Accurate fidelity analysis of the reverse transcriptase by a modified next-generation sequencing

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Accurate complementary DNA (cDNA) synthesis is crucial for molecular biology applications.
• Reverse transcriptases (RTs) are enzymes essential for converting RNA to cDNA.
• Assessing the fidelity of different RTs under various conditions is important for optimizing molecular assays.

Purpose of the Study:

• To develop and validate a novel next-generation sequencing (NGS)-based method for precise fidelity evaluation of reverse transcriptases (RTs).
• To quantify the error rates of multiple RTs and engineered DNA polymerases with RT activity under diverse reaction conditions.
• To identify specific reaction components that influence RT fidelity.

Main Methods:

• A modified NGS approach was employed, utilizing primers with unique molecular and reaction condition tags.
• This method allowed simultaneous analysis of multiple cDNA synthesis reactions within a single NGS run.
• Error rates were quantified for 44 different cDNA synthesis reactions involving various retroviral RTs and engineered DNA polymerases.

Main Results:

• High concentrations of magnesium chloride (MgCl2), manganese(II) acetate (Mn(OCOCH3)2), and deoxynucleotide triphosphates (dNTPs) were found to decrease RT fidelity.
• These fidelity-reducing effects were more pronounced when using RT from human immunodeficiency virus type 1.
• The study successfully demonstrated precise fidelity monitoring through direct sequence determination.

Conclusions:

• The developed NGS method provides a robust platform for accurate fidelity assessment of reverse transcriptases.
• Reaction conditions, particularly high concentrations of specific divalent cations and dNTPs, significantly impact RT fidelity.
• Understanding these factors is critical for selecting appropriate RTs and optimizing protocols for sensitive molecular applications.