Design tiling PCR primers to achieve complete coverage of the target sequences. Tiling PCR involves dividing a long genomic region into a series of short/long, overlapping amplicons, each of which is amplified by a dedicated primer pair. Adjacent amplicons share a defined overlap to ensure there are no gaps in the coverage. This makes the approach ideal for the high-throughput sequencing of large genes, the validation of variants across entire coding regions and the targeted resequencing of regions.
Primer Design Options
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- Sequences use standard IUB/IUPAC codes:
B=CGT,D=AGT,H=ACT,K=GT,M=AC,N=ACGT,R=AG,S=GC,V=ACG,W=AT,Y=CT. - 3′-end pattern: Use
Nfor any, or specify patterns likeWSS. - Linguistic Complexity (LC%) measures sequence vocabulary richness; 100% = maximum diversity.
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- Target marking:
[target region]for single or multiple targets - Exclude regions:
/excluded/to skip specific areas - Set the desired amplicon size range based on your sequencing platform (NGS vs long-read).