Fig. 2, lane 3). In -50% of the assays
performed, rearranged cells were detected down to a dilution of
106 (Fig. 2, lanes 1 and 2). In a large series of experiments, we
did not see any false positive results in our negative controls as a
consequence of product carry-over.
The number of cycles in the first round of PCR has to provide
a basis for reliable detection of rare target sequences after the
To whom correspondence should be addressed
+Present address: 2nd Department of Pediatrics, University of Prague, Czech Republic
Nucleic Acids Research, 1995, Vol. 23, No. 22 4737
12 3 4 5 6
Figure 2. Sensitivity of the ST-2R-PCR assay, demonstrated on samples
containing different proportions of BCR-ABL rearranged cells. Cells from the
CML cell line K562 serially diluted into normal leukocytes were analyzed by
ST-2R-PCR using the 12 + 37 cycle protocol described in the text. Samples
containing 106 rearranged cells (lanes 1 and 2), showed detectable signals in
-50% of the experiments performed, whereas higher proportions of CML cells
were detectable in all instances. Lane 3, 105 dilution; lane 4, 104 dilution; lane
5, 1i-3 dilution; lane 6, 123 bp ladder.
second round of enzymatic amplification. On the other hand, the
second PCR round should not have excessively high molar
concentrations oftemplate when samples containing large amounts
of specific target molecules are analyzed (5). To determine the
optimum number of cycles in the first round of PCR, 100 ng of
cDNA derived from specimens with different proportions of
BCR-ABL rearranged cells (10-1 and 10-5) were taken out of the
cycler after 3-12 cycles of amplification with first round primers.
Subsequently, all specimens were centrifuged to introduce the
second round primer pair into the reaction, and additional 37 cycles
were performed. The samples containing 10-1 rearranged cells
showed signals of equal intensity regardless of the number of
cycles performed in the first PCR round. The samples with i05
rearranged cells revealed increasing signal intensities corresponding to the number of cycles in the first round (not shown).
To determine the number of cycles in the second round required
for adequate sensitivity, 100 ng of cDNA derived from specimens
with different proportions of BCR-ABL rearranged cells (10-1
and 10-5) were amplified in the first round of PCR for 12 cycles
and the reaction was terminated after a variable number of cycles
in the second round ranging from 5 to 37 (Fig. 3). Our results
indicate that a minimum of 37 cycles in the second round were
necessary to yield a clearly identifiable signal in samples with the
lowest proportion of rearranged cells tested. However, in the
presence of high template concentrations, the reactions reached
a plateau after a lower number of cycles. In the latter case, PCR
cycling beyond the plateau phase has not resulted in the
occurrence of non-specific products.
The principle of our ST-2R-PCR protocol is based on the
physical separation of the reaction mixtures for the first and
second round of PCR by a layer of silicon oil. The amount of oil
used is critical to prevent premature mixing of the first and second
round reagents. The minimum volume of oil needed is dependent
on the diameter of the reaction vessels used. The MicroAmp
reaction tubes require 150 gl of oil to stably separate the PCR
mixes until the reaction tubes are centrifuged. The reaction mix
for the second round (MIX-2) should be rather quickly released
onto the oil layer. If pipetted slowly, small droplets may be formed
which rapidly sink through the oil layer. After the first round of
PCR, the cycler was held at a high temperature (85°C) to prevent
5
9
13
17
21
25
29
33
37
Figure 3. Optimization of the number of cycles in the second round of
ST-2R-PCR analysis. Samples containing 10-5 (upper panel) and 10-1 (lower
panel) BCR-ABL rearranged cells were amplified for 12 cycles in the first
round, followed by 5-37 cycles in the second round. The PCR products were
electrophoresed in a 3% agarose gel, transfered by Southern blotting to a nylon
membrane and hybridized to a 32P-labeled, internal oligonucleotide probe
(5'-CAgggTCTgAgTgAAgCCgCTCgTT-3'). The numbers indicate the
number of cycles performed in the second round of PCR. In the upper panel,
the reaction remains exponential and yields a clearly identifiable signal after 12
+ 37 amplification cycles. In the lower panel, a plateau is reached after 12 + 29
- 33 cycles.
cooling of the reaction mixes while individual tubes were
centrifuged. This measure prevented the primers from non-specific annealing. Due to the high annealing temperatures of
primers 3 and 4, the second round of PCR could be performed at
a two-step temperature profile with annealing and extension at
70-71 °C without loss of sensitivity.
The sequences of the sense primers 1 and 3, as well as those of
the antisense primers 2 and 4, were designed to contain an overlap
of 18 nucleotides at the 5' end (Fig. 1). Therefore, the
amplification products generated during the first and second
round of PCR have identical length. This approach prevents the
formation of additional signals in samples containing large
amounts of target sequences. The primers 3 and 4 contain seven
additional nucleotides at the 3' end. This difference was sufficient
to prevent amplification of non-specific products possibly
generated during the first round of PCR. The selection of primers
and the cycling conditions could, however, be modified to permit
individual visualization of the first and second round amplification products by revealing signals of different size. The technique
presented is easily reproducible and can be readily adapted to a
number of different applications.
ACKNOWLEDGEMENT
This work was supported by a grant from the Hochschuljubilaumsstiftung der Stadt Wien to T.L.
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