Multiplex PCR Primer Design for Long Target Regions with MP-Ref

Multiplex PCR Primer Design for Long Target Regions with MP-Ref

July 21, 2025
Application, MP-Ref
Primer Design, Multiplex PCR, MP-Ref

What’s “Long Target Regions” #

If next-generation sequencing (NGS) or short-read sequencing (e.g., PE150) is used to validate amplicons, target regions ​longer than 200 bp​ should be considered ​long target regions (LTRs)​. This is because the total amplicon length (including primer sequences) should not exceed ​300 bp.

The Primer Design Strategy for “Long Target Regions” #

For ​long-range genotyping (LRG) targets > 200 bp, a ​tiling primer design strategy​ is typically required, often necessitating ​amplification in two separate tubes. MP-Ref automatically group the primer pairs into two different pools.

STR mode

Demo 1: Whole mitochondrial genome #

  • Input: chrM 0 16569
  • Genome: hg38
  • Other options: default

Demo 2: BRCA1/2 whole CDS region #