Complete decontamination and regeneration of DNA purification silica columns

Analytical Biochemistry 385 (2009) 182–183

Contents lists available at ScienceDirect

Analytical Biochemistry j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y a b i o

Notes & Tips

Complete decontamination and regeneration of DNA purification silica columns Marcello Tagliavia *,1, Aldo Nicosia 1, Fabrizio Gianguzza Di­par­ti­men­to di Bi­o­lo­gia Cel­lu­lare e dello Sviluppo, Uni­ver­sità di Palermo, 90128 Palermo, Italy

a r t i c l e

i n f o

Article history: Received 22 September 2008 Available online 1 November 2008  Key­words: Geno­mic DNA puri­fi­ca­tion Sil­ica col­umns regen­er­a­tion

a b s t r a c t Sil­ica col­umns are among the most used DNA puri­fi­ca­tion sys­tems, allow­ing a good yield of high-qual­ity nucleic acids with­out organic extrac­tions. Sil­ica col­umn regen­er­a­tion pro­to­cols reported up to now to remove DNA traces are time-con­sum­ing, and their effec­tive­ness on geno­mic DNA has not been dem­on­ strated. Here we report a very rapid regen­er­a­tion pro­ce­dure that ensures no DNA car­ry­over, inde­pen­ dent of its size, with­out impair­ing col­umn effi­ciency. The method takes advan­tage of the improved DNA removal by low con­cen­tra­tions of Tri­ton X-100. © 2008 Else­vier Inc. All rights reserved.

Sil­ica and glass fiber col­umns are among the most used DNA puri­fi­ca­tion sys­tems, ensur­ing the recov­ery of high-qual­ity DNA with­out any organic extrac­tion. Such col­umns are com­mer­cially avail­able for the puri­fi­ca­tion of either small mol­e­cules (e.g., PCR frag­ments, plas­mids) or geno­mic DNA suit­able for dif­fer­ent appli­ ca­tions. How­ever, their major dis­ad­van­tage is the cost given that they can be used only once because after elu­tion substantial amounts of DNA remain in the sil­ica matrix. Thus, the pos­si­bil­ity of recy­cling them quickly might be desir­able. The main chal­lenge in every regen­er­a­tion pro­ce­dure is the ­com­plete removal of any detect­able DNA trace. In the past, dif­ fer­ent meth­ods for col­umn regen­er­a­tion have been pro­posed, but they either do not avoid car­ry­over con­tam­i­na­tion [1–3] or are time-con­sum­ing (>24 h) [4] and have not been dem­on­strated to be effec­tive in the elim­i­na­tion of geno­mic DNA [4,5]. Sil­ica-bound DNA could be the­o­ret­i­cally expected to be ­effi­ciently dep­u­ri­nat­ed and removed by strong acids even after short expo­sures, mak­ing longer treat­ments (as pro­posed in other pro­to­cols [4]) dis­pens­able. How­ever, after such a short regen­er­ a­tion pro­ce­dure, small amounts of ampli­fi­able DNA can still be detected (Fig. 1, lane 1). We have hypoth­es­ ized that such fail­ure might be due to an incom­plete per­me­ation of the acidic solu­tion into the sil­ica matrix, where the nucleic acid might still be bound to sil­ica or trapped because of its high molec­u­lar weight [5]. This might allow var­i­ able amounts of DNA to escape the dep­u­ri­nat­ing agent, result­ing in resid­ual ampli­fi­able traces. This lim­i­ta­tion can be over­come by the pro­ce­dure described below. It can be com­pleted in approx­i­mately 45 min and allows not only regen­er­a­tion of sil­ica col­umns con­tam­i­nated by DNA of any size but also substantial time sav­ings.

