A high-throughput genome-walking method and its use for cloning unknown flanking sequences

Analytical Biochemistry 381 (2008) 248–253

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A high-throughput genome-walking method and its use for cloning unknown flanking sequences Palakolanu Sudhakar Reddy, Srikrishna Mahanty, Tanushri Kaul, Suresh Nair, Sudhir K. Sopory, Malireddy K. Reddy * International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India

a r t i c l e

i n f o

Article history: Received 19 May 2008 Available online 22 July 2008 Keywords: Whole genome amplification (WGA) Phi29 DNA polymerase Multiple displacement amplification (MDA) PCR-based genome walk

a b s t r a c t We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 50 ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 50 end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 50 flanking regions/promoters of selected plant genes. Ó 2008 Elsevier Inc. All rights reserved.

The availability of complete genome sequence of Arabidopsis [1] and rice [2] has provided a reference platform for plant genomics. However, there exists a tremendous amount of biological diversity among different plant species that requires sampling for an understanding of the diversity of gene content and expression. The larger genomes of many other plants are currently precluded from complete sequencing either due to the technical issues associated with complex plant genomes or due to the prohibitive costs associated with such enormous sequencing projects [3]. This has led to the evolution of alternative methods such as EST sequencing, to access actual coding portions of the genome, and genome-walking protocols to access the 50 flanking promoters and other regulatory elements from cDNA sequences of interest. Genome walking is a relatively fast and reliable procedure that enables the identification of unknown regions flanking a known DNA sequence, based on PCR1 amplification, without going through laborious and timeconsuming genomic library preparation and screening with cDNA/ DNA clones or probes obtained from prior screenings. Several successful genome-walking strategies have been developed that have

* Corresponding author. Fax: +91 11 26742316. E-mail address: [email protected] (M.K. Reddy). 1 Abbreviations used: MDA, multiple displacement amplification; PCR, polymerase chain reaction; WGA, whole genome amplification. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.07.012

their respective strengths and limitations for the characterization of a sequence adjacent to a known region. They include inverse PCR [4–6], ligation-mediated PCR [7–19], and randomly primed PCR [20–29]. Both inverse and ligation-mediated PCR-based genome-walking methods rely on the restriction fragmentation of genomic DNA. The plant genomic DNA digested with any given restriction enzyme yields 107 to 108 (depending on the size of the genome) fragments ranging from <300 to >10,000 bp due to uneven distribution of the restriction enzyme recognition sites in the genome. It is also very difficult to efficiently circularize or ligate oligonucleotide cassettes to these genomic DNA fragments for successful PCR-based genome walk. Other limitations in these methods being amplification of very small DNA fragments or no DNA amplification at all because the restriction site is located very close to or very distant from the locus-specific primer. Also, in most cases, the information on the distribution of restriction enzyme sites in a region of interest is unavailable before the start of the walking experiment. Therefore, combinations of several different enzymes must be tried in order to increase the probability of generating convenient DNA fragments for a successful genome walk using PCR. In the case of randomly primed PCR walk [20–29] the ability of oligonucleotide primers to bind nonspecifically in the unknown region, under low stringency annealing conditions in addition to

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the known region, facilitates the PCR amplification of target DNA fragment. The multiple nonspecific priming of the oligonucleotide primer during PCR at low stringency results in amplification of large number of ‘‘off targets,” and often with little or no amplification of target sequence. Several primer annealing temperatures must be tried to obtain the proper amplification of the target sequence and/or enrichment procedures must be introduced for specific fragments [27] which entail further costs. Even then these methods remain a low-throughput system and a time-consuming exercise, as enzymatic modifications of the target DNA (i.e., restriction digestion and ligation) and/or intensive screening for the specific PCR-amplified fragments is required. Also, it is impractical to perform such optimizations with each locus-specific primer for high-throughput promoter isolation experiments using the above method. In order to overcome the above-noted limitations for successful directional genome walk we developed a very simple PCRbased high-throughput genome-walking protocol in this investigation. The novelty of this protocol is in the introduction of a unique walker primer-binding site randomly into different regions of the genome without any restriction digestion and ligation of DNA cassettes, by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase [30], after annealing partially degenerate walker-adapter primers to the denatured genomic DNA. This leads to the comprehensive nonbiased whole genome amplification (WGA) into long overlapping fragments by a strand-displacement DNA synthesis approach by the Phi29 DNA polymerase [31] with the unique walker-adapter sequence attached to the 50 end of all the newly synthesized genomic DNA fragments. Subsequently, directional genome walking can be performed using a locus-specific primer and the walker primer, specific to walker adapter, and using the library of Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primer and their corresponding nested primers effectively amplify the flanking DNA fragment. This entire process can be completed in a single working day. The desired fragment can be either cloned for further use or directly sequenced with another nested locus-specific primer. We show here the successful use of this protocol to isolate 50 flanking regions/promoters of selected plant genes using the cDNA sequence information. Also, we discuss the potential use of this technique in gap closing in genome sequence assembly projects where the missing clones are difficult to obtain by conventional library screening and also in the identification of transgene integration sites and/or T-DNA-tagged sites in any genome.

