Single-stranded DNA binding protein facilitates specific enrichment of circular DNA molecules using rolling circle...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Pure nucleic acids, especially circular DNA (plasmids, mitochondrial DNA), are crucial for various molecular biology techniques.
• Rolling Circle Amplification (RCA) is widely used for amplifying DNA templates, particularly for sequencing.
• Nonspecific DNA products can be a challenge in RCA reactions, necessitating improved methods for target enrichment.

Purpose of the Study:

• To develop an efficient method for separating circular DNA molecules from complex nucleic acid mixtures.
• To enhance the amplification of circular DNA targets over background and linear DNA using RCA.
• To leverage a specific mutant single-stranded DNA binding protein (SSB) for improved RCA specificity and yield.

Main Methods:

• Utilized rolling circle amplification (RCA) with random hexamers and bacteriophage Phi29 DNA polymerase.
• Employed a mutant single-stranded DNA binding protein (SSB) from Thermus thermophilus (TthSSB) HB8.
• Applied the method to mixtures containing different species of nucleic acids, including circular and linear DNA.

Main Results:

• The mutant TthSSB effectively eliminated nonspecific DNA products in RCA reactions.
• Plasmid or mitochondrial DNA content in the amplified product was enhanced by approximately 500x.
• The mutant TthSSB promoted selective amplification of circular DNA targets over background and linear DNA.

Conclusions:

• The developed method using mutant TthSSB provides a significant improvement in separating and amplifying circular DNA.
• This technique enhances the purity and yield of circular DNA, facilitating downstream molecular biology applications.
• The mutant TthSSB is a valuable tool for enriching circular DNA targets in RCA, overcoming limitations of previous methods.