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• W83487037 abstract "BioTechniquesVol. 37, No. 6 BenchmarksOpen AccessOne-tube restriction enzyme digest and fluorescent labeling for restriction endonuclease fingerprinting single-strand conformational polymorphismOve Bruland & Per Morten KnappskogOve Bruland*Address correspondence to: Ove Bruland, Center of Medical Genetics and Molecular Medicine, Haukeland University, Hospital, N-5021 Bergen, Norway. e-mail: E-mail Address: ove.bruland@helse-bergen.noHaukeland University Hospital, BergenSearch for more papers by this author & Per Morten KnappskogHaukeland University Hospital, BergenUniversity of Bergen, Bergen, NorwaySearch for more papers by this authorPublished Online:6 Jun 2018https://doi.org/10.2144/04376BM04AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Single-stranded conformational polymorphism (SSCP) has been widely used for detecting mutations and single nucleotide polymorphisms (SNPs) since the method was first described in 1989 by Orita et al. (1,2). Although the original protocol has been optimized with respect to running conditions and gel composition (3,4), the major drawbacks of this method have been the limited fragment length that can be separated on a standard polyacrylamide slab gel and the simultaneous detection of weak and strong radioactively labeled conformers. This has been partly overcome by restriction endonuclease fingerprinting single-strand conformational polymorphism (REF-SSCP; Reference (5), in which large radioactively labeled PCR fragments are digested by restriction enzymes before separation on polyacrylamide slab gels. Fluorescence SSCP (6), with separation and detection performed on DNA sequencers, both enhanced the sensitivity and reproducibility of the method (7,8). In addition, SSCP has been successfully adapted to capillary electrophoresis (9–11). Fluorescence labeling and detection make it possible to add an internal size standard to correct for lane-to-lane or run-to-run variations. There are several different approaches to the fluorescence labeling of the PCR fragments, including prelabeling of PCR primers (6), incorporation of fluorescently labeled nucleotides (12,13), and multicolor post-labeling of the PCR products (14). With fluorescence labeling and detection using SSCP analysis, it is important that all products are uniformly labeled and that the labeled nucleotide is present at the end of the PCR product and not randomly distributed within the PCR product. We have designed a fluorescence REF-SSCP protocol for mutation or SNP detection by combining widely used technologies (i.e., restriction digest, DNA minisequencing, and SSCP analysis) and have tested it by performing a blind test on a collection of mutated cDNAs of human phenylalanine hydroxylase (PAH). When performing the blind test, we detected gel shifts in all mutated samples. Following PCR amplification, the PCR product (1544 bp) was digested with two restriction enzymes (HinfI and BanI), producing DNA fragments with 5′ protruding overhangs ranging from 291 to 20 bp (291, 230, 206, 199, 174, 111, 99, 95, 66, 51, and 22 bp; Figure 1A). The 5′ protruding overhang is essential for the next step, labeling by minisequencing of each digested fragment by including Thermo Sequenase™ (Amersham Biosciences AB, Uppsala, Sweden) and fluorescence-labeled ddNTPs. The restriction enzyme digest and the minisequencing are performed simultaneously in one tube. Because only fluorescence-labeled ddNTPs are present, only one fluorescence-labeled ddNTP will be incorporated at the end of each digested DNA fragment in a sequence-dependent manner. The blind test comprised one no-template control (no DNA) and 39 cDNAs (cloned and sequence verified) of human PAH. Within the 39 cDNA samples, 36 contained one or more specific mutations while 3 samples were normal controls (no sequence variants). The mutated samples were chosen to represent sequence variants distributed throughout the PAH cDNA (Figure 1A). One normal control was added as a reference of known identity against which each of the 40 samples was