Will preamplification introduce amplification bias?
By limiting both the primer concentration and the number of PCR cycles, the preamplification protocol enables unbiased amplification of specific DNA targets.
Limiting the primer concentration in a multiplex reaction ensures that all primer sets have ample access to other reaction components, such as deoxyribonucleotide triphosphates (dNTPs) and polymerase, regardless of potential differences in amplification efficiency.
To avoid any SNP allele bias at a given locus, the primers used in the preamplification reaction should be target-specific but not allele-specific. It is important that preamplification primer binding sites do not contain additional variants that may be linked to the SNP allele of interest, as this may indirectly lead to allele-specific differences in amplification.
Any preamplification primers used must also be unique. Thus, to preserve SNP allele balance when amplifying from genomic DNA template, a preamplification primer must not target multiple regions of the genome.