Accurate and Efficient One-Pot Reverse Transcription and Amplification of 2'-Fluoro-Modified Nucleic Acids by Commercial DNA Polymerases.

DNA is a foundational tool in biotechnology and synthetic biology but is limited by sensitivity to DNA-modifying enzymes. Recently, researchers have identified DNA polymerases that can enzymatically synthesize long oligonucleotides of modified DNA (M-DNA) that are resistant to DNA-modifying enzymes. Most applications require M-DNA to be reverse transcribed, typically using a RNA reverse transcriptase, back into natural DNA for sequence analysis or further manipulation.

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates.

Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more-diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2'-modified DNAs; however, it is limited in the length of the products it can synthesize.

Taq DNA Polymerase Mutants and 2'-Modified Sugar Recognition.

Chemical modifications to DNA, such as 2' modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2'-modified DNA nucleotides. While these mutant enzymes recognize these modified nucleotides, they are not capable of synthesizing full length modified DNA; thus, further engineering is required for these enzymes.