RT-based Sanger sequencing of RNAs containing complex RNA repetitive elements.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Sanger sequencing is the gold standard for DNA sequence validation due to its accuracy, simplicity, and low cost.
• Early RNA sequencing using reverse transcriptase (RT) was limited by enzyme processivity and template structure handling.
• Avian Myeloblastosis Virus (AMV) RT struggles with repetitive sequences and stable secondary/tertiary structures common in RNA.

Purpose of the Study:

• To upgrade the Sanger sequencing method for direct RNA analysis.
• To overcome limitations of traditional RT enzymes in sequencing complex RNA molecules.
• To introduce MarathonRT as a superior enzyme for robust RNA sequencing.

Main Methods:

• Replaced AMV RT with MarathonRT in the Sanger sequencing protocol.
• Utilized MarathonRT's ultra-processive nature to copy RNA molecules of diverse sequences and structures.
• Developed a simple and robust method for direct RNA sequence analysis.

Main Results:

• MarathonRT successfully copies RNA molecules with complex features, including repetitive sequences and stable structures.
• The upgraded method provides a feasible approach for chain termination-based RNA sequencing.
• The new protocol is robust and offers guidance for troubleshooting and optimization.

Conclusions:

• MarathonRT enables a simplified and robust Sanger RNA sequencing method.
• This approach overcomes previous technical barriers in direct RNA sequencing.
• The enhanced method holds promise for basic research and clinical applications requiring accurate RNA analysis.