W654 Nucleic Acids Research, 2006, Vol. 34, Web Server issue will avoid any SNPs within the primer and any repeats overlapping with the 30-part of the primer. An example of such PCR-speciﬁc masking is shown in Figure 2B. This example demonstrates a case where the investigator is interested in ampliﬁcation of the region around third SNP (rs851320). SNP together with the 25 bp of the ﬂanking region is deﬁned as a target for the ampliﬁcation (shown in italic). GenomeMasker is used in a strand-speciﬁc mode which means that only the upper strand is masked on the left side of the target and only the lower strand is masked on the right side of target region. The strand-speciﬁc masking allows ﬁnding more potential PCR primer candidates in this repeat-rich region. For hybridization probe design or for other purposes it may be useful to mask SNPs together with RepeatMasker (Figure 2C). Figure 2. Examples of different masking styles. Masked repeats are shown in boldface and SNPs are highlighted in red for the visualization in this Figure. (A) Original sequence from the human genome sequence, assembly NCBI35.1. (B) The same sequence masked for PCR primer design with the GenomeMasker using parameter ‘target’. Asymmetrical masking is used—on the left side of target the upper strand is masked, on the right side of target the lower strand is masked. The middle part around the third SNP (shown in italic) is the target region which is chosen to be amplified. (C) The sequence masked with the RepeatMasker. (D) Population-specific masking of SNPs. The original nucleotides in the genome sequence have been substituted with a population-specific (lower-case) nucleotides using HapMap frequency information.