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Recent advances in PCR have made this ports spurning the use of polyamines in .S_permidine technique one of the most powerful PCR, ~12~ we determined that micromolar Facilitates PCR tools for a wide spectrum of molecular concentrations of spermidine enhanced analyses, such as genome mapping, mo- PCR amplification significantly from Amplification of lecular evolution, diagnosis of genetic plant DNA and that inclusion of spermi- disease, and forensic sciences. ~ Many dine to reactions was vastly superior Target DNA PCR applications involve the specific to the compensatory effects of hot- and reproducible amplification of ge- start PCR. Reactions supplemented with nomic DNA from biological samples. the PCR adjuvants dimethylsulfoxide However, inconsistent PCR amplifica- (DMSO), formamide, or Tween 20 failed Ching-Yi Wan and tion results are linked primarily to the to amplify target genes, suggesting that Thea A. Wilkins quality and quantity of the template, as acidic polysaccharides were not a con- well as other parameters, such as compo- tributing factor to PCR amplification Department of Agronomy and Range nents in the reaction mixture, cycling problems encountered with the DNA Science, University of California, Davis, conditions, and the type of thermal cy- used in this study. California 95616-8515 cler used. Genomic DNA isolated from plants is known to contain higher levels of phenolic compounds and polysaccha- MATERIALS AND METHODS rides than DNA purified from animal Cotton Genomic DNA Isolation cells. Phenolic compounds are especially troublesome because they oxidize readily Young expanding cotton leaves were during homogenization, irreversibly in- harvested from diploid (Gossypium her- teract with proteins and nucleic acids baceum L. and G. laxum Phillips, genome and, consequently, hinder molecular designation A 1 and D 8, respectively) and analysis. (2~ Inclusion of adjuvants such tetraploid [G. hirsutum L., genome desig- as DMSO and Tween 20 are reported to nation (AD)I] plants grown in the green- counteract the inhibitory effects on PCR house. The leaves were frozen in liquid by some plant acidic polysaccharides. r nitrogen and stored at -80~ until use. Under conditions where the nucleotide Genomic DNA was isolated according to composition or the quality of DNA is a the procedures described by Galau et limiting factor, techniques such as "hot- al. ~13~ except for the following modifica- start" PCR, ~1'4's~ "GC clamp, ''~1~ the ad- tions. The extraction buffer contained dition of single-stranded DNA-binding 0.5% sodium deoxycholate (wt/vol), and protein, ~6~ or formamide (7~ to the reac- 0.5% NP-40 (vol/vol), and 20 mM dithio- tion mixture have been found to im- threitol (DTT), which was substituted for prove the specificity of amplification 2-mercaptoethanol. Nucleic acid pellets from genomic DNA. recovered after isopropanol precipita- Spermidine [N-(3-aminopropyl)- 1,4- tion were washed gently with 70% etha- butanediamine] is a polyamine that is nol, lyophilized, and resuspended in 1.5 routinely included in restriction enzyme ml of TE buffer [10 mM Tris-HCl (pH digestions to improve the cleavage effi- 8.0), 1 mM EDTA]. The resuspended ge- cacy of the DNA. Spermidine counteracts nomic DNA was purified further by CsC1 the inhibitory effects of contaminants density gradient centrifugation as de- coisolated with DNA and consequently scribed by Maniatis et al. (14~ For non- permits complete digestion of the DNA CsCl purified DNA, the nucleic acid at lower enzyme concentrations. Experi- pellet was resuspended in TE buffer con- ments in vitro show that spermidine has taining RNase A at a concentration of 1.0 a high affinity for nucleic acids and neu- mg/ml and incubated overnight at 4~ tralizes at least part of the negative Proteinase K (U.S. Biochemical 20818) charges in the phosphate backbone, was added to the DNA to a final concen- thereby stabilizing DNA and RNA. (8~ tration of 50 i~g/ml of TE buffer and in- Polyamines are also known to stimulate cubated at 42~ for 1.5 hr. The genomic the activities of the enzymes involved in DNA was extracted once with phenol/ nucleic acid metabolism, such as DNA chloroform/isoamyl alcohol (25:24:1, and RNA polymerases ~9'~~ and topoi- vol/vol/vol), followed by another extrac- somerases. (~) In this paper we examined tion with chloroform/isoamyl alcohol the stimulating effect of spermidine and (24:1, vol/vol). The size of the genomic several PCR enhancers on amplification DNA was estimated by agarose gel elec- of the 69-kD vacuolar H+-ATPase cata- trophoresis, and DNA concentrations lytic subunit (subunit A) genes from cot- were determined in a DNA fluorometer ton genomic DNA. Contrary to earlier re- or spectrophotometer.

