AutoCloner

AutoCloner automates homeologue-specific primer design for polyploid organisms to enable targeted PCR amplification and gene cloning by placing single-nucleotide polymorphisms (SNPs) at primer 3' ends to ensure specificity.

Key Features:

• Homeologue-specific primer design: Designs primers that use SNPs at the 3' end to discriminate between highly similar homeologous sequences in polyploid genomes.
• BLAST search against genome assemblies: Performs a BLAST search of the input sequence against the specified polyploid genome assembly to identify homologous sequences.
• Multiple sequence alignment and SNP mining: Compiles homologous sequences with the input sequence into a multiple sequence alignment and mines that alignment for single-nucleotide polymorphisms (SNPs).
• Primer combination generation: Generates multiple combinations of potential primers to provide coverage across the entire gene of interest.
• Primer evaluation with Primer3: Evaluates candidate primer sets using Primer3 and selects the highest-scoring set.
• Comprehensive primer output: Returns all possible primers at every identified SNP location to supply alternative primer options for PCR.
• Targeted to polyploid crops: Specifically addresses high sequence similarity issues common in polyploid crops such as wheat to reduce non-specific amplification.

Scientific Applications:

• Gene cloning in polyploids: Enables cloning of genes from polyploid organisms by providing homeologue-specific PCR primers.
• Homeologue-specific PCR amplification: Facilitates targeted amplification of a single homeologue among closely related genomic copies.
• Application to wheat genomics: Has been applied to cloning of various genes in the Apogee wheat variety.
• Large-scale primer design: Has been applied to approximately 80,000 high-confidence gene models from a wheat genome assembly.

Methodology:

Starting from a user-provided sequence, AutoCloner performs a BLAST search against the specified genome assembly, compiles identified homologous sequences into a multiple sequence alignment, mines that alignment for SNPs, generates combinations of potential primers across the gene, evaluates primers with Primer3 selecting the highest-scoring set, and returns all possible primers at each SNP location.