Bisulfite treatment of CG-rich track of trinucleotide repeat expansion disorder: Make the sequence less CG rich

Abstract

Background: Trinucleotide repeat (TNR) expansion is a kind of mutation with instability in the number of microsatellite repeats. This nature of mutation leads to the different kinds of neurological and neuromuscular disorders; among them, fragile-X syndrome is the main cause of intellectual disability in which the increasing number of CGG TNR in 5' untranslated region is the main reason for epigenetic silencing of Fragile X mental retardation 1 gene. The aim of this study is to decrease the CG content of the candidate region to facilitate amplification by conventional polymerase chain reaction (PCR). Bisulfite treatment of the genomic DNA results in conversion of unmethylated cytosine to uridine and may overcome the diagnostic pitfalls. Materials and Methods: The whole blood DNA was extracted and bisulfite treated. Then any simplification in PCR process of desire sequence were assayed through following conventional PCR using specifically designed primers for converted sequence. Bisulfite-treated PCR product of a nearby sequence confirmed our results as a conversion control. Results: Both the control and the candidate sequences undergoing bisulfite treatment were successfully amplified by PCR. Conclusions: Decreasing the GC content of the sequence by bisulfite treating could be a new approach to overcome difficulties in amplifying GC-rich sequences.