Identification of Leukemia-Specific Fusion Gene Transcripts with a Novel Oligonucleotide Array
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Identification of Leukemia-Specific Fusion Gene Transcripts with a Novel Oligonucleotide Array Sung-Min Chun,1 Yoo-Li Kim,2 Hee Baeg Choi,3 Yong-Taek Oh,4 Yoo-Jin Kim,2 Seok Lee,2 Tai-Gyu Kim,3 Eun Gyeong Yang,1 Yong-Keun Park,5 Dong-Wook Kim2 and Byoung-Don Han4 1 2 3 4 5
Life Sciences Division, Korea Institute of Science and Technology, Seoul, Korea Department of Hematology, St. Mary’s Hospital, Seoul, Korea Catholic Medical School, Catholic Hematopoietic Stem Cell Bank, Seoul, Korea Koma Biotech Research Center, Seoul, Korea Graduate School of Biotechnology, Korea University, Seoul, Korea
Abstract
Background: Identification of specific chromosomal translocations is essential for the diagnosis and prognosis of leukemia. In this study, we employ DNA microarray technology to detect chromosomal aberrations in patients with chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), as well as in leukemic cell lines. Methods: Reverse transcription using a random 9-mer primer was performed with total RNA from patients and leukemic cells lines. Multiplex PCR reactions using four groups of primer sets were then performed for amplification of cDNA from reverse-transcribed total RNA samples. Normal and fusion sequences were distinguished by hybridization of the amplified cDNA to a selective oligonucleotide array (SOA) containing 20–30mer synthetic probes. A total of 23 sets of oligomers were fabricated on glass slides for the detection of normal and fusion genes, as follows: BCR/ABL, AML/EAP, AML/ETO, AML/MDS, PML/RARA, NUMA1/RARA, PLZF/RARA, and CBFB/MYH. Results: Gene translocation in leukemia was effectively identified with the SOA containing various leukemiaspecific fusion and normal control sequences. Leukemic fusion sequences from patients and cell lines hybridized specifically to their complementary probes. The probe sets differing by ≈50% at their 5′ or 3′ ends could distinguish between normal and fusion sequences. The entire process of detection was completed within 8 hours using the SOA method. Conclusions: Probe sets on SOA can effectively discriminate between leukemia-specific fusion and normal sequences with a chip hybridization procedure. The oligonucleotide array presents several advantages in identifying leukemic gene translocations, such as multiplex screening, relatively low cost, and speed.
1. Background Identification of specific chromosomal abnormalities in patients with leukemia or lymphoma is essential for diagnosis and prognosis. The chimeric proteins resulting from gene translocations rearrange the regulatory and coding regions of various transcription factors and interfere with cell growth control.[1] Several diagnostic methods have been developed to identify these translocations. Karyotyping, Southern blot analysis, fluorescence in situ hybridization (FISH), reverse transcription PCR (RT-PCR), and
real-time quantitative RT-PCR (RQ-PCR) are among the technologies currently applied in the diagnosis of chromosomal
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