Phi29-based amplification of small genomes

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 354 (2006) 154–156 www.elsevier.com/locate/yabio Notes & Tips Phi29-based ampliWcation of small genomes Ivan Brukner a,¤, Damian Labuda a,b, Maja Krajinovic a,b a Centre de Recherche, Hôpital Sainte-Justine, Montréal, Québec, Canada H3T 1C5 b Département de Pédiatrie, Université de Montréal, Montréal, Québec, Canada H3T 1C5 Received 5 January 2006 Available online 2 May 2006 Whole genome ampliWcation (WGA),1 based on the 2686 bp, with a long carrier DNA, such as poly(dA–dT) or isothermal reaction of the enzyme Phi29 and random human DNA, before performing Phi29-based WGA. primers, is one of the most popular methods currently We compared WGA performance of serial dilutions of used in genomic research [1–3]. However, there is one (i) HindIII-cleaved, blunt end, religated pUC19; (ii) Hin- major drawback limiting applicability of this technique in dIII-cleaved, blunt end pUC19 (no ligase); (iii) HindIII- certain Welds such as forensics, microbiology, and envi- cleaved, blunt end pUC19 ligated with a high-molecular ronmental monitoring. The WGA method is hardly appli- DNA; and (iv) HindIII-cleaved, blunt end pUC19 with a cable to small genomes and/or degraded DNA, especially high-molecular DNA (no ligase). BrieXy, 10 pg of plasmid if the number of genome copies is approaching 100–1000 DNA was linearized by 1 U of HindIII (NEB, Beverly, molecules. Here, we describe a simple procedure in which MA, USA) for 3 h at 37 °C in 10 l. The 3⬘ overhang was WGA ampliWes 1 fg of the linear pUC19 DNA suYciently removed by incubation for 30 min at 37 °C in 10 l total for hundreds of downstream (i.e., post-WGA) PCR reac- volume of reaction buVer (40 mM Tris–HCl [pH 7.5], tions. Thus, the ampliWcation of limited quantities of 50 mM KCl, 10 mM MgCl2, 5 mM (NH4)2SO4, and 4 mM other unknown small genomes or genomic fragments dithiothreitol [DTT]) with 1 U of T4 DNA Polymerase should be possible as well. (Amersham Biosciences, Canada), and the 5⬘ overhang It was observed previously that self-priming of degener- Wll-in was performed in the presence of 0.25 mM dNTPs ate 6-mers might compete with the priming of the template by incubation for another 30 min at 37 °C. Six tubes of 10- DNA, especially if the quantity and/or length of the DNA fold serial dilutions, starting from 10 pg of pUC19 per template are small [2–5]. However, resolving self-priming reaction, were prepared, and 1 U of T4 Ligase and 1£ problem did not remove performance limitations of the Ligation BuVer (USB, Cleveland, OH, USA) were added Phi29-based WGA procedure [5]. For example, in our labo- in the total volume of 5 l (12 h at 37 °C). When ligation of ratory, 300 copies of a single copy gene could be WGA the template DNA with high-molecular weight DNA pre- ampliWed in the context of human DNA but not as an iso- ceded WGA, the samples were prepared as follows: lyoph- lated plasmid DNA. It seemed that the technical bottleneck ilized poly(dA–dT), approximate average length 8778 bp was due to a low hybridization of the degenerate 6-mers to (Amersham Biosciences), was dissolved in 1£ Ligation the template, suggesting a better WGA performance with BuVer to a Wnal concentration of 500 ng/l. The 500 ng of an equivalent number of copies of longer templates or poly(dA–dT) was treated in the same mode as cleaved embedded within longer templates, thereby favoring a plasmid DNA to create blunt ends. The equivalent dilu- higher number of priming events to occur. Therefore, we tions of blunt end pUC19 samples (starting from 10 pg) decided to ligate a small template, such as pUC19 of were religated or ligated with 10 ng of blunt end poly(dA– dT) by incubation with 1 U of T4 DNA Ligase in 5 l of total volume. The reaction steps, which do not include T4 * DNA ligation, were done as a control. The WGA was per- Corresponding author. Fax: +1 514 345 4731. E-mail address: [email protected] (I. Brukner). formed for each pUC19 dilution as described previously 1 Abbreviations used: WGA, whole genome ampliWcation; DTT, dithio- [1] or using primers with 5⬘ attached C3 and C18 spacers threitol. (Integrated DNA Technologies, Coralville, IA, USA) [5]. 0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.04.017  Notes & Tips / Anal. Biochem. 