Visual genotyping of thalassemia by using pyrrolidinyl peptide nucleic acid probes immobilized on carboxymethylcellulose-modified paper and enzyme-induced pigmentation

• Main Authors: Nuttapon Jirakittiwut, Thongperm Munkongdee, Kanet Wongravee, Orapan Sripichai, Suthat Fucharoen, Thanit Praneenararat, Tirayut Vilaivan
• Other Authors: Chulalongkorn University
• Format: Article
• Published: 2020
• Subjects: Chemistry
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/54516
• Institution: Mahidol University

© 2020, Springer-Verlag GmbH Austria, part of Springer Nature. A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. [Figure not available: see fulltext.].