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High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay
Osama A Alsmadi1, Carole J Bornarth, Wanmin Song
1Molecular Staging, Inc, 300 George Street, Suite 701, New Haven, CT 06511, USA. osamaa@molecularstaging.com
BMC Genomics
|June 5, 2003
Summary
This study presents a highly accurate, single-tube genotyping assay using ligation and exponential rolling circle amplification (ERCA). The method achieves over 99% accuracy for clinically relevant mutations directly from genomic DNA in under an hour.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Rolling circle amplification (RCA) of ligated probes offers a sensitive method for direct genotyping from genomic DNA.
- Open circle probes (OCP) are circularized by DNA ligase upon matching a specific genotype, initiating signal amplification.
- Exponential rolling circle amplification (ERCA) generates a detectable signal from the circularized probe, enabling high-throughput applications due to cost-effectiveness and scalability.
Purpose of the Study:
- To develop and validate a highly accurate, single-tube genotyping assay.
- To optimize ligation and ERCA for direct analysis of genomic DNA.
- To assess the assay's performance for clinically relevant mutations.
Main Methods:
- A single-tube assay combining ligation and ERCA was developed.
- Open circle probes (OCP) interrogated specific genotypes in human genomic DNA samples.
- Fluorescent detection using quenched-peptide nucleic acid (Q-PNA) and multiple ERCA primers accelerated signal generation.
- Probes with hairpin structures were designed to minimize misamplification.
Main Results:
- The assay achieved greater than 99% accuracy in genotyping four clinically relevant mutations (Factor V Leiden, Factor II prothrombin, C282Y, and H63D).
- The entire process, from ligation to fluorescent signal detection, was completed in under one hour directly from genomic DNA.
- Genotyping accuracy remained high (>99%) irrespective of whether purified genomic DNA or whole genome amplified (WGA) DNA was used.
- Both real-time and endpoint measurements of fluorescent signal output were utilized.
Conclusions:
- The optimized single-tube ligation/ERCA assay provides highly accurate genotyping directly from genomic DNA.
- The assay demonstrated over 99% accuracy for four distinct probe sets targeting clinically significant mutations.
- No genotyping errors were observed when using either standard genomic DNA or whole genome amplified DNA samples.