Spermidine facilitates PCR amplification of target DNA.

Department of Agronomy and Range Science, University of California, Davis, California 95616-8515 Recent advances in PCR have made this technique one of the most powerful tools for a wide spectrum of molecular analyses, such as genome mapping, molecular evolution, diagnosis of genetic disease, and forensic sciences. ~ Many PCR applications involve the specific and reproducible amplif icat ion of genomic DNA from biological samples. However, inconsistent PCR amplificat ion results are l inked primari ly to the quality and quanti ty of the template, as well as other parameters, such as components in the reaction mixture, cycling conditions, and the type of thermal cycler used. Genomic DNA isolated from plants is known to contain higher levels of phenol ic compounds and polysaccharides than DNA purified from animal cells. Phenolic compounds are especially troublesome because they oxidize readily during homogenizat ion, irreversibly interact with proteins and nucleic acids and, consequently, h inder molecular analysis. (2~ Inclusion of adjuvants such as DMSO and Tween 20 are reported to counteract the inhibi tory effects on PCR by some plant acidic polysaccharides. r Under condit ions where the nucleotide composi t ion or the quality of DNA is a l imi t ing factor, techniques such as "hotstart" PCR, ~1'4's~ "GC clamp, ''~1~ the addit ion of single-stranded DNA-binding protein, ~6~ or formamide (7~ to the react ion mixture have been found to improve the specificity of amplif icat ion from genomic DNA. Spermidine [N-(3-aminopropyl)1,4butanediamine] is a polyamine that is routinely included in restriction enzyme digestions to improve the cleavage efficacy of the DNA. Spermidine counteracts the inhibi tory effects of contaminants coisolated with DNA and consequent ly permits complete digestion of the DNA at lower enzyme concentrations. Experiments in vitro show that spermidine has a h igh affinity for nucleic acids and neutralizes at least part of the negative charges in the phosphate backbone, thereby stabilizing DNA and RNA. (8~ Polyamines are also known to stimulate the activities of the enzymes involved in nucleic acid metabolism, such as DNA and RNA polymerases ~9'~~ and topoisomerases. (~) In this paper we examined the s t imulat ing effect of spermidine and several PCR enhancers on amplif icat ion of the 69-kD vacuolar H+-ATPase catalytic subuni t (subunit A) genes from cotton genomic DNA. Contrary to earlier reports spurning the use of po lyamines in PCR, ~12~ we de te rmined that micromolar concentrat ions of spermidine enhanced PCR amplif icat ion signif icantly from plant DNA and that inclus ion of spermidine to reactions was vastly superior to the compensatory effects of hotstart PCR. Reactions supplemented with the PCR adjuvants d imethylsul foxide (DMSO), formamide, or Tween 20 failed to ampli fy target genes, suggesting that acidic polysaccharides were not a contr ibuting factor to PCR ampl i f ica t ion problems encountered with the DNA used in this study.