Materials and methods Genomic DNA isolation and whole genome amplification Genomic DNA was isolated from Pennisetum and Salicornia seedlings using the CTAB method [32]. Approximately, 100 ng of the genomic DNA was heat-denatured and annealed to 150 ng of each of the four different walker-adapter primers (Table 1) at 30 °C separately in four individual tubes in the presence of 1X Phi29 DNA polymerase reaction buffer and 200 lM dNTPs. To each of the tube 10 units of Phi29 DNA polymerase (New England Biolabs, USA) was added in a 20-ll final reaction volume and incubated at 30 °C for 90 min to initiate multiple primer extension events. The reaction was terminated by incubating the reaction mix at 65 °C for 10 min. The unincorporated walker-adapter primers were removed from the Phi29 DNA polymerase-amplified

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Table 1 The nucleotide sequences of different primers used in this study Primer name

Primer sequence

Walker adaptor 1 Walker adapter 2 Walker adapter 3 Walker adapter 4 Walker primer 1 Walker primer 2 Apx primer 1 Apx primer 2 Hsp10 primer 1 Hsp10 primer 2 Hsp70 primer 1 Hsp70 primer 2 Gst primer 1 Gst primer 2

50 -GTGAGCGCGCGTAATACGACTCACTATAGGGNNNNATGC-30 50 -GTGAGCGCGCGTAATACGACTCACTATAGGGNNNNGATC-30 50 -GTGAGCGCGCGTAATACGACTCACTATAGGGNNNNTAGC-30 50 -GTGAGCGCGCGTAATACGACTCACTATAGGGNNNNCTAG-30 50 -GTGAGCGCGCGTAATACGA-30 50 -GTAATACGACTCACTATAGGG-30 50 -AGTGCCACGCGAGACGGAGCATG-30 50 -CTTCTCGACGGCCTCCTGGTACTC-30 50 -TTAGCTGCTTTGACGTCTCG-30 50 -GCGGTCTTCTTGGGCTGCAC-30 50 -AGGTCGTGCCGAGGTCGAGGTCGATCC-30 50 -TCTCGCTCGTCGGGAATCTGC-30 50 -TCTTCAATGAATTCATAATC-30 50 -ATGCTCCTAGAACTTTCACCT-30

Primers with suffix ‘2’ are nested primers.

genomic DNA using PCR purification columns (Qiagen Cat. No. 28104) and the DNA was eluted using 40 ll of TE buffer prior to its use as genome-walking template. Approximately 30 ng of Phi29 DNA polymerase-amplified genomic DNA was used as template for PCR amplification with 150 ng each of locus-specific and walker primer 1 (Table 1) in a 50-ll reaction volume. The PCR conditions were 94 °C, 1 min; 55 °C, 30 s; and 72 °C, 1 min, for 30 cycles unless otherwise specified. Nested PCR was carried out using 1 ll of 50 times diluted primary PCR products, from each of the tubes, as template along with nested locus-specific primer 2 and walker primer 2 in four separate tubes. The primer concentration and the PCR cycling conditions were the same as those noted earlier. Five microliters of the products from the second round of PCR amplification was electrophoresed on a 1% agarose gel and visualized after ethidium bromide staining. Cloning and sequencing of PCR fragments The selected DNA fragment(s) that were generated by the nested PCR-amplified products were gel-purified using Qiagen gel extraction columns (Cat. No. 28104) and cloned into a TA cloning vector as per the manufacturer’s instructions (Invitrogen, USA). Either the recombinant plasmid and/or the purified PCR products were sequenced using a cycle-sequencing protocol.

