PROBER

PROBER designs Tiling Oligonucleotide Probes (TOPs) and PCR primer pairs for fluorescence in-situ hybridization (FISH) and related in‑situ labeling techniques by masking repetitive genomic sequences to target essentially unique genomic regions and enable amplification of 100–2000 base pair fragments.

Key Features:

• Tiling Oligonucleotide Probes (TOPs): Generates tiling probe sets composed of multiple oligonucleotides to cover user-defined genomic intervals.
• Repeat masking: Masks repetitive genomic sequences to restrict probe design to essentially unique regions of the genome.
• Unique genomic block delineation: Identifies essentially unique genomic blocks as targets for probe and primer design.
• Multiple primer pair design: Designs multiple PCR primer pairs that collectively amplify small DNA fragments to produce a robust fluorescent signal.
• Amplification size range: Targets PCR amplicons in the 100–2000 base pair range.
• Small-region targeting: Optimized for compact loci including single genes and small (10–100 kb) genomic regions.
• Alternative to BACs for FISH: Provides probe sets suitable when bacterial artificial chromosomes (BACs) are unstable, chimeric, unavailable, or insufficiently specific for small loci.
• Minimum unique-sequence requirement: Requires loci to contain at least 10 kb of essentially unique sequence for reliable probe generation.
• Application to cancer genomic events: Facilitates probe design for regions involved in genomic amplifications and hemizygous deletions identified by Representational Oligonucleotide Microarray Analysis (ROMA) in breast cancer tumors.

Scientific Applications:

• FISH probe design for small intervals: Produces probe sets for fluorescence in-situ hybridization targeting small genomic intervals and single genes.
• BAC replacement in FISH experiments: Serves as an alternative to BAC-based probes for loci where BACs are unsuitable.
• Validation of copy-number events: Enables validation and localization of genomic amplifications and hemizygous deletions detected by ROMA in breast cancer samples.
• In-situ labeling of 100–2000 bp targets: Supports in-situ amplification and labeling of small DNA fragments within tissues or cells.

Methodology:

Masks repetitive sequences, delineates essentially unique genomic blocks, and designs multiple PCR primer pairs to assemble Tiling Oligonucleotide Probes (TOPs) that amplify 100–2000 bp fragments.