Genome walking by Klenow polymerase

Analytical Biochemistry 430 (2012) 200–202

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Genome walking by Klenow polymerase Mariateresa Volpicella a,1, Claudia Leoni a,1, Immacolata Fanizza a, Sebastian Rius b, Raffaele Gallerani a, Luigi R. Ceci c,⇑ a

Department of Biosciences, Biotechnologies, and Pharmacological Sciences, University of Bari, 70126 Bari, Italy Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI, CONICET), Rosario 2000, Argentina c Institute of Biomembranes and Bioenergetics – CNR, 70126 Bari, Italy b

a r t i c l e

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Article history: Received 19 July 2012 Received in revised form 10 August 2012 Accepted 11 August 2012 Available online 23 August 2012 Keywords: Genome walking Klenow enzyme Maize Endochitinase A gene P1 gene

a b s t r a c t Genome walking procedures are all based on a final polymerase chain reaction amplification, regardless of the strategy employed for the synthesis of the substrate molecule. Here we report a modification of an already established genome walking strategy in which a single-strand DNA substrate is obtained by primer extension driven by Klenow polymerase and which results suitable for the direct sequencing of complex eukaryotic genomes. The efficacy of the method is demonstrated by the identification of nucleotide sequences in the case of two gene families (chiA and P1) in the genomes of several maize species. Ó 2012 Elsevier Inc. All rights reserved.

In any given genome, the identification of nucleotide sequences flanking already known DNA regions can be directly carried out by one of the available genome walking (GW)2 methods [1]. GW methods are based on different strategies devised to obtain a proper substrate for a final polymerase chain reaction (PCR) amplification in which a specific primer for the known sequence is coupled with a primer dictated by the GW strategy. Three main GW strategies can be identified: (i) restriction-based GW, in which a restriction digestion of the genomic DNA is required before ligation of restriction fragments to DNA cassettes; (ii) primer-based GW, characterized by the use of variously designed combinatorial (random or degenerate) primers to be coupled to sequence-specific primers in the final PCR step; and (iii) extension-based GW, based on the extension of a sequence-specific primer and subsequent tailing of the resulting single-strand DNA (ssDNA) molecule. A critical overview of the GW strategies available and their possible applications was recently reported [1]. In the course of direct sequencing experiments of genomes of several origins (human and plants) by a previously developed extension-based GW procedure [2], in some cases we observed the occurrence of aspecific products. To overcome this problem,

⇑ Corresponding author. Fax: +39 080 5443317. E-mail address: [email protected] (L.R. Ceci). These authors contributed equally to this study. Abbreviations used: GW, genome walking; PCR, polymerase chain reaction; ssDNA, single-strand DNA; Ta, annealing temperature; Tm, melting temperature. 1 2

0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.08.008

we focused our attention on the first (and probably more critical) step of the procedure: the extension of the sequence-specific primer. In this article, we show a substitutive procedure for this step, based on the use of Klenow DNA polymerase, to be adopted when an alternative gene-specific primer cannot be easily found. The procedure has been tested in the case of the maize ChiA and P1 genes, resulting in the direct sequencing of several members of the multigene families in different wild and inbred plant subspecies. Maize seeds were obtained from the International Maize and Wheat Improvement Center (CIMMYT, Mexico City, Mexico). B73 seeds were obtained from the Instituto Nacional de Tecnología Agropecuaria (INTA, http://www.inta.gov.ar). Genomic DNA was purified from 2-week-old maize seedlings using the GenElute Plant Genomic Kit (Sigma–Aldrich). The following oligonucleotides (Sigma–Aldrich) were used (positions of 50 - and 30 termini of each oligonucleotide on the ChiA gene [EBI accession number AY532774] and P1 gene [accession number EF165349] are also reported): chiA_GSP: 50 -AAGTGAGGTTGGGCCCTGGGTC-30 ; coordinates: 971, 950 chiA_GSP_A: 50 -CTGGCAGTACTGCTTGTAGTAGCC-30 ; coordinates: 937, 914 P1_GSP: 50 -CGTCCACCTCCCTCGCTT-30 ; coordinates: 6651, 6633 P1_GSP_A: 50 -CTTCTCGCAGCACGGCGCCCTCCC-30 ; coordinates: 6624, 6601