Results and discussion Whole genome amplification into long overlapping fragments having a unique 50 walker-adapter sequence We have utilized the capacity of Phi29 DNA polymerase to carry out highly reproducible comprehensive and unbiased in vitro whole genome amplification by multiple displacement amplification (MDA) resulting in hyperbranching of strand-displacement DNA by the rolling circle mode (Fig. 1). Unlike in other WGA methods where random hexamers are used for multiple priming, in this protocol we used partially degenerate long primers (Table 1). The four walker-adapter primers that were used in this study differ from each other at their 30 end. The initial four bases from the 30 end were arbitrarily fixed for each walker-adapter primer and the following four bases were completely degenerate (to reduce the permutations and combinations universal base(s) can also be used instead of introducing degeneracy). The remaining 50 bases of these walker-adapter primers were identical and arbitrarily fixed as 50 -GTGAGCGCGCGTAATACGACTCACTATAGGG-30 . The target genomic DNA is denatured and annealed to four different walker-adapter primers in four separate reaction tubes. Theoretically,

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Fig. 1. Diagrammatic representation of whole genome amplification (WGA) by multiple strand-displacement DNA synthesis using Phi29 DNA polymerase and subsequent PCR amplification of unknown flanking regions. Partially degenerate primers randomly prime DNA synthesis and the Phi29 DNA polymerase concurrently extends the primers as it displaces downstream DNA products resulting in repeated replication of DNA fragments by a ‘‘hyperbanching” mechanism of strand-displacement synthesis. The newly synthesized DNA fragments that carry a unique single-stranded walker adapter on their 50 ends are used for subsequent PCR amplification of 50 or 30 flanking regions using locus-specific, walker, and their corresponding nested primers in two rounds of PCR amplifications (for details see Materials and methods). The solid and dashed lines are the two complementary strands of DNA. The arrowheads represent the direction of DNA synthesis and the solid circles at the start of each line represent the walker-adapter sequence. ( ) and ( ) represent the walker and the nested walker primers, respectively. ( ) and ( ) represent locus-specific and nested locus-specific primers, respectively.

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the 4-base sequence combination present on the 30 end of each of the walker-adapter primer can exist once in every 256 bp in the genomic DNA. Moreover, the preceding 4 bases being degenerate can also form a perfect complement on the same template DNA. Therefore, there is perfect annealing of at least 8 bases from the 30 end of these walker-adapter primers randomly to different regions of the genomic DNA. The Phi29 DNA polymerase will initiate multiple primer extensions in the presence of nucleotides and the inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 50 ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that covers the whole genome. The exceptionally tight binding of Phi29 DNA polymerase to the DNA template allows it to add up to 70,000 nucleotides on an average for each priming reaction [32] without being affected by GC content and other secondary structures of the template DNA. This is the highest processivity reported for any DNA polymerase in the absence of cellular multisubunit complexes and therefore accounts for its ability to generate extremely long DNA fragments even up to 100 kb with an average length of 12–15 kb compared to 100–1000 bp for PCR-based WGA methods [33]. Besides, the high fidelity (proofreading activity) of Phi29 DNA polymerase allows it to accurately copy genomic sequences. This is a more efficient and reliable way to introduce walker primer-binding sites into the genome randomly without employing restriction and ligation.

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cloned fragments whether they actually flank the selected cDNA sequence, the insert DNA was sequenced completely and compared with the corresponding cDNA sequence for the 50 end overlap, as the nested locus-specific primer was designed such that there was at least a 40-bp overlap with the 50 end of the known cDNA sequence (Table 2). A detailed analysis of these promoter regions will be published elsewhere. Alternatively, direct sequencing of PCR-amplified fragments can be carried out with another nested sequencing primer. This is the preferred approach over cloning of specific fragment(s) fol-

Directional genome walking using PCR Following primer extension using Phi29 DNA polymerase, all the newly synthesized genomic DNA fragments carry a unique single-stranded walker-adapter sequence on their 50 ends which serve as binding sites for the walker and nested walker primers after the locus-specific primer initially synthesizes the complementary sequence in the walker-adapter region after the first cycle of PCR. This is because the walker and the nested walker primers are designed to be identical and not complementary to the walker-adapter region. The locus-specific primer will determine the position and direction of the genome walk. Because the walker primer-binding site is generated only after the extension primed by the locus-specific primer and also due to high stringency annealing temperature that the PCR is carried out in, it effectively circumvents the problem of nonspecific background amplification. However, nonspecific ‘‘off-target” amplification products cannot always be excluded even after hot-start PCR or touchdown PCR because of the complexity of the template DNA (whole genome). Thus, use of the nested locus-specific primer 2 ensures the selective amplification of the target fragment in the second round of PCR, thereby increasing the probability of only amplifying the desired flanking DNA sequence while suppressing the off-target PCR amplifications. A higher probability of obtaining longer flanking DNA fragment amplification products can be achieved by using long-range PCR amplification enzyme mixes. This is because the genomic template is not cleaved into smaller fragments using restriction enzyme (as in other WGA methods) but instead the whole genome is copied into very long overlapping fragments with Phi29 DNA polymerase. To test the efficacy of the methodology we PCR-amplified promoter regions of different genes, from genomic DNA of highly complex Pennisetum and Salicornia plant genomes. Using locus-specific primer(s), synthesized in the antisense orientation toward the 50 end (Table 1) and based on the cDNA sequence of different genes, together with walker primer(s), we successfully amplified 50 flanking genomic regions of all the genes that were attempted. Figs. 2A and B show that the PCR amplification products were discrete fragments in all the cases after nested PCR. The desired PCR fragments were gel-purified and cloned. To verify the authenticity of the

