ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2017, Vol. 43, No. 5, pp. 544–551. © Pleiades Publishing, Ltd.

, 2017.
Original Russian Text © K.V. Bogdanov, T.S. Nikulina, E.G. Lomaia, M.N. Slyadnev, A.Y. Zaritskey, 2017, published in Bioorganicheskaya Khimiya, 2017, Vol. 43, No. 5, pp. 523–531.

Identification of Oncogene Mutations in Leukemia Patients Using

Microchip-Based PCR Analysis
K. V. Bogdanova, b, 1, T. S. Nikulinaa, E. G. Lomaiaa, M. N. Slyadnevb, and A. Y. Zaritskeya
aInstitute of Hematology, Almazov Federal Medical Research Center, St. Petersburg, 197341 Russia
bLumex Group, St. Petersburg, 192029 Russia

Received September 2, 2016; in final form, September 13, 2016

Abstract⎯At present, cytogenetic and PCR-based techniques are generally used in the diagnosis of leukemia.
Due to high accuracy, specificity, and sensitivity of PCR assays, they have the advantage over traditional cyto-
genetic tools. As a rule, the classical real time PCR is carried out in a 25-μL reaction mixture. It requires a
large volume of each reagent and takes a long time to finish the test. The molecular genetic assay presented
here is microchip-based real-time PCR that is optimized for simultaneous analysis of 15 oncogene mutations
and one housekeeping gene abl. Moreover, this diagnostic tool requires a minimal amount of cDNA and
PCR reagents (10-fold less) and a short time (2-fold less) for quantitative estimation of more than five copies
of gene-target. Thus, microchip-based PCR analysis can improve the detection of oncogene mutations in
leukemia patients and may be used for both early diagnostics and long-term monitoring of leukemia.

Keywords: PCR, microchip, oncogene, leukemia

DOI: 10.1134/S1068162017040033

One of the possible reasons for leukemia develop-
ment is the activation of proto-oncogenes often
induced by chromosomal mutations accompanied by
gene rearrangements [1, 2]. It is known that many
chromosomal abnormalities, including deletions,
inversions, and translocations, are associated with
particular subtypes of leukemia. The most abundant of
them are presented in Table 1. It should be noted that
rise of these mutations in as little as a single hemato-
poietic stem cell leads to leukemia initiation. Appear-
ance of additional mutations in the genome is associ-
ated with progression of the hematological malignan-
cies. Hematopoietic cells gain the ability for unlimited
proliferation and/or loose the ability for differentia-
tion, exhibiting all phenotypic properties of tumor
cells [3].
To detect chromosomal rearrangements, as a rule,
cytogenetic and molecular genetics approaches are
used. Cytogenetic analysis of the patient’s bone mar-
row sample is based on confocal microscopy for visu-
alization of metaphase plates (n = 20) or interphase
1 Corresponding
author: phone: +7 (911) 289-26-88; e-mail:
kvbogdanov@yandex.ru.
Abbreviations: MCT, molecular colonies technique; MRD,
minimal residual disease; RT, reverse transcription; ALL, acute
lymphoblastic leukemia; B-ALL, B-cell ALL; AML, acute
myeloid leukemia; APML, acute promyelocytic leukemia;
CML, chronic myelogenous leukemia; FISH, fluorescent in
situ hybridization; MALDI-TOF, time-of-flight matrix-assisted
laser desorption/ionization mass spectrometry; NGS, next gen-
eration (whole-genome) sequencing.
and metaphase nuclei (n = 100) after carrying out flu-
orescence in situ hybridization (FISH). Due to insuf-
ficient specificity and sensitivity of cytogenetic tests
(one leukemia cell per 102 normal cells), including
FISH (one leukemia cell per 103 normal cells), pro-
longed biomaterial preparation procedure, and dura-
tion of the whole analysis, PCR is favored. High sen-
sitivity of PCR allows for detection of as little as one
leukemia cell per 10 4–106 normal cells. Moreover,
sample preparation before PCR takes a short time. It
includes RNA extraction from patients' biopsy and
carrying out the reverse transcription (RT) reaction
promoting cDNA synthesis on the RNA template.
Today, to detect oncogene mutations in leukemia
patients, several PCR-based methods are used. First,
these include classical PCR in a tube with 25- or 50-μL
sample, which requires the consumption of large
amount of reagents [4]. The total duration of PCR is
210–300 min. Moreover, PCR hybridization with
melting curve plotting, PCR-based biochip test with
dot-blot hybridization, molecular colonies technique
(MCT) with amplification of the target gene in a poly-
acrylamide gel layer, direct sequencing, and mass
spectrometry (MALDI-TOF) are also used [5–8].
Recently, next generation sequencing (NGS)
method has been used for identification of defects in
the whole human genome. In spite of the good results
obtained using this test and mass spectrometry, both
of them are rather labor consuming and require bioin-
formatics data analysis [9]. Methods based on dot-blot

