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Published on: June 25, 2014
Optical PCR: genomic analysis by long-range PCR and optical mapping
1W.M. Keck Laboratory for Biomolecular Imaging, New York University, Department of Chemistry, Room 866, 31 Washington Place, New York, New York 10003, USA.
Summary
Optical mapping combined with long-range PCR enables rapid, automated DNA restriction mapping of large genomic regions. This optical PCR technique generates high-resolution physical maps without extensive prior sequencing, useful for complex gene loci.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Optical mapping provides rapid, automated DNA restriction mapping of single molecules.
- Previous applications focused on large insert clones like BACs and genomic DNA.
Purpose of the Study:
- To combine optical mapping with long-range PCR for automated restriction mapping of long PCR products.
- To demonstrate the utility of this 'optical PCR' method for human genomic loci.
Main Methods:
- Amplification of three long PCR products (avg. 14.6 kb) spanning the human tissue plasminogen activator (TPA) gene.
- Surface mounting of PCR products in gridded arrays for parallel mapping.
- Restriction digestion and optical mapping using enzymes like ScaI, XmnI, HpaI, ClaI, and BglII.
Main Results:
- Construction of a contig of overlapping, high-resolution restriction maps.
- Generated maps closely matched predictions from existing sequence data.
- Demonstrated successful mapping of 37-kb human TPA gene locus.
Conclusions:
- Optical PCR enables automated physical mapping of genomic loci with minimal prior sequence information.
- This method allows rapid, high-resolution mapping of large DNA segments without cloning vectors.
- The approach is practical for genomic regions where sequence data is limited.
