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Clonal polymerase chain reaction–single-strand conformational polymorphism analysis: An effective approach for identifying cloned sequences
Analytical Biochemistry 378 (2008) 111–112
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Clonal polymerase chain reaction–single-strand conformational polymorphism analysis: An effective approach for identifying cloned sequences H. Zhou, J.G.H. Hickford * Gene-Marker Laboratory, Agriculture and Life Sciences Division, Lincoln University, Canterbury 7647, New Zealand
a r t i c l e
i n f o
Article history: Received 12 February 2008 Available online 7 April 2008
a b s t r a c t The amplification of target sequences from genomic DNA can result in more than one amplicon sequence being produced even when highly specific primers are used. Here we present a clonal polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) approach for screening cloned amplicons and identifying particular clones prior to sequence determination. Comparison of the PCR–SSCP patterns of the cloned amplicons with the PCR–SSCP patterns observed for the DNA templates from which the clones were derived allows PCR artifacts, different alleles, and even different loci to be differentiated prior to sequencing. Using this approach, the number of clones required for reliable sequence determination is minimized, and complex ‘‘mixed” amplicons can be resolved easily, cost-effectively, and reliably. Ó 2008 Elsevier Inc. All rights reserved.
Although the direct sequencing of polymerase chain reaction (PCR)1 amplicons can be fast, it is normally reliable only with homogeneous sequences because it can be confounded if nucleotide insertions, deletions, sequence rearrangements, or more than one nucleotide substitution (single nucleotide polymorphism [SNP]) are present in one (or more) of the amplified sequences (e.g., if multiple targets are amplified). This leads to a requirement to clone and then sequence amplicons. However, this approach is laborious, especially if numerous clones need to be screened prior to sequencing so as to be able to ensure that all of the amplified sequences have been identified. The cloning of PCR amplicons also has the drawback that sequence artifacts generated during PCR amplification (e.g., nucleotide mismatches [1], template rearrangements [2]) can be perpetuated through cloning and, hence, are difficult to resolve with the actual sequence of the template. Although the use of proofreading DNA polymerases can, to some extent, reduce the introduction of nucleotide mismatches during PCR, errors may still occur [3]. Another potential problem associated with the cloning approach is the preferential amplification of a particular sequence or sequences during PCR [4], making the identification of other ‘‘minor” clones difficult, especially if more than two sequences are amplified. Therefore, a technique allowing the rapid identification of cloned amplicons would be valuable.
* Corresponding author. Fax: +64 3 3253851. E-mail address: [email protected] (J.G.H. Hickford). 1 Abbreviations used: PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; SSCP, single-strand conformational polymorphism; TBE, Tris– borate–EDTA; EDTA, ethylenediaminetetraacetic acid. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.04.005
Here we describe an approach for identifying cloned amplicons using what we term clonal PCR–SSCP (single-strand conformational polymorphism) analysis. This approach involves three simple steps after the overnight culturing of clones derived from the transformation of Escherichia coli with ‘‘mixed amplicon” constructs, as are created when a PCR amplicon (or mixed amplicons) is inserted into a plasmid vector. Step 1 is to rapidly recover plasmid DNA from overnight cultures of the clones. Step 2 is to do a second round of PCR using both the crude plasmid isolates and the original DNA template from which the clones were derived. Step 3 is to run an SSCP analysis on these amplicons so as to compare the banding pattern derived from the individual clones with that derived from the original DNA. Only those clones that produce a PCR–SSCP pattern that matches part of the pattern produced by the original DNA template are chosen for sequencing, and only when clones that can explain all of that original PCR–SSCP pattern are identified can the full extent of variation in the original amplicon be understood. We used this approach to clone a new ovine DQA2 allele that exhibited a PCR–SSCP pattern different from that of the 23 previously reported sequences [5]. This allele was detected from a sheep that was revealed to have three DQA2 sequences derived from two different loci. Briefly, DNA was purified from an ovine blood sample collected on an FTA card (Whatman, Middlesex, UK) using a two-step procedure [6], and the polymorphic region of ovine DQA2 exon 2 was amplified by Pwo SuperYield DNA Polymerase (Roche Applied Science, Mannheim, Germany) using PCR primers (DQA2s-up and DQA2s-dn) and conditions described previously [5]. The PCR amplicon was ligated into the pCR4 Blunt–TOPO vector (Invitrogen,
112
Notes & Tips / Anal. Biochem. 378 (2008) 111–112
1101 08011 08011 New allele 1101 1101 New allele 08011 Genomic DNA New allele ? 1101 08011 08011 New allele 08011
1101
Fig. 1. Comparison of PCR–SSCP patterns derived from clones and sheep genomic DNA. Five clones (corresponding to ovine DQA2 allele 1101) had a banding pattern that matched part of the genomic DNA pattern (labeled as genomic DNA), and six clones (corresponding to ovine DQA2 allele 08011) had a pattern that matched another part of the genomic DNA pattern. Four clones (labeled as new allele) produced a PCR–SSCP pattern that matched the remainder of the genomic DNA pattern. One clone (labeled as ?) generated a banding pattern that was not observed for the genomic DNA and is thought to be the result of a PCR artifact.
