DNAdetection of chronic myelogenous leukemia by magnetic nanoparticles

Abstract

A novel tool for the detection of BCR/ABL fusion gene in chronic myelogenous leukemia (CML) was developed by a magneto-polymerase chain reaction (PCR)-enzyme linked gene technique. The forward primers covalently bound to the surface of magnetic nanoparticles allowed a convenient separation of PCR products with high sensitivity (0.5 pg ml(-1)) and high specificity using K562 cell line and CML patients. The results were obtained when the biotinylated-reverse primer bound to streptavidin-horseradish peroxidase (HRP) and hydrolysed the substrate. This novel readout system was approximately 1000-fold more sensitive than the conventional agarose gel electrophoresis. The present technique is practical and useful for following up CML patients and for providing appropriate treatment, particularly to patients in remote areas.

Magnetic Nanoparticles PCR Enzyme‐Linked Gene Assay for Quantitative Detection of BCR/ABL Fusion Gene in Chronic Myelogenous Leukemia

“…Briefly, carboxylated MNPs with superparamagnetic properties (hydrodynamic diameter 250 nm) (Chemicell, Berlin, Germany) (1 mg) were washed twice with a 2‐( N ‐morpholino)ethanesulfonic acid (MES) buffer (Fluka, USA) (25 mM, pH 6.0) under an external magnetic field. The carboxylated MNPs were immobilized using an NH 2 ‐modified BCR forward primer (5′‐NH 2 ‐C 6 ‐CTG ACC AAC TCG TGT GTG AAA C‐3′) (Bio Basic Inc.) using the carbodiimide method . The primer‐immobilized MNPs were then resuspended in Tris‐EDTA buffer (Sigma, USA) (10 mM Tris‐HCl (Sigma, USA) and EDTA 1 mM pH 8.0) and maintained at 4°C.…”

“…To immobilize the forward primer onto the MNP surface, we employed a previously reported procedure . Briefly, carboxylated MNPs with superparamagnetic properties (hydrodynamic diameter 250 nm) (Chemicell, Berlin, Germany) (1 mg) were washed twice with a 2‐( N ‐morpholino)ethanesulfonic acid (MES) buffer (Fluka, USA) (25 mM, pH 6.0) under an external magnetic field.…”

“…cDNA was amplified by PCR using primers specific for BCR/ABL as previously reported . The PCR reaction mixture was prepared using Taq DNA polymerase (RBC Bioscience, New Taipei City, Taiwan) (5 U/μl, 0.25 μl), reaction buffer (10×, 5 μl), dNTP mix (10 mM, 0.5 μl), forward primers ( BCR forward primer, 5′‐CTG ACC AAC TCG TGT GTG AAA C‐3′) (10 mM, 1 μl) or primer‐immobilized MNPs (2 μg/μl, 1 μl), reverse primer with or without biotin tag ( ABL reverse primer, 5′‐(Biotin)‐TGT GAT TAT AGC CTA AGA CCC GGA‐3′, Bio Basic Inc.) (10 mM, 1 μl), DEPC‐treated water (up to 50 μl), and cDNA (1 μl).The temperature for denaturation, annealing, and elongation were 95ºC, 63ºC, and 72ºC, respectively, for 35 cycles . The PCR product was determined using agarose gel electrophoresis (Research Organics Inc., OH).…”

“…After PCR amplification using the primer‐immobilized MNPs and biotinylated reverse primer , the biotinylated‐PCR products bound to MNPs were washed twice with DEPC‐treated water under an applied external magnetic field. Horseradish peroxidase‐conjugated streptavidin (SA‐HRP) (KPL, USA) (0.1 mg/ml, 1 μl) in distilled H 2 O (50 μl) was then added to the biotinylated‐PCR products bound to MNPs, and the mixture was incubated at room temperature for 30 min per the manufacturer's recommendation.…”

“…In a standard PCR‐ELISA, amplicons are immobilized on a plastic ELISA plate and then detected by hybridization with an amplicon‐specific primer. Recently, the novel gene detection assay has been reported used in combination with MNPs, molecular techniques, and enzyme–substrate systems to provide increased specificity and sensitivity (<1 pg/ml of total RNA) . The assay consists of PCR using two primers, that is, forward and reverse primers immobilized on MNP surfaces and biotin.…”