Multiply-primed rolling circle amplification of human papillomavirus using sequence-specific primers

Area of Science:

• Molecular Biology
• Virology
• Genomics

Background:

• Rolling circle amplification (RCA) is effective for amplifying circular DNA.
• Human papillomavirus (HPV) identification can benefit from efficient whole genome amplification.

Purpose of the Study:

• To develop an efficient RCA method for whole genome amplification of HPV using HPV-specific primers.
• To establish a protocol for Sanger sequencing of amplified HPV genomes from clinical samples.

Main Methods:

• Optimized multiply-primed RCA using HPV-specific primers versus random hexamers.
• Whole genome amplification of HPV DNA from clinical samples.
• Sanger sequencing of RCA products without cloning.

Main Results:

• HPV-specific primers increased RCA effectiveness 15.2-fold and HPV enrichment 62.2-fold compared to random hexamers.
• Successful whole HPV genome sequencing from 4 patient samples on FTA cards.
• Generated high-quality and quantity DNA suitable for sequencing and downstream analyses.

Conclusions:

• Optimized RCA with HPV-specific primers is highly effective for whole genome amplification and sequencing of HPV.
• This method enables efficient HPV genome analysis from low-input clinical samples, including those on FTA cards.