544
 IDENTIFICATION OF ONCOGENE MUTATIONS IN LEUKEMIA 545

Table 1. Most frequent chromosomal rearrangements in leukemia [4, 12, 13]

Chromosomal Mutation frequency
Diagnosisa Oncogene variant
rearrangementb in adults (children), %

CML, Ph+ t(9;22)(q34;q11) bcr-abl (p210), Mbcr 95

bcr-abl (p190), mbcr 1–2
ALL, Ph+ t(9;22)(q34;q11) bcr-abl (p190), mbcr 65 (80)
bcr-abl (p210), Mbcr 5–35
В-ALL t(12;21)(p13;q22) tel-aml1 20–25 (16)
t(4;11)(q21;q23) mll-af4 (a) 5
mll-af4 (b)
pre-В-ALL t(1;19)(q23;p13) e2a-pbx1 3–5
Т-ALL del(1)(p32p32) sil-tal1 5–25
AML (М1, М2, etc.) t(8;21)(q22;q22) aml1-eto 5–12
inv16(p13;q22) cbfb-myh11 (A) 5–7
cdfb-myh11 (D)
cbfb-myh11 (E)
APML (M3) t(15;17)(q22;q21) pml-rara (bcr1) 3–9
pml-rara (bcr2)
pml-rara (bcr3)
AMKL (M7) t(1;22)(p13;q13) rbm15-mkl1 3–14
aALL, acute lymphoblastic leukemia; В-ALL, B-cell ALL; Т-ALL, Т-cell ALL; AML, acute myeloid leukemia; AMKL, acute
megakaryoblastic leukemia; APML, acute promyelocytic leukemia; CML, chronic myelogenous leukemia.
b t, translocation; del, deletion; inv, inversion.

hybridization are typically multistep and take much
time and/or are insufficiently specific. Besides, their
application for quantitative analysis is hampered by
the elevated background level after blot washing from
the unbound components. On the other hand, the
MCT application allows for quantitative determina-
tion of oncogene expression, particularly, aml1-eto;
notably, it requires no standard dilution of DNA plas-
mids containing nucleotide sequences of control
genes. However, one needs to prepare polyacrylamide
gel pads for PCR. Moreover, reagent consumption for
such amplification is comparable with the classical
PCR in a tube, where the sample volume is 50 μL.
In the last decade, methods promoting gene
expression evaluation on microchip analyzers gained
wide application [10, 11]. They are characterized by
high specificity, the possibility of 100 to 1000 reactions
in 50–300 nL of the reaction mixture, as well as high
performance, and automation. Yet, highly specialized
microchips are required for such an analysis. Mean-
while, full loading of even widely specialized micro-
chips is possible only in large research centers, and the
analysis takes much time.
Therefore, the aim of the research was to carry out
the modification and optimization of the classical
PCR conditions to amplify 15 wide-spread target
oncogenes in leukemia patients using native reagents
and microchips under conditions of real-time PCR.
Moreover, it would be important to evaluate the pos-
sibility of quantitative analysis of fusion oncogene bcr-
abl (p190) often detected in ALL patients, using the
microchip-based PCR and compare the results of the
test with classical PCR.
RESULTS AND DISCUSSION
Before microchip PCR was carried out to detect
the 15 wide-spread oncogene mutations in leukemia
patients (Table 1), amplification conditions were
modified and optimized using native reagents and
plasmid DNA containing fragments of nucleotide
sequences of relevant oncogene targets. Figure 1 pres-
ents the scheme of sample introduction into the
microchip wells for detection of oncogene mutations.
In the current study, the amount of oligonucle-
otides necessary for amplification of all 15 oncogene
targets and the abl control gene was selected after the

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 43 No. 5 2017