Mw. Carmody et Cph. Vary, AN EMPIRICAL-METHOD FOR THE ESTIMATION OF NONSPECIFIC PCR PRIMER DERIVED GENOMIC REACTIVITY, BioTechniques, 16(6), 1994, pp. 1044-1050
A total of 36 PCR primers were subjected to thermal cycling, in a reac
tion composed of a single primer only, in order to assess their indivi
dual tendencies toward ''nonspecific'' amplification of human genomic
DNA background Nonspecific amplification of genomic DNA was estimated
by Southern hybridization of the amplification products with an Alu-sp
ecific DNA probe. The yield of amplified Alu-hybridizing material was
estimated by scanning densitometry. In this way a quantitative estimat
e of ''mispriming'' was obtained for the primers tested Human Alu and
total primate GenBank(R) databases were scanned in order to obtain an
estimate of the prevalence of homologous primer binding sites for each
primer. Database searches were conducted at both 70% primer binding s
ite homology and 100% homology at the 3'-terminal third of the primer.
The observed levels of amplified, Alu-hybridizing material correlated
best, but only marginally, with homology-based estimates of potential
cross-reactivity at the 3' terminus of the printer (R = 0.193). A set
of p53 tumor suppressor gene primers, exhibiting low and high extreme
s of background amplification, were tested for sensitivity in the pres
ence and absence of added genomic DNA. The primer that gave the highes
t level of background amplification was the one whose performance was
most severely affected by added genomic DNA. Empirical assessment of t
he tendencies of individual primers to amplify irrelevant DNA at low l
evels of the intended target may permit a useful ''noise-specific'' ad
junct to primer design by computational methods