Fig. 2. Amplified products after the second round of PCR. (A) The different DNA fragments amplified after two rounds of PCR-based genomic walk from four (1, 2, 3, 4) different walker-adapter primed whole genome-amplified templates are on each lane. (B) Selected DNA fragments (white arrows) were cloned into TA-Topo cloning vector and PCR-amplified using M13 (forward and reverse) primers. The names of the promoters amplified are indicated on the top. Lane M represents the DNA size standard markers (1-kb ladder). The numbers on the left represent the DNA fragment length in kb.

Table 2 Details of the 50 flanking sequence amplified in this study Promoter name

DNA fragment size (bp)

Length of sequence overlap with respective cDNA (bp)

Accession No.

Apx promoter Hsp10 promoter Hsp70 promoter Gst promoter

886 967 610 1270

54 71 94 65

EU492461 EU620495 EU620496 EV295484

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lowed by subsequent sequencing of the cloned fragment. The analysis of the second-round PCR-amplified products on the agarose gel showed, in most cases, fragments of different sizes (Fig. 2A). This can be explained by different priming sites of Phi29 DNA polymerase during the initial WGA after random

annealing of the walker-adapter primer followed by MDA to generate overlapping genomic fragments. This results in amplification of a mixture of specific PCR fragments that are heterogeneous in length. Also, a fraction of nonspecific fragments is expected; nevertheless, the nonspecific products do not inter-

Fig. 3. Portion of electropherogram(s) showing the quality of sequence data. Panels A1–A4 show the quality of sequence generated using recombinant plasmid DNA templates and panels B1–B4 show the sequence generated by direct sequencing of the same PCR products with specific internal sequencing primer.

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fere with cycle sequencing using another internal locus-specific sequencing primer. The quality of the electropherogram(s) generated from direct sequencing of the PCR fragments is comparable to those generated when sequencing the cloned fragment (Fig. 3). The simultaneous high-throughput two-step PCR-based genome walking followed by direct sequencing of PCR-amplified fragments with nested specific sequencing primer could enable many new research strategies for gap closing in genome assembly projects and also in the identification of transgene integration site(s) and/or transposon-tagged sites. Merits of the method Unlike earlier methods the present method does not rely on the restriction of genomic DNA and therefore does not suffer due to uneven distribution of the restriction enzyme sites, in the region of interest, to generate convenient fragments (template) for PCR amplification. It is a fast and reliable way of introducing unique walker primer-binding sites randomly into different regions of the genome, without restricting and ligating any adapter cassettes, by annealing long synthetic oligonucleotides with a partial degeneracy at the 30 end for proper annealing and extension by the DNA polymerase. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 50 ends of downstream strands and DNA synthesis continues resulting into a large number of overlapping fragments that cover the whole genome. The binding site for the adapter primer will only be formed as the result of the locus-specific primer extension after the first cycle of PCR. The locus-specific primer will determine the position and direction of the genome walk during the PCR. Although we have focused on obtaining promoter regions of selected genes from plant systems, this rapid PCR-based DNA walking can be performed with any genomic DNA to walk bidirectionally from any sequence-tagged site. This protocol can also be used to locate the transgene integration site. This can be achieved by using two gene-specific primers, one each for each end of the gene and then amplifying the regions flanking the gene insertion site. Sequence analysis of the amplified flanking region would identify the transgene integration site. This has great relevance for the development of an accurate transgenic signature, for each transgenic event generated, to protect the investigator from unauthorized transfer of transgene, generated by the investigator, into other varieties by simple breeding by competitors. Therefore, this method can logically be used for protecting the intellectual property of the investigator. After the initial Phi29 DNA polymerase amplification this template DNA can be used to amplify and clone any unknown flanking sequences using PCR and to achieve this only the locus-specific primer needs to be changed. As all this can be done in a single working day; it is thereby suitable for high-throughput gap closing in genome assembly programs. Acknowledgment This work was supported in part by a Department of Biotechnology (Ministry of Science and Technology, Government of India) research grant. References [1] Arabidopsis Genome Initiative, Analysis of the genome sequence of the flowering plant Arabidopsis thaliana, Nature 408 (2000) 796–815. [2] International Rice Genome Sequencing Project, The map-based sequence of the rice genome, Nature 436 (2005) 793–800. [3] D. Adam, Now for the hard ones, Nature 408 (2000) 792–793.

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