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P1_GSP2: 50 -ATAGCAGCGGACGGAGTACG-30 ; coordinates: 30, 49 P1_GSP2_A: 50 -GCGTTTAGTAGAGTGCACGTTACGGACCG-30 ; coordinates: 60, 88 AAP: 50 -CCACGCGTCGACTACTACGGGGGGGGGGGGGGGGGGGG GGG-30 . The primer extension reactions were carried out as follows. Using the TripleMaster Polymerase Mix (Eppendorf, Hamburg, Germany), two reactions were performed for chiA_GSP primer at an annealing temperature (Ta) of either 57 or 60 °C according to the previously established protocol [2]. Alternatively, for reactions carried out by Klenow polymerase, 2 ll of 10 U/ll Klenow Fragment Polymerase (Fermentas, Burlington, Ontario, Canada) were used in the presence of 0.05 lg of genomic DNA, 60 pmol of gene-specific primers (chiA_GSP, P1_GSP, and P1_GSP2), and 0.4 mM of each deoxynucleotide triphosphate (dNTP) in a total 100-ll reaction volume. The reaction was performed at 37 °C overnight. All of the extension products were purified through GFX MicroSpin columns (GE Healthcare, Chalfont St. Giles, UK). The subsequent tailing reaction was performed according to the established procedure [2] using 18.5 ll of purified ssDNA from the extension reaction. Amplification reactions of tailed ssDNA were carried out as already described [2]. The first, PCR amplification was run at two different Ta values (51 or 57 °C) for chiA_GSP primer, 54 °C for P1_GSP primer, and 55 °C for P1_GSP2 primer. After column purification, amplification products were subjected to nested PCR using Ta values of 57 °C for the chiA_GSP_A primer, 67 °C for the P1_GSP_A primer, and 62 °C for P1_ GSP2_A primer. Purified nested PCR products were cloned in pGEM-T Easy Vector (Promega, Madison, WI, USA) according to standard procedures [2]. Colonies showing a plasmid insert longer than 350 bp were analyzed by sequencing. Here we report first the improvements applied to the original GW method using the maize ChiA as working material. Then the results obtained by a single run of GW for both ChiA and P1 in several maize plants are shown. A workflow of the original GW procedure [2] is reported in the supplementary material (Fig. S1). ChiA-specific primers, chiA_GSP and chiA_GSP_A, were selected in the 30 -terminal region of the Zea mays (subsp. parviglumis) chiA gene (Fig. S2 in supplementary material) in a region conserved in most of the maize ChiA and ChiB genes. GW was applied to the genomic DNA extracted from the teosinte Zea perennis (AMES 21875), the maize landrace Arrocillo amarillo (AMES 19880), and the B73 inbred line. Unfortunately, sequencing results showed the occurrence of aspecific PCR amplification fragments due to mis-annealing of extension primer (chiA_GSP) during the extension step, with the synthesis of unpredictable exponential amplification products (not shown). Therefore, substantial modifications were introduced to the original protocol. They are discussed below and summarized in Table 1. The primer extension step of the GW procedure consists in the linear amplification by Taq polymerase of a primer designed toward the end of the known region and externally directed. In the original protocol, optimal conditions for primer annealing were established by taking into account the melting temperature (Tm) values of the genespecific primer. Schematically, the extension reaction is carried out using a Ta intermediate among calculate oligonucleotide Tm values [2]. The reaction is intrinsically prone to produce aspecific amplification fragments if the primer finds the conditions to act as either forward or reverse primer. Indeed the moderately stringent conditions of the extension reaction dictated by the GW procedure and the large dimension of genomes can result in unwanted amplifications. The extension step, therefore, has been modified by replacing Taq polymerase with Klenow DNA polymerase. Fig. 1 reports the

Table 1 Main differences between GW methods used in this work. Method

Step

Extension

Tailing PCR

Taq GW

Klenow GW

Taq enzyme (7.5 U); Ta intermediate among extreme Tm values; 35 cycles 5 ll of ssDNA Ta below lowest Tm

Klenow enzyme (20 U); 37 °C overnight 18.5 ll of ssDNA Ta intermediate among extreme Tm values

elongation products of the chiA_GSP primer obtained from 50 ng of Z. perennis genomic DNA by using either Klenow polymerase or Taq polymerase. In the first case, the reaction product is not detectable (Fig. 1A); a faint amplification fragment of approximately 400 bp is visible for the reaction carried out by Taq polymerase (Fig. 1B). When used in the successive GW steps, the products from the Taq extension step gave aspecific sequences (not shown), whereas those obtained with the Klenow enzyme proved to be suitable for the GW procedure with some minor modifications. The successive tailing step was performed essentially as described in the original procedure with the exception that the maximum amount of extension product was used in the reaction mix, that is, 18.5 ll instead of 5.0 ll. This was to ensure the production of a suitable amount of substrate for the successive PCR step. The PCR conditions differ from those described in the original GW protocol for the Ta that is raised to values between the extreme Tm values. Fig. 1C shows the electrophoretic pattern of the PCR amplification carried out on the Klenow extension product. The

Fig.1. Electrophoretic characterization of GW intermediate products. Lane M: GeneRuler 1-kb Plus DNA Ladder (Fermentas); lane A: Klenow polymerase extension products with primer chiA_GSP (Ta = 57 °C); lane B: Taq polymerase extension products with primer chiA_GSP (white arrow indicates a faintly visible fragment); lane C: PCR amplification products obtained from poly-dC-tailed ssDNA using AAP and chiA_GSP primers; lane D: nested PCR amplification products obtained using DNA from the first PCR as substrate and AAP and chiA_GSP_A primers.

