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Cloning of deleted sequences (CODE): A genomic subtraction method for enriching and cloning deleted sequences
Abstract:
The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of d7TP After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.
Insights
Researchers developed a new method, cloning of deleted sequences (CODE), to identify specific genomic sequence deletions in cancer. This technique aids in discovering potential therapeutic targets for disease treatment.
Area of Science:
- Genomics
- Molecular Biology
- Cancer Research
Background:
- Genomic sequence deletions are implicated in cancer pathogenesis.
- Identifying these deletions can reveal new therapeutic strategies.
Purpose of the Study:
- To introduce a novel method for selectively cloning deleted genomic sequences.
- To facilitate the identification of disease-associated genomic alterations.
Main Methods:
- Developed the cloning of deleted sequences (CODE) method.
- Utilized restriction enzyme digestion, linker ligation, and PCR amplification.
- Employed selective DNA amplification and purification using uracil-DNA glycosylase (UDG) and mung bean nuclease treatment, followed by streptavidin magnetic bead purification.
Main Results:
- Successfully isolated and cloned genomic sequences homozygously deleted in tumor DNA but present in normal DNA.
- Demonstrated a simple and robust technique for analyzing complex human genomes.
Conclusions:
- The CODE method enables efficient identification of specific genomic deletions.
- This technique holds promise for advancing cancer research and therapeutic development.