Journal of Proteomics & Bioinformatics
Open Access

Abstract

Amplicon Secondary Structure Formation and Elongation during the Process of Sequencing

Elham Davoudi-Dehaghani, Sirous Zeinali, Nejat Mahdieh, Atefeh Shirkavand and Hamideh Bagherian

Understanding the exact mechanism involved in different molecular processes such as replication, transcription and recombination, as well as improving different applications of single stranded DNA, requires increasing of our knowledge regarding different properties of single stranded DNA secondary structure. Despite some efforts in this field, our understanding of the exact mechanism is still very limited. During our investigation on hearing loss, we encountered an unusual sequencing result for exon 5 of TMC1 gene in a patient suspected of having mutation in this gene. BLASTN was used to find out the origin of extra segment seen in the sequencing result. The Mfold web server was used to investigate if secondary structure formation of the amplicon during sequencing can be the cause of this elongation. Our investigation on the DNA sequence revealed that the extra sequence was an inverted repeat of the first 270 nucleotides inserted just after the sequence of the reverse primer. To investigate the origin of this repeat, we run the original PCR product on an agarose gel and no size increase was observed. This suggested that the inverted repeat has been formed during sequencing as a result of secondary structure formation and subsequent extension during sequencing. Despite of the above the Mfold web server did not predict this hypothetical structure. However, when we submitted RNA equivalent for this sequence, the above server suggested formation of several secondary structures in which one of them was very similar to the one which had been predicted. Inability of the Mfold to predict DNA secondary structure for our sequence may lie in its thermodynamic parameters. This finding, during DNA sequencing, suggests that single stranded DNA elongation in the presence of dNTPs and DNA polymerase can be used as a new way for studying the secondary structure in the single stranded DNA.