A Pax-5a gene analysis approach enabled by selective digestion with lambda exonuclease.

ABSTRACT

Owing to the rapid increase in acute leukemia patients, the detection of Pax-5a, which is a tumor marker, is very important for the early diagnosis of patients. Therefore, by combining the selective digestion function of lambda exonuclease and the hybridization chain reaction (HCR) enzyme-free amplification system, we design a biosensor to detect the Pax-5a gene with high sensitivity. Lambda exonuclease can cleave the blunt end formed by the hairpin probe and the Pax-5a gene, which exposes the nucleic acid sequence that can initiate the HCR. When the HCR is triggered, the fluorophore and quencher on H1 and H2 move away from each other, so that the fluorescence signal of the quenched fluorophore can be recovered. Under optimal experimental conditions, a good linear relationship was established between the fluorescence intensity and the logarithm of the target concentration, and the limit of detection (LOD) of Pax-5a was calculated to be 7.6 pM. In addition, the biosensor can not only discriminate the base mismatch sequences of the Pax-5a gene, but also be suitable for target detection in complex human serum samples. Therefore, this biosensor, with the advantages of simple operation, high sensitivity, and good selectivity, has a good application prospect and guiding role in the diagnosis of acute B lymphocytic leukemia and the design of biosensors.