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`26 Clin Pathol: Mol Pathol 1997;50:266-268
`266
`
`A rapid RT-PCR based method for the detection
`of BCR-ABL translocation
`
`Janna Yu Sidorova, Liudmila B Saltykova, Anton A Lyschov, Andrey Yu Zaritskey,
`Kudrat M Abdulkadyrov, Michail N Blinov
`
`Abstract
`Aims-To optimise a one step reverse
`transcriptase polymerase chain reaction
`(RT-PCR) protocol for BCR-ABL chi-
`maera detection.
`Methods-Compared with published RT-
`PCR procedures, this novel approach has
`at least two advantages. First, the same
`enzyme is used for both reverse transcrip-
`tion and PCR. Second, amplification of
`the target (BCR-ABL chimaera) and con-
`trol gene (ABL) is performed simultane-
`ously in the same tube.
`Results-On testing 40 chronic myelo-
`genous leukaemia patients and 10 healthy
`donors there was a specificity for the
`newly developed technique. In addition,
`dilution experiments demonstrated that
`the protocol was highly sensitive.
`Conclusions-The suggested one step
`PCR strategy is a simple and reliable way
`to reveal BCR-ABL chimaeras.
`(J Clin Pathol: Mol Pathol 1997;50:266-268)
`
`Keywords: chronic myelogenous leukaemia; reverse
`transcriptase polymerase chain reaction; BCR-ABL
`
`BCR-ABL translocation t(9,22) occurs in 95%
`of chronic myelogenous leukaemia (CML)
`cases and in 15-25% of acute lymphocytic ieu-
`kaemia (ALL) cases, often resulting in the
`appearance of the so called Philadelphia (Ph)
`chromosome. Detection of this alteration plays
`an important role in the differential diagnosis
`malig-
`and monitoring of haematological
`nancies."1 In particular, testing for the translo-
`cation allows one to identify the type of
`haematoblastosis at the time of onset and thus
`choose the appropriate treatment. Once diag-
`nosed, BCR-ABL translocation can be used as
`a marker of a malignant clone to monitor the
`efficacy of treatment and to predict relapse.
`For some years, this chromosomal damage
`has been detected cytogenetically. However,
`recently, cytogenetic procedures have been
`extensively replaced by polymerase chain reac-
`tion (PCR) based protocols. The advantages of
`PCR detection of BCR-ABL translocation lie
`in its high specificity and unique sensitivity5-7:
`PCR is able to detect the mutated gene even in
`some cytogenetically normal (Ph negative) leu-
`kaemic clones and can even detect it in the
`presence of thousands of normal genomes.
`Thus, PCR is much more informative than
`cytogenetics alone as a means of t(9,22) detec-
`tion.
`
`Many PCR based protocols for the diagnosis
`of the BCR-ABL chimaera have been publi-
`shed.8'-1 Most of them include the detection of
`the translocation at the level of the RNA tran-
`script. The method usually consists of two
`steps. The first reaction is that of obtaining
`cDNA from RNA by means of reverse
`transcription (RT). Subsequently, an aliquot of
`cDNA is transferred to another tube and sub-
`jected to the second reaction, that is, PCR
`amplification. Two different enzymes are used,
`namely, reverse transcriptase for the RT step
`and Taq polymerase for the PCR.
`RT-PCR can be simplified significantly if
`another thermostable enzyme, namely Tth, is
`used instead. Tth is known to possess both RT
`and DNA polymerase activity.'2 Thus, only
`minimal adjusting of the reaction mixture in
`the tube is necessary to switch the process from
`RT to PCR. Such "one tube" RT-PCR
`protocols have been developed already for
`many widely used procedures.'3 '4 However,
`there are no published accounts of the potential
`benefits of this rapid RT-PCR strategy for the
`detection of BCR-ABL translocation.
`In this paper, we present a one tube protocol
`for BCR-ABL chimaera detection. The meth-
`od is cheap and time saving. Also, the reduction
`in the numbers of manipulations minimises the
`risk of contamination. Finally, this modifica-
`tion combines both the specificity and sensitiv-
`ity of traditional PCR procedures.
`
`Materials and methods
`RNA ISOLATION
`White blood cells were pelleted by soft
`centrifugation and RNA was isolated according
`to a standard protocol.'5
`
`PCR CONDITIONS
`Multiplex RT-PCR was used in the study.
