Molecular breeding of polymerases for resistance to environmental inhibitors

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Polymerase chain reaction (PCR) assays are crucial for molecular biology but are often inhibited by complex environmental substances found in various specimens.
• The presence of inhibitors such as humic acid, bone dust, and soil components significantly reduces PCR efficiency and reliability.

Purpose of the Study:

• To engineer a DNA polymerase with enhanced resistance to a wide array of complex environmental inhibitors.
• To improve the applicability of PCR assays in challenging sample matrices relevant to fields like paleontology and forensics.

Main Methods:

• Molecular breeding of eight polymerase orthologues from the genus Thermus.
• Directed evolution using cyclic sequential recombination (CSR) in the presence of inhibitors.
• Selection for resistance against Neomylodon bone powder inhibitors.

Main Results:

• Isolated a chimeric DNA polymerase, designated 2D9, with engineered resistance.
• 2D9 exhibited significant resistance to diverse inhibitors including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment, and tar.
• Demonstrated broad-spectrum inhibitor resistance, overcoming limitations of standard PCR assays.

Conclusions:

• The engineered 2D9 polymerase offers a robust solution for PCR applications in the presence of complex inhibitors.
• This novel polymerase has potential applications in palaeobiology, archaeology, conservation biology, forensic science, and historic medicine.