Carlsbad, CA, USA), and 16 white colonies (clones) were picked and incubated overnight in Terrific broth (Invitrogen) at 37 °C. Bacteria from approximately 30 ll of overnight cultures were spun down and suspended in 40 ll of 0.8% Triton X-100 solution. After 10 min incubation at 95 °C followed by 1 min centrifugation at 12,000 g, 1 ll of the supernatant was used to prime a second PCR amplification using the same conditions as the first except for the use of Taq DNA polymerase (Qiagen, Valencia, CA, USA). A separate amplification was also performed on the original sheep genomic DNA sample. Amplicons derived from the 16 selected colonies and the genomic DNA were subjected to SSCP analysis in a 14% polyacrylamide gel at 380 V and 5 °C for 18 h in 0.5 TBE (Tris–borate–EDTA [ethylenediaminetetraacetic acid]) buffer. The PCR–SSCP patterns of individual clones were compared with the pattern derived from the original sheep DNA. Of the 16
colonies, 15 had PCR–SSCP patterns that matched part of the genomic DNA pattern (Fig. 1) and, therefore, were thought to contain allelic sequences of the ovine DQA2 gene. The remaining colony was considered to be the result of a PCR artifact because it exhibited a banding pattern that was not observed for the sheep genomic DNA. Based on the PCR–SSCP banding pattern, four clones containing the potentially new allele could be identified (Fig. 1). Three of these four clones were selected for further plasmid DNA purification, and sequencing subsequently confirmed that these clones contained an identical insert sequence (Fig. 2). A BLAST search revealed that this sequence was novel but shared high homology with other ovine DQA2 sequences in GenBank. This sequence was named ovine DQA2 *0603 and was deposited into GenBank with accession number EU479713. Using this approach, not only can clones containing genuine sequences be differentiated from those containing PCR artifacts, but also individual components of the amplicon can be rapidly identified and selected for sequence determination. This minimizes the numbers of clones required for plasmid DNA purification and sequencing, and it speeds up the isolation of specific target sequences. This technique spans more genomes than SNP analysis and, therefore, can resolve individual sequences through an extended haplotype, but its utility is normally limited to the 100- to 400bp fragment size ideally suited to SSCP analysis and it is only as sensitive as the SSCP technique typically is for detecting polymorphism.
References [1] P. Keohavong, W.G. Thilly, Fidelity of DNA polymerases in DNA amplification, Proc. Natl. Acad. Sci. USA 86 (1989) 9253–9257. [2] A. Meyerhans, J.P. Vartanian, S. Wain-Hobson, DNA recombination during PCR, Nucleic Acids Res. 18 (1990) 1687–1691. [3] J. Cline, J.C. Braman, H.H. Hogrefe, PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases, Nucleic Acids Res. 24 (1996) 3546–3551. [4] P.S. Walsh, H.A. Erlich, R. Higuchi, Preferential PCR amplification of alleles: Mechanisms and solutions, PCR Methods Appl. 1 (1992) 241–250. [5] J.G.H. Hickford, H. Zhou, S. Slow, Q. Fang, Diversity of the ovine DQA2 gene, J. Anim. Sci. 82 (2004) 1553–1563. [6] H. Zhou, J.G.H. Hickford, Q. Fang, A two-step procedure for extracting genomic DNA from dried blood spots on filter paper for PCR amplification, Anal. Biochem. 354 (2006) 159–161.