compared. The cDNAs were PCR amplified from pMAL-PAH (15) with 2 ng plasmid DNA, 5 µL GeneAmp® 10× PCR buffer (Applied Biosystems, Foster City, CA, USA), 4 µL 25 mM MgCl2, 1 µL 10 µM forward primer (5′-GGTCGTCAGACTGTCGATGA-3′), 1 µL 10 µM reverse primer (5′-TTTTCGGACTTTTTCTGATG-3′), 0.2 µL 25 mM dNTP, and 2.5 U AmpliTaq® polymerase (Applied Biosystems) in a final volume of 50 µL. The PCR parameters were 20 cycles for 94°C for 5 min, 94°C for 20 s, 58°C for 20 s, and 72°C for 10 s, followed by a final extension at 72°C for 4 min. The PCR products were then purified on QIAquick® PCR purification columns (Qiagen GmbH, Hilden, Germany), as described by the manufacturer. For fragmentation and labeling, 0.5–5.0 ng (1 µL) purified PCR product were digested in a total volume of 30 µL, containing 3 µL 10× restriction enzyme buffer (HinfI bf, 2 U HinfI, 2 U BanI, NEBuffer 2; all from New England Biolabs, Beverly, MA, USA), 1 U Thermo Sequenase, and 0.1 µL fluorescent labeled ddNTPs (0.05 µL each of R6G-labeled ddATP DyeDeoxy™ and R110-labeled ddGTP DyeDeoxy Terminators; Applied Biosystems). The reaction was incubated at 37°C for 3 h. Excess ddNTPs were removed by purification on QIAquick PCR purification columns (Qiagen). For SSCP analysis, the labeled DNA fragments were vacuumed dried and dissolved in 3 µL formamide/Blue Dextran 2000 (Pharmacia Fine Chemicals, Uppsala, Sweden), 2 µL GENESCAN®-500 ROX standard (Applied Biosystems), and 0.5 µL 0.1 M NaOH. Before loading the samples onto the polyacrylamide slab gel [10% urea, 6% polyacrylamide gel, 1% (w/v) bisacrylamide/19% (w/v) acrylamide, 1× TBE (Tris-borate-EDTA)], they were denatured at 98°C for 3 min and immediately cooled on ice. Each sample (1.5 µL) was loaded and separated on an ABI Prism® 377 DNA Sequencer (Applied Biosystems) at 2140 volts (V), 60 mA, and 200 W in 1× TBE for 6 h. The temperature was kept constant at 12°C by an external water bath. Fragment lengths of each individual conformer were determined by comparing the data point to that of the internal GENESCAN-500 ROX standard using the GENESCAN v.3.0 software (Applied Biosystems; Figure 2C). Any abbreviations in fragment length (shift) to that of the normal control sample were determined, resulting in all the mutated samples being detected and the three normal samples identified. Introducing more than one restriction enzyme enabled multicolor labeling and localization of specific restriction fragments containing sequence variants (Figure 2, A and B), depending on the recognition sequence and digestion of the particular restriction enzymes. We have successfully tested the protocol for SNP haplo-typing of selected families, in which a 5.1-kb genomic DNA fragment of the human hydroxymethylbilane synthase (HMBS) gene was screened for SNPs, with restriction enzyme fragment sizes from 600 to 20 bp. The shifts discovered were then used directly as intragenic markers for haplotyping (16).Figure 1. Overview of PAH cDNA samples and the fluorescence labeling step.(A) Identity and distribution of mutations in PAH cDNA samples included in the blind test. Each sample is numbered from 1 to 36 and represented by position and the specific mutation(s). No sample had mutations in more than one codon. The red line indicates the PAH coding sequence, the black line is the pMAL-E vector sequence. Numbering is according to the cDNA sequence of PAH (starting at the first ATG, GenBank® accession no. NM_000277). The location and identity of the restriction enzyme sites are marked below the red line. *21. (782 G > A) is the only sample in which the mutation disrupts a restriction enzyme site (HinfI 781), creating a restriction fragment length polymorphism (RFLP). None of the sequence variants creates additional HinfI or BanI sites. (B) Outlining the one-tube digestion and labeling by minisequencing. (1) Deoxy nucleotides from PCR must be removed by purifying the PCR product before (2) digestion and labeling, which are performed simultaneously in one tube. Because only fluorescent ddNTPs are present, only one single nucleotide will be incorporated in a sequence-dependent manner at the end of each digested fragment. PAH, phenylalanine hydroxylase.Figure 2. Fluorescence REF-SSCP of PAH cDNA samples.