The method con­sists in sequen­tial alka­line and acidic ­treat­ments that dena­ture and dep­u­ri­nate, respec­tively, any DNA still pres­ent in the col­umn. A fur­ther alka­line treat­ment hydro­lyzes long dep­u­ ri­nat­ed DNA mol­e­cules, reduc­ing them into very small frag­ments. These chem­i­cal treat­ments are per­formed in the pres­ence of a low con­cen­tra­tion of Tri­ton X-100, which has been shown to improve DNA removal. To test our method, exper­i­ments were car­ried out using sil­ica col­umns with resid­ual yeast geno­mic DNA. Each col­umn was first loaded with a solu­tion con­tain­ing 1 N NaOH and 0.15% (v/v) Tri­ton X-100, incu­bated for 5 min, and briefly cen­tri­fuged. Then a vol­ume of 1.5 N HCl/0.15% (v/v) Tri­ton X-100 solu­tion was added, and the col­umns were incu­bated for 30 min at room tem­per­a­ture. After a brief cen­tri­fu­ga­tion, the acid solu­tion was dis­carded and the col­ umns were treated with 1 N NaOH/0.15% (v/v) Tri­ton X-100, incu­ bated for 5 min, and briefly cen­tri­fuged. A final wash­ing was per­formed using a vol­ume of ster­ile ­dou­bly dis­tilled H2O (ddH2O)2 cor­re­spond­ing to the max­i­mum capac­ity of the col­umns (» 600 ll) to flush out both hydro­lyzed DNA frag­ments and NaOH traces. Any resid­ual DNA was then recov­ered by dou­ble elu­tion (2 £ 50 ll) with ster­ile ddH2O (the pH value was checked after elu­tion to con­firm the neu­tral­ity). Approx­i­mately half of the elu­ate was used as a tem­plate in a poly­mer­ase chain reac­tion (PCR) assay using the short ITS-2 region of the high-copy-num­ber ribo­somal gene clus­ters as a tar­get sequence so as to improve the detec­tion sen­si­tiv­ity of any trace of ampli­fi­able DNA. No PCR prod­ ucts were obtained in ana­lyz­ing elu­ates from the col­umns treated with Tri­ton X-100-con­tain­ing solu­tions (Fig. 1A, lane a2), thereby prov­ing the com­plete removal of DNA. In addi­tion, fur­ther assays were car­ried out to eval­ua ­ te the ­treat­ment effi­ciency even in the deg­ra­da­tion of large amounts of

* Cor­re­spond­ing author. Fax: +39-091-6577-430. E-mail address: m.ta­gli­avi­a@un­i­pa.it (M. Tagliavia). 1 These authors con­trib­uted equally to this work.

2 Abbre­vi­a­tions used: ddH2O, dou­bly dis­tilled H2O; PCR, poly­mer­ase chain reac­ tion; dsDNA, dou­ble-stranded DNA; IGS, inter­genic spacer.

0003-2697/$ - see front matter © 2008 Else­vier Inc. All rights reserved. doi:10.1016/j.ab.2008.10.021



Notes & Tips / Anal. Biochem. 385 (2009) 182–183

183

Fig. 1. PCR results of DNA sam­ples from regen­er­ated col­umns. (A) Anal­y­sis of PCR prod­ucts from elu­ates after col­umn regen­er­a­tion. Here 20 ll of each sam­ple (a–c, lanes 1 and 2) and 1 ll of con­trol PCR (a–c, lane 3) were loaded onto a 1.8% aga­rose gel and stained with ethi­dium bro­mide (1 lg/ml) after elec­tro­pho­re­sis. PCR reac­tions were car­ried out in a total vol­ume of 40 ll. M: 100-bp lad­der (Fer­men­tas); a–c, lane 1: PCR anal­y­sis of elu­ates from col­umns treated with deter­gent-free solu­tion; a–c, lane 2: PCR anal­y­sis of elu­ates from col­umns treated with solu­tions sup­ple­mented with Tri­ton X-100; a–c, lane 3: con­trols from elu­ates of untreated col­umns. (a) Col­umns con­tam­i­nated by geno­mic yeast DNA. (b,c) Col­umns treated after the load­ing of plasmi­dic DNA and a COX-1 PCR frag­ment. PCR prod­ucts cor­re­spond to the full-length COX-1 PCR frag­ment (b) and to 380 bp of the plasmi­dic b-lac­ta­mase gene (c). (B) Cap­il­lary elec­tro­pho­re­sis anal­y­ses of FAM-59-labeled PCR prod­ucts ampli­fied from geno­mic DNA sam­ples puri­fied with regen­er­ated col­umns. The same sil­ica col­umn was used to sequen­tially purify geno­mic DNA from S. coe­li­color M145, E. coli K12, and again S. coe­li­color M145, regen­er­at­ing it after each puri­fi­ca­tion cycle. PCR anal­y­ses were car­ried out using the lat­ter two DNA sam­ples. Cap­il­lary elec­tro­pho­re­sis was per­formed with an ABI Prism 310 Genetic Ana­lyzer in the pres­ence of Gene­Scan 500 LIZ Size Stan­dard (ABI) (marked peaks). DNA frag­ment anal­y­sis was per­formed with Gene­Scan 2.1 soft­ware (ABI).

