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Coincidence painting: a rapid method for cloning region specific DNA sequences
D M Bailey1, N P Carter, D de Vos
1Department of Pathology, Cambridge University, UK.
Nucleic Acids Research
|November 11, 1993
Summary
A new Coincidence Painting method rapidly generates specific DNA sequences using Degenerate Oligonucleotide Primed PCR and in situ hybridization. This technique aids in creating sequence tagged sites for genetic research.
Area of Science:
- Molecular Biology
- Genetics
- Genomics
Background:
- Generating region-specific DNA sequences is crucial for genetic mapping and analysis.
- Existing methods can be time-consuming and lack efficiency for large-scale applications.
Purpose of the Study:
- To develop a novel, rapid method for generating large numbers of region-specific DNA sequences.
- To enable efficient creation of sequence tagged sites (STSs) for genetic research.
Main Methods:
- Coincidence Painting strategy utilizing Degenerate Oligonucleotide Primed PCR (DOP-PCR) amplification.
- In situ hybridization of PCR products onto flow-sorted chromosomes for sequence selection.
- Novel insert end revelation and ligation technique for cloning.
Main Results:
- Successful generation of region-specific sequences with insert sizes from 150-1300 bps.
- Approximately 54% of cloned inserts were identified as single-copy sequences.
- Demonstrated utility in YAC and cosmid screening for genetic mapping.
Conclusions:
- Coincidence Painting offers a rapid and efficient approach for generating region-specific DNA probes.
- The methodology facilitates the rapid saturation of genomic regions with probes, particularly those defined by translocation breakpoints.
- Enables the generation of sequence tagged sites for advanced genetic studies.