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A rapid method to identify cosmids containing rare restriction sites.
Nucleic Acids Research
|February 25, 1987
Summary
Researchers developed a new method to find specific cosmid clones with rare-cutting restriction enzyme sites. This technique helps map rare cutter sites, simplifying the creation of DNA sequence milestones.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Identifying rare-cutting restriction enzyme sites in DNA is crucial for genome mapping.
- Cosmid cloning is a common method for storing large DNA fragments.
Purpose of the Study:
- To develop a procedure for identifying cosmid clones containing specific rare-cutting restriction enzyme recognition sites.
- To simplify the generation of rare cutter maps for human chromosomes or regions.
Main Methods:
- Developed a hybridization-based procedure using labeled oligonucleotide probes.
- Optimized melting characteristics to differentiate between perfect and mismatched DNA hybrids.
- Screened 100 cosmid clones containing human inserts.
Main Results:
- Successfully detected cosmids containing NotI (8 bp match) and XmaIII/EagI (6 bp match) recognition sites.
- The method allows for the identification of specific rare cutter sites within cosmid DNA.
Conclusions:
- This approach simplifies the mapping of rare cutter sites along DNA sequences.
- Enables the identification of "milestones" at intervals of several hundred kilobases (kb) for DNA sequencing.