208 PCR Methods ondApplicotions 3:208-2109 byCold Spring Harbor Laboratory Press ISSN 1054-9803/93 $5.00 Downloaded from genome.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

PCR Amplification of Genomic DNA unit A genes were amplified from non- CsC1- and CsCl-purified genomic DNA Two synthetic oligonucleotide primers isolated from three cotton species. Two (COT2 and COT8) were used to amplify genomic PCR fragments of 558 and 690 the nonhomologous region of the 69-kD bp were expected to be amplified by the vacuolar H +-ATPase subunit genes from selected primers as shown in Figure 1. cotton. (ls~ The PCR reactions contained The amplification reactions with non- the following components unless speci- CsCl-purified DNA were either unsuc- fied: 20 ng of genomic DNA per 25-~1 cessful (lane 5) or produced a low yield reaction and 0.2 ~M of each primer, 50 of only the 558-bp fragment (lanes 1,3). mM KC1, 10 mM Tris-HC1 (pH 8.8 at FIGURE 2 Comparison of the enhancing ef- CsCl-purified DNA greatly improved the 25~ 1.5 mM MgCl2, 0.1% (vol/vol) Tri- fect of hot-start PCR and spermidine. Slight PCR amplification of both fragments; ton X-100, 50 p~M of each dNTP, and 0.04 enhancement of PCR amplification by hot- however, variability in yield of the 690- start PCR (lanes 3,4) relative to conventional units of Taq DNA polymerase (Promega) bp fragment was observed from experi- PCR (lanes 1,2) was observed. Addition of per microliter of reaction. PCR mixtures ment to experiment (lanes 7,8). Supple- spermidine alone (lanes 5,6) and combina- were supplemented with 5% DMSO (vol! menting the reactions with 1 mM tion of hot start plus spermidine (lanes 7,8) vol), 5% formamide (volIvol), 0.5% spermidine significantly enhanced the showed significant improvement in yield of Tween 20 (vol/vol), (3) or 0.1-2 mM sper- amplification of both genomic frag- both 558- and 690-bp fragments of the vacu- midine. The reaction mixture was over- olar H § subunit A genes from cotton. ments from either non-CsCl- or CsCl~ layed with an equal volume of mineral purified templates (lanes 2,4,6,9). A titra- oil and submitted to denaturation for 2 tion of spermidine concentrations span- min at 94~ in a Ericomp thermal cycler, ning from 0.1 to 2 mM established an served by the increased yield of the 558- followed by 30 cycles of amplification (1 optimal range between 0.2 and 1 mM bp fragment (lane 3) and the presence of min at 94~ 1 min at 48~ and 1 min at (data not shown). the 690-bp fragment (lane 4). However, 72~ and an elongation cycle of 10 min Alternative approaches, such as hot- hot-start PCR was often ineffectual in en- at 72~ Hot-start PCR was performed by start PCR, have been developed to im- hancing PCR amplification, or the re- adding the Taq polymerase at 80~ after prove specificity of PCR amplification. sults were highly variable (data not denaturation for 2 min at 94~ followed Hot-start PCR is reported to minimize shown). The efficacy of using hot-start by PCR amplification with regular cy- nontarget amplification and the forma- PCR was presumably influenced by the cling conditions. The products were an- tion of primer-dimer. (1~ The