healthmedicinet

PNA-PCR clamping for BCR-ABL1 T315I mutation

The study was approved by the local ethic committee of San Luigi Hospital, Orbassano,
Turin. After written informed consent BM aspirates were obtained from 17 imatinib
resistant CML patient and 1 Ph?+?Acute Lymphoblastic Leukemia (ALL), all displaying
T315I mutation detected by Sanger Sequencing. In addition, as negative control, 25
CML patients without mutations and 15 healthy subjects were examined. Detection of
BCR-ABL1 was performed by capillary Sanger Sequencing method and analyzed by sequencing with
BigDye terminator v3.1 (Applied Biosystem, Foster City, California CA) and capillary
electrophoresis on ABI PRISM 3130XL (Applied Biosystem, Foster City, California CA).
The sensitivity of this method was previously estimated by serial dilutions experiments
to be approximately10 %.

Primer Sequences were as follow:

FWD: 5’-tatcatcactgagttcatgacc-3’;

REV: 5’-ggccaaaatcagctaccttcac-3’;

PNA competitor: OO-atatcatcactgagttcat-Lys

Competitor PNA sequence was designed to perfectly match WT template sequence.

PNA-PCR clamping conditions for BCR-ABL1 T315I mutation detection were as follow:

1st step PCR: 94 °C 3 min, (94 °C 30 s, 55 °C 30 s, 72 °C 1 min 40 s) for 40 cycles,
72 °C 5 min; 2nd step PCR: 94 °C 3 min, (94 °C 30 s, 65 °C 20 s, 55 °C 50 s, 72 °C
15 s) for 30 cycles. Sensitivity was assessed mixing, at different ratio, mutated
(T315I) and WT template. Dilutions were as follow: 100, 20, 10, 5, 1, 0.5 and 0 %
T315I mutated versus WT template. PCR amplification was carried-out in absence (?) or in presence (+)
of competitor PNA, at a concentration 3× greater than primer FWD. The amplification
performed without (?) PNA represents an internal positive control displaying the efficiency
of template amplification.

PNA FISH for the detection of BCR-ABL1 T315I mutation

In addition, for the detection of T315I mutation in progenitor cells we set up a method
based on fluorescently-labelled PNA probe (PNA FISH). CD34
⁺
cells were enriched by magnetic cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach,
Germany) according to the manufacturer’s protocol. PNA probe was designed on the human
BCR-ABL1 T315I cDNA. The single nucleotide mismatch falls just in the middle of the sequence. The
probe has been further tagged by fluorescinated dye at its amino-terminus. The sequence
is as follow: Alexa488-OO-TATCATTGAGT-Lys. Detection of BCR-ABL T315 by PNA FISH was performed as previously described 10]. At least 500 cells have been evaluated for each sample. A positive control is added
in each reaction to distinguish between negative results and lack of hybridization.