Sensitive detection of fusion transcripts with padlock probe-based continuous cascade amplification (P-CCA)

Area of Science:

• Molecular Biology
• Biotechnology
• Cancer Diagnostics

Background:

• Gene fusions, arising from chromosomal rearrangements, create novel functional genes.
• Fusion genes are highly specific to tumor cells, making them valuable diagnostic biomarkers and therapeutic targets.
• Sensitive detection of fusion transcripts is crucial for biological research and disease diagnosis.

Purpose of the Study:

• To develop a sensitive method for detecting PML-RARα gene fusion transcripts.
• To create a convenient and reliable platform for early clinical diagnosis of gene fusion-related cancers.

Main Methods:

• Utilized padlock probes for direct ligation at the gene fusion junction site.
• Employed a continuous cascade amplification strategy involving rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP).
• Integrated RCA and LAMP into a one-tube isothermal reaction with a single-step operation.

Main Results:

• Achieved sensitive detection of target fusion transcripts down to 1 fM concentration.
• Demonstrated high specificity due to the ligation reaction's ability to differentiate mismatched sequences.
• Successfully applied the method to analyze real biological samples.

Conclusions:

• The proposed padlock probe-based continuous cascade amplification (P-CCA) method provides a sensitive and specific approach for detecting gene fusion transcripts.
• This technique offers a convenient, fast, and reliable platform for the early clinical diagnosis of cancers associated with gene fusions.
• The integrated one-tube isothermal reaction simplifies the detection process.