User's Manual

Primer design guidelines

The Xdrop™ enrichment technology relies on carefully designed and highly specific PCR primer pairs. Two

sets of non-overlapping PCR primer pairs are required. The first one is called dPCR primer set and is

responsible for creating a fluorescent signal used for target selection. The second set of PCR primers, called

the qPCR QC primer set, is used to validate the assay and quantify the number of target fragments in the

pool of enriched fragments.

If the target spans over a region of 50 kb or longer, a combination of two or more sets of dPCR/qPCR primers

are recommended.

Help for designing primers can be found in the online primer design tool at samplix.com

General guidelines for primer design

Apart from the dPCR primer pair, a qPCR QC primer pair is required for validation and calculation of the

enrichment.

dPCR primers

qPCR QC primers

Amplicon length

120-160 bp

80-120 bp

Melting temperature

~ 60°C

Note: The qPCR QC primer pair must be different from the enrichment dPCR primer pair and placed within

2 kb distance from it. The qPCR QC and dPCR amplicons must not overlap.

The risk of false-negative enrichment prediction increases if the validation qPCR assay is placed further

away from the dPCR assay.

The following guidelines apply to both dPCR and qPCR QC primer pairs:

• Avoid primer pairs with more than 2°C difference in melting temperature between forward and

reverse primer.

• Avoid placing primers in low complexity regions.

• Primers need to be specific. Avoid primer pairs that amplify sequences not related to the target

sequence.

• Follow the general recommendations for PCR primer design: avoid self-complementarity, stable

secondary structures, hairpins etc.