`Detection ofthe BCR-ABL chimaeric transcript
`was performed using the CML A (5'-GGA
`GCTGCAGATGCTGACCMC-3' (bcr
`spe-
`cific))'0 and ABL3 (5'-CCAlY-l-ll-GGTF-I"
`GGGCTTCACACCAT TCC-3' (abl speci-
`fic))9 primers. To control the efficiency of PCR,
`a 233 base pair abl oncogene fragment was
`amplified simultaneously in the same tube using
`primers9 ABL2 (5'-CAGCG GCCAGTAG-
`CATCTAC TFFTG-3') and ABL3.
`The first step of the reaction included the
`reverse transcription of abl specific sequences.
`The RT mixture (10 gl) contained 67 mM Tris
`HC1 (pH 8.8), 16.6 mM (NH4),SO4, 0.2 mM
`500 nM ABL3 primer,
`MnCl1,
`0.2 mM
`glycerol,
`units
`Tth
`2.5%
`dNTPs,
`2.5
`thermostable polymerase (TET-Z polymerase;
`
`Institute of
`Haematology and
`Transfusiology, 2-ya
`Sovetskaya, 16, 193024,
`St Petersburg, Russia
`J Y Sidorova
`L B Saltykova
`K M Abdulkadyrov
`M N Blinov
`
`NN Petrov Institute of
`Oncology, Pesochny-2,
`189646, St Petersburg,
`Russia
`A A Lyschov
`
`Clinical Center of
`Advanced Medical
`Technologies, Hospital
`31, Dinamo 4, 197042,
`St Petersburg, Russia
`A Y Zaritskey
`
`Correspondence to:
`Dr Blinov, Biochemistry
`Laboratory, Institute of
`Haematology and
`Transfusiology, 2-ya
`Sovetskaya 16, 193024, St
`Petersburg, Russia.
`
`Accepted for publication
`26 June 1997
`
`Illumina Ex. 1119
`IPR Petition - USP 10,435,742
`
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`Rapid RT-PCR based method ofBCR-ABL translocation detection
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`267
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`_
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`4
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`5
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`8
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`9
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`10
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`110 -
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`Detection of the BCR-ABL chimaera by different PCR protocols. RNA samples
`Figure I
`were analysed by means of methods reported previously'0 (lanes 1, 2, 5, 6, and 7) and the
`newly developed RT-PCR approach (lanes 3, 4, 8, 9, and 10). Healthy donors yielded no
`product (lane 2) and a 233 base pair ABL specific product only (lane 4). The b3:a2
`BCR-ABL translocation resulted in extra bands of 200 base pairs (standard RT-PCR: lane
`1, K-562 cell line; lane 5, patient 1) or 397 base pairs (one tube protocol: lanes 3 and 8, the
`same RNA samples). The b2:a2 chimaera (patient 2) was recognised by the 125 base pair
`(lane 6) or 322 base pair (lane 9) additional fragments. Water negative controls are
`represented in lanes 7 and 10. Molecular weight markers (MspI digested pUC 19 plasmid)
`are on the left side of the gel.
`
`Biomaster, Moscow, Russia), and 0.1-0.7 pg of
`total leucocyte RNA. The tubes were overlaid
`with mineral oil and heated for two minutes at
`94°C for RNA denaturation. Annealing of the
`ABL3 primer was performed at 65°C for five
`minutes. The RT reaction was completed by
`specific synthesis of cDNA at 70°C for 20 min-
`utes.
`To perform the second step of the reaction,
`the mixture in the tubes was adjusted for PCR
`conditions by adding 2,l lOx stock solution
`comprising: 670 mM Tris HCI (pH 8.8),
`166 mM (NH4),2SO4, 7.5 mM EGTA, 0.25%
`(vol/vol) Tween 20, 25% glycerol, 2 mM
`dNTP, 20 mM MgC1I,
`500 nM ABL2,
`500 nM CML A, 2 mM dNTP, and sterile dis-
`tilled water up to 20 jl. PCR was started by
`cDNA denaturation (94°C for two minutes).
