Automation of a primer design and evaluation pipeline for subsequent sequencing of the coding regions of all human Refseq genes.

Daniel Lai,Donald R Love

doi:10.6026/97320630008365

Abstract

Screening for mutations in human disease-causing genes in a molecular diagnostic environment demands simplicity with a view to allowing high throughput approaches. In order to advance these requirements, we have developed and applied a primer design program, termed BatchPD, to achieve the PCR amplification of coding exons of all known human Refseq genes. Primer design, in silico PCR checks and formatted primer information for subsequent web-based interrogation are queried from existing online tools. BatchPD acts as an intermediate to automate queries and results processing and provides exon-specific information that is summarised in a spreadsheet format.

Highlights

The molecular confirmation of a clinical diagnosis in the context of human heritable and somatic mutation events largely involves sequencing-based technology
It would be desirable to incorporate other features in a primer design process: in-silico PCR evaluation; the identification of single nucleotide polymorphisms (SNPs) that lie in the human target sequence against which each primer anneals that might result in allele bias in genomic amplification; and optional primer tailing in order to allow for downstream sequencing applications
None of the currently available free primer design software or packages addresses the specific task of designing primers against all exons of a given gene accession input with the ability

Summary

Introduction

The molecular confirmation of a clinical diagnosis in the context of human heritable and somatic mutation events largely involves sequencing-based technology. It would be desirable to incorporate other features in a primer design process: in-silico PCR evaluation; the identification of single nucleotide polymorphisms (SNPs) that lie in the human target sequence against which each primer anneals that might result in allele bias in genomic amplification; and optional primer tailing in order to allow for downstream sequencing applications.

Results

Conclusion