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PCR step conducted at a Ta below the lowest calculated Tm resulted in the presence of aspecific products. The successive nested PCR (Fig. 1D), cloning, and sequencing procedures were conducted as in the original protocol. The described GW strategy allows identification of approximately 500 nucleotides. The Klenow GW method was validated by application on two genes that are known to be present as multigene families in maize: ChiA, coding for an endochitinase [3,4], and P1, coding for a regulator of 3-deoxy flavonoid biosynthesis [5]. Several maize species were also assayed. To identify possible variants of the ChiA genes present in the genomes of maize plants of different origins (teosinte, landraces, and inbred lines), we applied the Klenow GW procedure to four teosinte subspecies (Zea luxurians [PI 441933], Zea nicaraguensis [PI 615697], Z. perennis, and Zea diploperennis [PI 441932]), four maize Z. mays landraces (A. amarillo, Cacahuacintle [PI 280692], Confite puneño [PI 384063], and Conico norteño [NSL 2844]), and the B73 line. Fig. S3 in the supplementary material reports the multialignment of representative sequences obtained by a single run of GW on different maize genomes for some of the different polymorphic forms identified. Multialignments of sequences for single polymorphic forms are reported in Fig. S4. Interestingly, the adopted GW approach also succeeded in revealing genes of an additional class of chitinase occurring in maize, known as chitinase B [6], despite the primers do not match perfectly with the identified sequence. This result, instead of being a drawback, can be considered as a specific characteristic of this GW strategy useful for studying multigene families [7]. The Klenow GW approach was also adopted for the maize P1 gene. In this case, GW was conducted with a couple of primers located near the 50 end of the coding region as a possible strategy to identify different regulatory regions. Primers were designed on the sequence EF165349. Four maize variants were investigated: Z. nicaraguensis, Confite, Conico, and B73. For each plant, a multialignment of obtained sequences is available as supplementary material (see Fig. S5). In contrast to the ChiA and ChiB genes, in this case a generally higher diversity can be observed between sequences, maybe due to the lower functional constraints exerted on noncoding regions. Analysis of nucleotide sequences by a motif search program [8] allows the identification of conserved putative regulatory motifs (Fig. S5). For B73, a second round of GW was carried out, allowing the identification of an additional locus with a

slightly different 50 -untranscribed region. Interestingly, the two B73 sequences also differ in their 50 -flanking region from that recently reported by Rius and coworkers [9] (Fig. S6). In conclusion, the Klenow GW method proved to be suitable for the direct and rapid identification of multicopy genes and their regulatory regions in the complex genomes of maize species. Acknowledgments This work was partly supported by the Italian Ministry of Instruction, University, and Research (PRIN 20087ATS57, ‘‘Food Allergens’’). Claudia Leoni was the recipient of a fellowship from the Italian Consortium for Biotechnologies. The authors thank Erich Grotewold for his continuous interest in the P1 research program. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2012.08.008. References [1] C. Leoni, M. Volpicella, F. De Leo, R. Gallerani, L.R. Ceci, Genome walking in eukaryotes, FEBS J. 278 (2011) 3953–3977. [2] C. Leoni, R. Gallerani, L.R. Ceci, A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions, Biotechniques 44 (2008) 229. 232–235. [3] P. Tiffin, Comparative evolutionary histories of chitinase genes in the genus Zea and family Poaceae, Genetics 167 (2004) 1331–1340. [4] T.A. Naumann, D.T. Wicklow, Allozyme-specific modification of a maize seed chitinase by a protein secreted by the fungal pathogen Stenocarpella maydis, Phytopathology 100 (2010) 645–654. [5] E. Grotewold, B.J. Drummond, B. Bowen, T. Peterson, The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset, Cell 76 (1994) 543–553. [6] Q.K. Huynh, C.M. Hironaka, E.B. Levine, C.E. Smith, J.R. Borgmeyer, D.M. Shah, Antifungal proteins from plants: purification, molecular cloning, and antifungal properties of chitinases from maize seed, J. Biol. Chem. 267 (1992) 6635–6640. [7] C. Leoni, M. Volpicella, A. Placido, R. Gallerani, L.R. Ceci, Application of a genome walking method for the study of the spinach Lhcb1 multigene family, J. Plant Physiol. 167 (2010) 138–143. [8] M. Lescot, P. Dehais, G. Thijs, K. Marchal, Y. Moreau, Y. Van de Peer, P. Rouze, S. Rombauts, PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences, Nucleic Acids Res. 30 (2002) 325–327. [9] S. Rius, E. Grotewold, P. Casati, Analysis of the P1 promoter in response to UV-B radiation in allelic variants of high-altitude maize, BMC Plant Biol. 12 (2012) 92.