New allele clone-1 New allele clone-2 New allele clone-3 Ovine DQA2*08011 Ovine DQA2*1101 New allele clone-1 New allele clone-2 New allele clone-3 Ovine DQA2*08011 Ovine DQA2*1101 New allele clone-1 New allele clone-2 New allele clone-3 Ovine DQA2*08011 Ovine DQA2*1101 Fig. 2. Alignment of the nucleotide sequences from three clones that produced a novel PCR–SSCP pattern in amplified sheep DNA with the two previously identified ovine DQA2 sequences (alleles 1101 and 08011). Sequences exclude the PCR primer binding regions.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Clonal polymerase chain reaction–single-strand conformational polymorphism analysis: An effective approach for identifying cloned sequences H. Zhou, J.G.H. Hickford * Gene-Marker Laboratory, Agriculture and Life Sciences Division, Lincoln University, Canterbury 7647, New Zealand
a r t i c l e
i n f o
Article history: Received 12 February 2008 Available online 7 April 2008
a b s t r a c t The amplification of target sequences from genomic DNA can result in more than one amplicon sequence being produced even when highly specific primers are used. Here we present a clonal polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) approach for screening cloned amplicons and identifying particular clones prior to sequence determination. Comparison of the PCR–SSCP patterns of the cloned amplicons with the PCR–SSCP patterns observed for the DNA templates from which the clones were derived allows PCR artifacts, different alleles, and even different loci to be differentiated prior to sequencing. Using this approach, the number of clones required for reliable sequence determination is minimized, and complex ‘‘mixed” amplicons can be resolved easily, cost-effectively, and reliably. Ó 2008 Elsevier Inc. All rights reserved.
Although the direct sequencing of polymerase chain reaction (PCR)1 amplicons can be fast, it is normally reliable only with homogeneous sequences because it can be confounded if nucleotide insertions, deletions, sequence rearrangements, or more than one nucleotide substitution (single nucleotide polymorphism [SNP]) are present in one (or more) of the amplified sequences (e.g., if multiple targets are amplified). This leads to a requirement to clone and then sequence amplicons. However, this approach is laborious, especially if numerous clones need to be screened prior to sequencing so as to be able to ensure that all of the amplified sequences have been identified. The cloning of PCR amplicons also has the drawback that sequence artifacts generated during PCR amplification (e.g., nucleotide mismatches [1], template rearrangements [2]) can be perpetuated through cloning and, hence, are difficult to resolve with the actual sequence of the template. Although the use of proofreading DNA polymerases can, to some extent, reduce the introduction of nucleotide mismatches during PCR, errors may still occur [3]. Another potential problem associated with the cloning approach is the preferential amplification of a particular sequence or sequences during PCR [4], making the identification of other ‘‘minor” clones difficult, especially if more than two sequences are amplified. Therefore, a technique allowing the rapid identification of cloned amplicons would be valuable.
* Corresponding author. Fax: +64 3 3253851. E-mail address: [email protected] (J.G.H. Hickford). 1 Abbreviations used: PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; SSCP, single-strand conformational polymorphism; TBE, Tris– borate–EDTA; EDTA, ethylenediaminetetraacetic acid. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.04.005
Here we describe an approach for identifying cloned amplicons using what we term clonal PCR–SSCP (single-strand conformational polymorphism) analysis. This approach involves three simple steps after the overnight culturing of clones derived from the transformation of Escherichia coli with ‘‘mixed amplicon” constructs, as are created when a PCR amplicon (or mixed amplicons) is inserted into a plasmid vector. Step 1 is to rapidly recover plasmid DNA from overnight cultures of the clones. Step 2 is to do a second round of PCR using both the crude plasmid isolates and the original DNA template from which the clones were derived. Step 3 is to run an SSCP analysis on these amplicons so as to compare the banding pattern derived from the individual clones with that derived from the original DNA. Only those clones that produce a PCR–SSCP pattern that matches part of the pattern produced by the original DNA template are chosen for sequencing, and only when clones that can explain all of that original PCR–SSCP pattern are identified can the full extent of variation in the original amplicon be understood. We used this approach to clone a new ovine DQA2 allele that exhibited a PCR–SSCP pattern different from that of the 23 previously reported sequences [5]. This allele was detected from a sheep that was revealed to have three DQA2 sequences derived from two different loci. Briefly, DNA was purified from an ovine blood sample collected on an FTA card (Whatman, Middlesex, UK) using a two-step procedure [6], and the polymorphic region of ovine DQA2 exon 2 was amplified by Pwo SuperYield DNA Polymerase (Roche Applied Science, Mannheim, Germany) using PCR primers (DQA2s-up and DQA2s-dn) and conditions described previously [5]. The PCR amplicon was ligated into the pCR4 Blunt–TOPO vector (Invitrogen,
112
Notes & Tips / Anal. Biochem. 378 (2008) 111–112
1101 08011 08011 New allele 1101 1101 New allele 08011 Genomic DNA New allele ? 1101 08011 08011 New allele 08011
1101
Fig. 1. Comparison of PCR–SSCP patterns derived from clones and sheep genomic DNA. Five clones (corresponding to ovine DQA2 allele 1101) had a banding pattern that matched part of the genomic DNA pattern (labeled as genomic DNA), and six clones (corresponding to ovine DQA2 allele 08011) had a pattern that matched another part of the genomic DNA pattern. Four clones (labeled as new allele) produced a PCR–SSCP pattern that matched the remainder of the genomic DNA pattern. One clone (labeled as ?) generated a banding pattern that was not observed for the genomic DNA and is thought to be the result of a PCR artifact.