enhancing timing and degree of mechanical mixing alyzed by agarose gel electrophoresis and effects of hot start versus spermidine in of the enzyme in the reaction. However, visualized under UV light. PCR amplification from non-CsCl-puri- the supplement of 0.4 mM spermidine to fled cotton genomic DNA was investi- the reactions significantly enhanced the gated. As shown in Figure 2, amplifica- amplification of both fragments (lanes RESULTS AND DISCUSSION tion of non-CsCl-purified DNA with PCR 5,6). The combination of hot-start PCR Extraction of high-quality genomic DNA was either unsuccessful (lane 1) or pro- and spermidine produced results similar for molecular analysis from most plant duced low yields of only the 558-bp frag- to those with the addition of spermidine species frequently requires tedious puri- ment (lane 2). Slight enhancement of alone (lanes 5,6), indicating that spermi- fication procedures. Gossypium (cotton) PCR amplification by hot start was ob- dine was superior in ameliorating PCR species are especially recalcitrant be- specificity, especially when dirty DNA cause of interference by elevated levels was employed as a template. No poten- of endogenous polysaccharides and phe- tial synergistic effects were observed in nolic compounds. PCR amplification of any hot-start PCR amplification experi- genomic DNA isolated by different ments supplemented with spermidine. methods or on different dates was highly Relative to hot-start PCR, the initial ad- variable because of the quality of the dition of spermidine to reaction mix- template DNA. However, purity does not tures simplifies PCR amplification setup necessarily explain the occasional incon- procedures. sistency observed in PCR amplification Optimization of PCR amplification of CsCl-purified genomic DNA from the conditions by the addition of common same preparation. Spermidine is known laboratory reagents has gained wide- to enhance the ability of restriction en- FIGURE 1 The effect of 1 mM spermidine in spread attention. Inclusion of forma- zymes to digest "dirty" DNA (8~ and stim- PCR amplification of the nonhomologous re- mide as a denaturant increases the spec- ulate the enzyme activities of DNA and gion of vacuolar H § subunit A genes ificity during PCR amplification of RNA polymerases in vitro. ~9~ Therefore, from non-CsCl-purified (lanes 1-6) and CsCl- genomic DNA, especially GC-rich re- purified (lanes 7-9) cotton genomic DNA our goal was to examine the influence of gions. (7~ In particular, DMSO and Tween from three Gossypium species [genome desig- spermidine and several other PCR en- nations A1, Ds, and (AD)I]. Significant en- 20 counteract the inhibitory effects of hancers on amplification of DNA from hancement in yields of the expected 558- and some acidic polysaccharides on PCR am- various sources by Taq DNA polymerase. 690-bp fragments by spermidine (lanes plification of plant DNA, whereas forma- To investigate the effect of spermi- 2,4,6,9) was observed compared with reac- mide is ineffectual. (3~ However, despite dine in PCR, vacuolar H§ sub- tions without spermidine (lanes 1,3,5,7). the promise of these buffer adjuvants as