small DNA mol­e­cules as PCR prod­ucts and plas­mids. Here 2 lg of an 860-bp PCR frag­ment (from yeast COX-1) and 10 lg of a 4-kb plas­ mid (pBAD, Invit­ro­gen) were loaded onto the col­umns (max­i­mum bind­ing capac­ity of 25 lg dou­ble-stranded DNA [dsDNA]). After DNA bind­ing, no elu­tion was per­formed so as to improve the detec­tion sen­si­tiv­ity of any resid­ual ampli­fi­able mol­e­cules. Then the col­umns were treated as described ear­lier (except for the omis­sion of the ini­tial alka­line treat­ment) using solu­tions with or with­out Tri­ton X-100. After the final wash­ing, the elu­ates were ana­lyzed by PCR using the b-lac­ta­mase plasmi­dic gene and the COX-1 frag­ment as tar­get sequences. Ampli­fi­able DNA is shown to still be pres­ent in elu­ates from col­umns treated with detergent-free solu­tions, whereas no PCR prod­ucts are detect­able when Tri­ton X-100 is added (Fig. 1A, lanes b1, b2, c1, and c2). Finally, bac­te­rial 16S–23S inter­genic spacer (IGS) anal­y­ses (Fig. 1B) were car­ried out with geno­mic DNA sam­ples sequen­

tially ­puri­f ied from dif­fer­ent spe­cies (Esch­e­richia coli K12 and ­Strep­to­my­ces coe­li­color M145) using regen­er­ated col­umns. FAM-59-labeled PCR prod­ucts, ampli­f ied with prim­ers com­ple­ men­tary to eubac­te­rial 16S and 23S con­served sequences, were ana­lyzed by cap­il­lary elec­tro­pho­re­sis and no sig­nal result­ing from con­tam­in ­ at­ing DNA was observed besides the expected peaks cor­re­spond­ing to the ampli­f ied IGS of the ana­lyzed bac­ te­ria. Fur­ther assays were car­ried out using 32P-labeled DNA, which con­firmed that the hydro­lyzed DNA was com­pletely eluted after the chem­i­cal treat­ment (data not shown) and that no reduc­tion of the sil­ica matrix bind­ing capac­ity occurs even after sev­eral rounds of regen­er­a­tion (Fig. 2). Our data indi­cate that the pro­to­col described above is sub­stan­ tially faster and more effec­tive than oth­ers pro­posed pre­vi­ously, allow­ing to avoid tedious pro­ce­dures with long incu­ba­tions and sev­eral washes. More­over, this method ensures col­umn regen­er­a­ tion and reuse in less than 1 h inde­pen­dent of the size of the pre­ vi­ously puri­fied DNA mol­ec­ ules with­out car­ry­over con­tam­i­na­tion, giv­ing DNA suit­able for any appli­ca­tion. Acknowl­edg­ments We thank M. La Farina for allow­ing us to carry out most exper­ i­ments in his lab­o­ra­tory, V. Val­en­ti for the use of her Applied Bio­ sys­tems (ABI) Genetic Ana­lyzer, and R. Bar­bi­eri for the crit­i­cal read­ing of the man­u­script. Ref­er­ences

Fig. 2. Col­umn per­for­mances after 15 cycles of regen­er­a­tion. Col­umns were loaded with 1.8 £106 cpm of a 400-bp 32 P-labeled DNA frag­ment mixed with 10 lg of unla­ beled frag­ment, and the radio­ac­tiv­ity in either sil­ica matrix or elu­ates was mea­ sured with a Beck­man Coul­ter LS6500 MP Scin­til­la­tion Counter. The reported val­ues refer to the aver­ages of 10 exper­im ­ ents. Bind­ing and recov­ery refer to the amounts of DNA bound to the col­umn and recov­ered after elu­tion, respec­tively, and reten­tion indi­cates the amount of DNA not released by the col­umn after elu­tion.

[1] V.W. Chang, R. Wu, Y.S. Ho, Recy­cling of anion-exchange res­ins for plas­mid DNA puri­fi­ca­tion, Bio­Tech­ni­ques 26 (1999) 1056. [2] B.L. Fo­gel, M.T. McNal­ly, Trace con­tam­in ­ a­tion fol­low­ing reuse of anion-exchange DNA puri­fi­ca­tion res­ins, Bio­Tech­ni­ques 28 (2000) 299–302. [3] A.I. Kim, S.P. He­bert, C.T. Denny, Cross-con­tam­i­na­tion lim­its the use of recy­cled anion exchange res­ins for pre­par­ing plas­mid DNA, Bio­Tech­ni­ques 28 (2000) 298. [4] N.B. Sidd­ap­pa, A. Avi­nash, M. Ven­ka­tra­ma­nan, U. Ranga, Regen­er­a­tion of com­ mer­cial nucleic acid extrac­tion col­umns with­out the risk of carry-over con­tam­ i­na­tion, Bio­Tech­ni­ques 42 (2007) 186–192.