(A) Gel image with 26 samples showing all labeled fragments, ROX-500 standard in red, HinfI-digested ends in green (labeled with R6G-labeled ddATP), and BanI-digested ends in blue (labeled with R110-labeled ddGTP). The direction of electro-phoresis is from top to bottom. Half of the samples were loaded onto the gel in every second well (2, 4, 6–26) and subjected to electrophoresis for 1 min before loading the rest of the samples (1, 3, 5–25). Two lanes contained ROX-labeled DNA standard only (lanes 5 and 10). Shifts are readily visible in most sections of the gel image correlating to the specific location of each specific sequence variant. A yellow box indicates a section of the gel image (enlarged in panel B) showing co-migration of two individual fragments, BanI(306)-HinfI(401) and HinfI(1324)-HinfI(1390). Three of the 16 samples displayed were mutated within the double-labeled fragment (HinfI-BanI), as shown in lane 4 (331 C > T); lane 8 (358 T > A, 359 G > T, 360 G > C); and lane 9 (359 G > T, 360 G > C). One sample with a mutation in the single-labeled (HinfI-HinfI) fragment is shown in lane 12 (1333 T > G, 1334 G > A). The other samples shown here carried no mutations in this particular region: (1) 1241 A > G; (2) 611 A > G; (3) 1285 C > T; (5) 709 T > G, 710 G > A; (6) 709 T > A; (7) normal sample; (10) 560 G > T, 561 G > C; (11) 977 G > T, 978 G > C; (13) normal sample. Lanes 14–15 were the reference samples. (C) To avoid lane-to-lane and run-to-run variations, analyzing shifts were done by comparing chromatograms to that of the reference samples after normalizing each individual sample to the internal size standard. Here we show four samples, (1) 776 C > T; (2) 810 A > T; (3) 814 G > T; and (4) 964 G > A, carrying mutations in the region or restriction enzyme fragments displayed in this chromatogram. The red line represents the R6G-labeled normal reference, and blue line represents four of the R6G-labeled mutant samples displaying shifts in this particular region. REF-SSCP, restriction endonuclease fingerprinting single-strand conformational polymorphism; PAH, phenylalanine hydroxylase.The one-tube restriction enzyme digest and fluorescent ddNTP labeling of 5′ protruding ends created by restriction enzymes (Figure 1B) proved to be highly efficient, reliable, and cost-efficient. It is not time-consuming, the concentration of fluorescent ddNTPs required is very low (0.05 µL/sample), and all fragments were reproducibly and uniformly labeled (Figure 2A). We believe that the ability to investigate large PCR fragments makes this method suitable for investigating SNPs located in genomic DNA, without requiring any knowledge of existing SNPs in the region or their frequencies. We therefore propose that this protocol should be useful for investigating sequence variants in model organisms as well as nonmodel organisms, in which sequence data and database information of SNPs are limited.Competing Interests StatementThe authors declare no competing interests.References1. Orita, M., H. Iwahana, H. Kanazawa, K. Hayashi, and T. Sekiya. 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86:2766–2770.Crossref, Medline, CAS, Google Scholar2. Orita, M., Y. Suzuki, T. Sekiya, and K. Hayashi. 1989. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879.Crossref, Medline, CAS, Google Scholar3. 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Haplotype analysis of Norwegian and Swedish patients with acute intermittent porphyria (AIP): extreme haplotype heterogeneity for the mutation R116W. Dis. Markers 19:41–46.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetails Vol. 37, No. 6 Follow us on social media for the latest updates Metrics Downloaded 310 times History Received 13 May 2004 Accepted 15 July 2004 Published online 6 June 2018 Published in print December 2004 Information© 2004 Author(s)Competing Interests StatementThe authors declare no competing interests.PDF download" @default.
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