A new method to evaluate trinucleotide repeats length polymorphism

• A new method to evaluate trinucleotide length polymorphism has been developed.
• The method is based on the secondary structure with doxorubicin as fluorescent probe.
• With doxorubicin as fluorescent probe, the method introduces no fluorophores and quenchers.

Trinucleotide repeats (TNRs) are involved in a number of debilitating diseases and disorders, the length of which usually indicates disease severity at gene level. Herein we have developed a novel fluorescent method in this work to evaluate TNRs length polymorphism based on its DNA secondary structure with doxorubicin (Dox) as fluorescent probe. This new method makes use of the fact that TNRs rich in guanine (G) and cytosine (C) are susceptible to forming stable intramolecular structures, resulting in the formation of double-stranded 5’-GC-3’ or 5’-CG-3’ sequences. So, intercalating of these sequences by Dox, fluorescence quenching of Dox occurs. Consequently, the length polymorphism of TNRs can be evaluated. Taking the study of CAG for an example, a linear relationship between fluorescence intensity and the sequences ranging from 10 to 35 CAG repeats has been obtained, and the assay of the TNRs length polymorphism for PCR products has been realized. Therefore, without the necessity to introduce fluorophores and quenchers by chemical modifications, this new method is simple, cost-effective and convenient to be operated, so it may hold great promise in the diagnosis of diseases arouse from the triplet expansion.

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