A Cost-Effective Approach for Single-Stranded DNA Amplification Using Primer-Blocked Asymmetric PCR

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• In vitro amplification of single-stranded oligonucleotide libraries is hampered by significant byproduct formation.
• Existing methods like asymmetric PCR and lambda exonuclease digestion often yield low-quality ssDNA.
• These limitations impact the utility of ssDNA libraries in various downstream applications.

Purpose of the Study:

• To develop an improved protocol for efficient and cost-effective in vitro amplification of ssDNA libraries.
• To alleviate byproduct formation during ssDNA library generation without compromising sequence diversity.
• To provide a universally applicable method for producing high-quality ssDNA sequences.

Main Methods:

• Utilized primer-blocked asymmetric PCR (PBA-PCR) incorporating a 3'-phosphate-blocked limiting primer to reduce mispriming.
• Combined PBA-PCR with emulsion PCR for enhanced amplification efficiency.
• Implemented a cost-effective downstream process involving biotin-streptavidin separation for dsDNA removal and ssDNA purification.

Main Results:

• The PBA-PCR protocol significantly reduced DNA byproduct formation compared to conventional methods.
• The combined approach yielded purified ssDNA libraries with preserved sequence space.
• Demonstrated a universally applicable, simple, and cost-effective method for ssDNA production.

Conclusions:

• The developed PBA-PCR based protocol offers a robust solution for high-quality ssDNA library amplification.
• This method is adaptable for various applications including aptamer generation, unique molecular identifiers, and CRISPR-Cas9 systems.
• The protocol facilitates on-demand labeling and production of unique ssDNA sequences.