Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction

BMC Bioinformatics

Table 2 Summary of potential unintended targets for primer pairs reported by QuantPrime and PRIMEGENES ^a

	QuantPrime	PRIMEGENS ^b
Total number of test cases	52	24
Number of cases where QuantPrime or PRIMEGENS was able to generate primers	38	15
Number of cases with potential unintended targets	12	14
Percentage of cases having at least one primer pair with potential unintended target	31.5% (12/38)	93.3% (14/15)
Number of total primer pairs generated	373	138
Number of primer pairs with potential unintended targets	50	60
Percentage of primer pairs with potential unintended targets	13.4% (50/373)	43.3% (60/138)
Number of potential unintended targets	162	116
Number of potential unintended targets with one or two mismatches to forward or reverse primer	30	4
Percentage of potential unintended targets with one or two mismatches to forward or reverse primer	18.5% (30/162)	3.4% (4/116)

^a: Primer pairs generated by QuantPrime or PRIMEGENES from randomly selected templates (see Additional files 1 and 2 for details) were fed into Primer-BLAST for target specificity checking using the matching organism (i.e., Human for QuantPrime and Arabidopsis thaliana for PRIMEGENS) and matching database (RefSeq mRNA database for all cases). Default search parameters were used for Primer-BLAST searches which can detect targets with up to 5 mismatches to primers. Since QuantPrime and PRIMEGENES still use databases that are a few years old and Primer-BLAST uses the regularly updated NCBI database, we only counted those unintended targets found by Primer-BLAST that are also present in QuantPrime database (RefSeq 04/30/09 (reference assembly)(genome+) (splice variants)) or PRIMEGENS database (Arabidopsis TAIR9 cDNA) . The searches were performed between 2/3/2012 and 2/6/2012
^b: Since PRIMEGENS adds non-specific primer pairs (i.e., pairs having more than one hybridization targets, see Additional file 2) to the primer result if it does not find sufficient number of target-specific primer pairs (i.e., pairs having only one hybridization target), we excluded all non-specific primer pairs in our analysis. In essence, our analysis only includes primer pairs that are deemed to be specific for the test template sequences by PRIMEGENS.

ISSN: 1471-2105