Carlsbad, CA, USA), and 16 white colonies (clones) were picked and incubated overnight in Terrific broth (Invitrogen) at 37 °C. Bacteria from approximately 30 ll of overnight cultures were spun down and suspended in 40 ll of 0.8% Triton X-100 solution. After 10 min incubation at 95 °C followed by 1 min centrifugation at 12,000 g, 1 ll of the supernatant was used to prime a second PCR amplification using the same conditions as the first except for the use of Taq DNA polymerase (Qiagen, Valencia, CA, USA). A separate amplification was also performed on the original sheep genomic DNA sample. Amplicons derived from the 16 selected colonies and the genomic DNA were subjected to SSCP analysis in a 14% polyacrylamide gel at 380 V and 5 °C for 18 h in 0.5 TBE (Tris–borate–EDTA [ethylenediaminetetraacetic acid]) buffer. The PCR–SSCP patterns of individual clones were compared with the pattern derived from the original sheep DNA. Of the 16
colonies, 15 had PCR–SSCP patterns that matched part of the genomic DNA pattern (Fig. 1) and, therefore, were thought to contain allelic sequences of the ovine DQA2 gene. The remaining colony was considered to be the result of a PCR artifact because it exhibited a banding pattern that was not observed for the sheep genomic DNA. Based on the PCR–SSCP banding pattern, four clones containing the potentially new allele could be identified (Fig. 1). Three of these four clones were selected for further plasmid DNA purification, and sequencing subsequently confirmed that these clones contained an identical insert sequence (Fig. 2). A BLAST search revealed that this sequence was novel but shared high homology with other ovine DQA2 sequences in GenBank. This sequence was named ovine DQA2 *0603 and was deposited into GenBank with accession number EU479713. Using this approach, not only can clones containing genuine sequences be differentiated from those containing PCR artifacts, but also individual components of the amplicon can be rapidly identified and selected for sequence determination. This minimizes the numbers of clones required for plasmid DNA purification and sequencing, and it speeds up the isolation of specific target sequences. This technique spans more genomes than SNP analysis and, therefore, can resolve individual sequences through an extended haplotype, but its utility is normally limited to the 100- to 400bp fragment size ideally suited to SSCP analysis and it is only as sensitive as the SSCP technique typically is for detecting polymorphism.
References [1] P. Keohavong, W.G. Thilly, Fidelity of DNA polymerases in DNA amplification, Proc. Natl. Acad. Sci. USA 86 (1989) 9253–9257. [2] A. Meyerhans, J.P. Vartanian, S. Wain-Hobson, DNA recombination during PCR, Nucleic Acids Res. 18 (1990) 1687–1691. [3] J. Cline, J.C. Braman, H.H. Hogrefe, PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases, Nucleic Acids Res. 24 (1996) 3546–3551. [4] P.S. Walsh, H.A. Erlich, R. Higuchi, Preferential PCR amplification of alleles: Mechanisms and solutions, PCR Methods Appl. 1 (1992) 241–250. [5] J.G.H. Hickford, H. Zhou, S. Slow, Q. Fang, Diversity of the ovine DQA2 gene, J. Anim. Sci. 82 (2004) 1553–1563. [6] H. Zhou, J.G.H. Hickford, Q. Fang, A two-step procedure for extracting genomic DNA from dried blood spots on filter paper for PCR amplification, Anal. Biochem. 354 (2006) 159–161.
New allele clone-1 New allele clone-2 New allele clone-3 Ovine DQA2*08011 Ovine DQA2*1101 New allele clone-1 New allele clone-2 New allele clone-3 Ovine DQA2*08011 Ovine DQA2*1101 New allele clone-1 New allele clone-2 New allele clone-3 Ovine DQA2*08011 Ovine DQA2*1101 Fig. 2. Alignment of the nucleotide sequences from three clones that produced a novel PCR–SSCP pattern in amplified sheep DNA with the two previously identified ovine DQA2 sequences (alleles 1101 and 08011). Sequences exclude the PCR primer binding regions.