Screening Experimental Studies Of DNA Electrochemical Probes For Detection Of Relative Genes In Chronic Myelogenous Leukemia

Electrochemical DNA biosensor is a novel biosensor which developed rapidly in recent years. As it has advantages of fast detection, simplity, sensitivity, low price, electrochemical DNA biosensor has far-reaching significance and applicable value in the study of clinical medicine and genetic project. Nucleic acid technology evolves with the development of genetic engineering technology to become a kind of new biological high technology. As it has advantages of good selectivity, high sensitivity and fast accurate detection, nucleic acid technology has exerted huge effect in biological macromolecule research, clinical diagnosis and treatment.Two novel DNA electrochemical probes (hairpin probe and locked nucleic acid) were respectively designed and developed for detection of BCR/ABL fusion gene in chronic myelocytic leukemia (CML) in this article. The stability and the specificity of these DNA biosensors were also been investigated.Therein, hairpin DNA electrochemical probe (HP) designed according to CML BCR/ABL fusion gene fragment was fixed onto the GCE through the bond of amino group in 5' end of the probe with the sulfonic group of 4-ABSA immobilized on the GCE by cyclic voltammetry (CV). A novel electrochemical DNA biosensor for detection of BCR/ABL fusion gene in CML using methelyne blue (MB) as the hybridization indicator was developed.The result indicated that the synthetical short sequence of BCR/ABL fusion gene would be recognized and quantitated according to the changeable peak current of MB. In the optical condition, the relationship between the oxidation peak current of MB and the concentration of complementary strand was linear in the range of 8.0×10-11 ~ 4.0×10-10 mol/L. The detection limit had been estimated as 9.4×10-13 mol/L. Otherwise, the biosensor has a good specificity to distinguish the complementary sequence, single-base mismatch sequence and non-complementary sequence. It is possible to qualitatively and quantitatively detect BCR/ABL fusion gene in CML.Besides, based on the combination of locked nucleic acid (LNA) probe and electrochemical enzyme immunoassay, the sandwich-type electrochemical enzymes-based LNA-modified DNA biosensor was developed for detection of BCR/ABL fusion gene in CML in this study. Under the optimal condition of the hybridization, while detecting the complementary DNA standard concentration range of the BCR/ABL from 1.0×10 -14 to 2.5×10–13 mol/L, there was a good linear relationship between the current signal and the concentration of complementary DNA, the lower limit was 5.0×10 -15 mol/L. Otherwise, the biosensor shows a good specificity to distinguish the complementary sequence, single-base mismatch sequence and non-complementary sequence. The obvious difference of results after hybridization demonstrated this biosensor could detect BCR/ABL fusion gene qualitatively and quantitatively.In the initial study of CML practical samples, the PCR analysis of cDNA of patients' blood samples was conducted using the design of primers. After electrophoresis, the products of PCR were detected using electrodchemical methods and compared with the results of electrophoresis. It provided a good basis of futher detection research in practical samples.