MSP-HTPrimer

MSP-HTPrimer designs and selects bisulfite- and MSRE-compatible PCR primers to enable high-throughput DNA methylation analysis.

Key Features:

• Integrated Approach: Combines BS- and MSRE-based methods to enhance sensitivity and specificity in quantifying DNA methylation status.
• High-Throughput Capability: Handles numerous target sequences without limitations on their number or size.
• Comprehensive Primer Design: Converts target sequences into bisulfite-treated templates for both forward and reverse strands and designs potential primer pairs while filtering based on single nucleotide polymorphisms (SNPs) and known repeat regions.
• Annotation and Selection: Annotates each primer pair with upstream and downstream RefSeq genes, CpG islands, and COBRA and MSRE cut sites, and applies user-defined hierarchical criteria to select primers from both strands.
• Output Formats: Generates a summary output table in TXT and HTML formats and UCSC custom tracks in GTF format for genomic visualization.
• Experimental Validation: Demonstrated high validation rate, with 63 of 66 BSP primer pairs validated without further optimization under the same qPCR conditions.

Scientific Applications:

• Epigenetic research: Supports precise DNA methylation analysis in studies of epigenetic regulation.
• Assay design: Supports Methylation-Specific PCR (MSP), Bisulfite Sequencing (BSP), pyrosequencing, Combined Bisulfite Restriction Analysis (COBRA), and MSRE assays by designing primers on both genomic strands to increase likelihood of successful assay development.

Methodology:

Converts target sequences into bisulfite-treated templates for both strands, designs potential primer pairs, filters primers based on SNPs and repeat regions, annotates primer pairs with RefSeq genes, CpG islands, and COBRA/MSRE cut sites, applies user-defined hierarchical selection criteria, and outputs results in TXT, HTML, and GTF (UCSC custom tracks) formats.