`The thermal cycler settings for the initial 10
`cycles were: 94°C for 50 seconds (denatura-
`tion), and 65°C for one minute (annealing +
`
`-1I
`
`n
`
`-z
`in2c)
`
`1
`
`-
`
`-4
`n
`
`-5
`in
`
`_ A
`
`n
`
`_/
`
`s
`
`n
`
`- 4
`
`in0
`
`-5
`
`i nC
`
`501 -
`
`331 -
`
`242-
`
`190-
`
`bp
`
`Comparison of the sensitivity of RT-PCR protocols. Serially diluted samples of
`Figure 2
`total RNA from K-562 cells were mixed with different amounts of leucocyte RNA from
`healthy donors and subjected to RT-PCR analysis. Numbers at the top represent the ratio
`between concentrations of K-562 and normal RNA. Results of the one tube protocol are
`shown on the left of the figure; results of the basic method are shown on the right. Details of
`the fragments are given in the legendforfig 1. Msp digested pUC19 plasmid DNA was
`used as a marker.
`
`synthesis). The next 25 cycles consisted of: 40
`seconds at 94°C (denaturation), 50 seconds at
`55°C (annealing), and 60 seconds at 72°C
`(synthesis). The reaction was completed by a
`final elongation at 72°C for seven minutes. The
`PCR products were separated electrophoreti-
`cally on a 6% polyacrylamide gel and visualised
`by routine ethidium bromide staining.
`
`Results
`Forty CML patients (38 Ph positive and two
`Ph negative) and 10 healthy donors were cho-
`sen for the comparison of the specificity of
`standard and novel PCR based methods for
`BCR-ABL chimaera detection. RNA samples
`standard
`subjected,
`parallel,
`in
`to
`were
`RT-PCR'0 and to one tube RT-PCR tests. As
`shown in fig 1, both PCR variants gave the
`same results, revealing the translocation in all
`CML patients but not in control samples; the
`b3:a2 translocation variant was observed in 28
`cases, whereas the remaining 12 CML patients
`carried the b2:a2 type of abnormality.
`The sensitivity of the new approach was
`tested by dilution experiments. K-562, a Ph
`positive chronic leukaemia cell line, was used as
`a source of the BCR-ABL chimaeric transcript.
`Total RNA from these cells was mixed in
`different proportions with RNA from healthy
`donors and subjected to PCR analysis. Where-
`as the standard RT-PCR'° revealed the translo-
`cation at a dilution of 1 in 10-5, our technique
`was limited to 1 in 10- (fig 2).
`
`Discussion
`In this study, we have adapted one tube Tth
`based RT-PCR methodology'2 for the detection
`of BCR-ABL translocation. Unlike published
`procedures for the detection of BCR-ABL chi-
`maeras, both steps of this test were performed
`in the same Eppendorf tube and required the
`same enzyme.
`The simplification of BCR-ABL diagnosis is
`unlikely to increase the frequency of false
`results. In our hands, both rapid and standard
`RT-PCR methodologies revealed the transloca-
`tion in all CML cases, indicating the lack of
`false negative detection. The method appears,
`therefore, to be most useful in the detection of
`the mutation. However, our procedure demon-
`strated an approximately 10-fold decrease in
`sensitivity in dilution experiments. Although
`the estimated threshold (one mutated gene
`copy in 10 000 normals) of the one tube proto-
`col remains sufficient for monitoring purposes,
`the reliable detection of residual malignant
`cells in peripheral blood or bone marrow might
`need to rely on the more laborious standard
`RT-PCR protocol. Conversely, the reduction in
`the numbers of manipulations and reagents
`positive
`decreases the probability of false
`results caused by contamination.
`In conclusion, we have described a rapid
`RT-PCR procedure which is advantageous in
`the routine detection of BCR-ABL chimaeras
`owing to its simplicity and reliability. This
`method is recommended as a laboratory test
`for CML diagnosis and assessment.
`
`

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`268
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`Sidorova, Saltykova, Lyschov, Zaritskey, Abdulkadyrov, Blinov
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`

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`
`Downloaded from mp.bmj.com on November 18, 2013 - Published by group.bmj.com
`
`
`
`
`
`
`
`A rapid RT-PCR based method for the
`detection of BCR-ABL translocation.
`Sidorova JYu, L B Saltykova, A A Lyschov, et al.
`
`Mol Path
` 1997 50: 266-268
`doi: 10.1136/mp.50.5.266
`
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