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PCR enhancers, reactions supplemented additives on PCR. BioTechniques 12: 332- with DMSO, formamide, or Tween 20 334. failed to produce the expected PCR prod- 4. D'Aquila, R.T., L.J. Bechtel, J.A. Videler, ucts in our laboratory (data not shown), J.J. Eron, P. Gorczyca, and J.C. Kaplan. 1991. Maximizing sensitivity and specific- indicating that polysaccharides were not ity of PCR by preamplification heating. a consideration. Nucleic Acids Res. 19: 3749. Spermidine enhancement of PCR was 5. Mullis, K. B. 1991. The polymerase chain consistent and reproducible in indepen- reaction in an anemic mode: How to dent experiments using assorted primer avoid cold oligodeoxyribonuclear fusion. pairs and different sources of DNA. For PCR Methods Applic. 1: 1-4. instance, vacuolar H § subunit A 6. Oshima, R.G. 1992. Single-stranded DNA genes from other plant species were not binding protein facilitates amplification evident following PCR amplification in of genomic sequences by PCR. BioTech- the absence of spermidine, whereas the niques 13: 188. 7. Sarkar, G., S. Kapelner, and S.S. Sommer. expected PCR products were amplified 1990. Formamide can dramatically im- successfully in the presence of spermi- prove the specificity of PCR. Nucleic Acids dine as detected by DNA hybridization Res. 18: 7465. (data not shown). Thus, the increased 8. Bouch~, J.P. 1981. The effect of spermi- quality, yield, and specificity of PCR dine on endonuclease inhibition by aga- products generated in a broad spectrum rose contaminants. Anal. Biochem. 115: of applications in our laboratory indicate 42-45. that the routine supplement of spermi- 9. Tobor, C.W. and H. Tobor. 1976. 1,4-Di- dine to the PCR reactions apparently aminiobutane (putrescine), spermidine compensates for contaminants in DNA and spermine. Annu. Rev. Biochem. 53" 749-790. that tend to inhibit PCR amplification. 10. Fisher, P.A. and D. Korn. 1979. Enzymo- The enhancing effect of spermidine was logical characterization of KB cell DNA also observed on PCR amplification of polymerase-c~. II. Specificity of the pro- phage DNA, phage libraries, plasmid tein-nucleic acid interaction. J. Biol. DNA, cDNAs, and DNA in low-melting- Chem. 254: 11033-11039. point agarose (data not shown). 11. Pommier, Y., D. Kerrigan, and K. Kohn. Spermidine is an inexpensive chemi- 1989. Topological complexes between cal that exerts a significant enhancing ef- DNA and topoisomerase II and effects of fect in PCR amplification of DNA with polyamines. Biochemistry 28: 995-1002. higher specificity and reproducibility, 12. Blanchard, M.M., P. Taillon-Miller, P. No- wotny, and V. Nowotny. 1993. PCR buffer yet allows simplified DNA isolation and optimization with uniform temperature PCR reaction setup procedures to be em- regimen to facilitate automation. PCR ployed. The addition of spermidine in Methods Applic. 2: 234-240. the PCR amplification reactions may 13. Galau, G.A., H.W. Bass, and D.W. Hughes. also be advantageous in random ampli- 1988. Restriction fragment length poly- fied polymorphic DNA-PCR (RAPD- morphisms in diploid and allotetraploid PCR), as well as other general PCR appli- Gossypium: Assigning the late embryogen- cations and genetic analysis. esis-abundant (Lea) alloalleles in G. hirsu- tum. Mol. Gen. Genet. 211: 305-314. 14. Maniatis, T., E.F. Fritsch, and J. Sambrook. ACKNOWLEDGMENTS 1982. Molecular cloning: A laboratory man- ual. Cold Spring Harbor Laboratory, Cold This work was supported by grants from Spring Harbor, New York. the U.S. Department of Energy (DE- 15. Wilkins, T.A. 1993. Vacuolar H+-ATPase FG03-92ER20067) and Cotton Incorpo- 69-kilodalton catalytic subunit cDNA rated to T.A.W. from developing cotton (Gossypium hirsu- turn) ovules. Plant Physiol. 102: 679-680.

REFERENCES Received August 2, 1993; accepted in 1. Erlich, H.A., D. Gelfand, and J.J. Sninsky. revised form October 18, 1993. 1991. Recent advances in the polymerase chain reaction. Science 252: 1643-1651. 2. Loomis, W.D. 1974. Overcoming prob- lems of phenolics and quinones in the isolation of plant enzymes and or- ganelles. Methods Enzymol. 31: 528-545. 3. Demeke, T. and R.P. Adams. 1992. The ef- fects of plant polysaccharides and buffer

210 PCR Methods and Applications Downloaded from genome.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

Spermidine facilitates PCR amplification of target DNA.

C Y Wan and T A Wilkins

Genome Res. 1993 3: 208-210

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