354 (2006) 154–156 155 A similar type of experiment was performed using sponding WGA was successful when using 100 times less HindIII-cleaved pUC19 and 10 ng of HindIII-cleaved template (0.1 pg) than the current minimal template human DNA as a universal carrier (data not shown), requirement [6], but only if template ligation with 10 ng thereby eliminating the need for the creation of blunt of high-molecular weight DNA was performed before ends. Of note is that 10 ng of high-molecular weight car- WGA reaction. rier DNA always produces WGA product (even if there is Blunt end ligation at a limited concentration of no input of additional template DNA) and that down- pUC19 (<0.1 pg/reaction) did not improve WGA perfor- stream PCR must be used to identify the presence or mance, as estimated by speciWc downstream PCR. absence of the ampliWed small template. Although blunt end ligation of small template DNA Two control PCR reactions were performed with improves WGA performance and overcomes terminal pUC19 and 1 l of 10-fold diluted WGA product using underrepresentation of Phi29-based ampliWcation of lin- the following pUC19-speciWc primer pairs: (A) 5⬘-gagc ear DNA [7] (apparent Kd of T4 Ligase for DNA is in the cggaagcataaagtgta-3⬘ and 5⬘-aactctgtagcaccgcctac-3⬘, low-micromolar to high-nanomolar range [8]), it seems producing a 750-bp amplicon (Fig. 1), and (B) cagg that eYciency of T4 ligation below attomolar concentra- ctgcgcaactgttgggaa and 5⬘-ccgtcgttttacaacgtcgta-3⬘, pro- tions of the substrate might compromise the ligation step ducing a 139-bp fragment using pUC19 (data not shown). of template alone and subsequent success of WGA The PCR reaction contained 1 M of each of the primers, reaction. 100 M of each dNTP, 10 mM Tris–HCl (pH 8.3), 2 mM The technique presented here goes one step further, MgCl2, 50 mM KCl, and 0.5 U of Taq polymerase (Plati- allowing the ampliWcation of only 1 fg of linear pUC19, num, Invitrogen, Carlsbad, CA, USA) in a volume of corresponding to 3.4 £ 102 plasmid copies. We believe that 50 l (30 cycles consisting of 30 s at 94 °C, 30 s at 52 °C, our procedure increases the applicability of Phi29-based and 30 s at 72 °C). The identity of PCR product from the ampliWcation by extending the repertoire of ampliWable WGA reaction was conWrmed by cleavage with HaeII templates to include small genomes and/or fragments of (Fig. 1C). As shown in Fig. 1B, the PCR from the corre- degraded DNA. Fig. 1. (A) PCR reactions from pUC19. (B) PCR reactions from WGA of pUC19 with or without religation and/or ligation to poly(dA–dT). (C) Digestion of PCR by HaeII, conWrming amplicon identity. In (A) lanes 1–4 are PCR products from serial dilutions of pUC19 (starting from 0.1 pg), and lane 5 is neg- ative control. In (B) lanes 1–6 are PCR products performed on 10-fold serial dilutions of plasmid DNA (HindIII-cleaved, blunt end, and religated, starting from 10 pg of pUC19), lanes 7–12 are PCR products of WGA of serial dilutions of pUC19 cleaved with HindIII and blunt-ended (starting from 10 pg), and lane 13 is PCR positive control (from plasmid DNA). Lanes 14–19 are PCRs from WGA of serial dilutions of pUC19 (starting from 10 pg) ligated with 10 ng of poly(dA–dT), lanes 20–25 are PCRs from WGA of serial dilutions of pUC19 (starting from 10 pg) done in the presence of 10 ng of poly(dA–dT) where the ligation step was omitted, and lane 26 is PCR negative control. (C) PCR product ampliWed from WGA (lane UC) and cleaved by HaeII (lane C), thereby conWrming amplicon identity by expected digestion pattern. Numbers on the left and right are length of DNA in base pairs (bp); M is 100-bp ladder (Invitrogen). 156 Notes & Tips / Anal. Biochem. 354 (2006) 154–156 Acknowledgments ply-primed rolling circle ampliWcation, Genome Res. 11 (2001) 1095– 1099. [4] D.L. Barker, S.T. Mark, M.S.T. Hansen, F. Faruqi, D. Giannola, O.R. This work was supported by the Centre de Recherche, Irsula, R.S. Lasken, M. Latterich, V. Makarov, A. Oliphant, J.H. Pin- Hôpital Sainte-Justine (Montréal, Québec, Canada). Part ter, R. Shen, I. Sleptsova, W. Ziehler, E. Lai, Two methods of whole- of the procedure is patent-pending technology (Univalor, genome ampliWcation enable accurate genotyping across a 2320-SNP Montréal). M. Krajinovic is a scholar of the Fonds de la linkage panel, Genome Res. 14 (2004) 901–907. Recherche en Santé du Québec. [5] I. Brukner, B. Paquin, M. Belouchi, D. Labuda, M. Krajinovic, Self- priming arrest by modiWed random oligonucleotides facilitates the quality control of whole genome ampliWcation, Anal. Biochem. 339 References (2005) 345–347. [6] J.M. Reagin, L.T. Giesler, L.A. Merla, M.J. Resetar-Gerke, M.K. [1] L. 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