Universal Method for Producing ROX-Labeled Size Standards Suitable for Automated Genotyping
2004; Future Science Ltd; Volume: 37; Issue: 3 Linguagem: Inglês
10.2144/04373bm02
ISSN1940-9818
AutoresJ. Andrew DeWoody, Jim Schupp, Leo J. Kenefic, Joseph D. Busch, Lisa M. Murfitt, Paul Keim,
Tópico(s)Plant biochemistry and biosynthesis
ResumoBioTechniquesVol. 37, No. 3 BenchmarksOpen AccessUniversal method for producing ROX-labeled size standards suitable for automated genotypingJ. Andrew DeWoody, Jim Schupp, Leo Kenefic, Joseph Busch, Lisa Murfitt & Paul KeimJ. Andrew DeWoody*Address correspondence to: Andrew DeWoody, Department of Forestry & Natural Resources, Purdue University, West Lafayette IN 47905-1159, USA. e-mail: E-mail Address: dewoody@purdue.eduPurdue University, West Lafayette, IN, Jim SchuppNorthern Arizona University, Flagstaff, AZ, USA, Leo KeneficNorthern Arizona University, Flagstaff, AZ, USA, Joseph BuschNorthern Arizona University, Flagstaff, AZ, USA, Lisa MurfittPurdue University, West Lafayette, IN & Paul KeimNorthern Arizona University, Flagstaff, AZ, USAPublished Online:6 Jun 2018https://doi.org/10.2144/04373BM02AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Fluorescently labeled size standards are indispensable for genotyping via an automated DNA sequencer. Microsatellites, amplified fragment-length polymorphisms (AFLPs), single nucleotide polymorphisms (SNPs), and random amplified polymorphic DNA (RAPD) can all be assayed on platforms such as the ABI Prism® 377 DNA Sequencer or the ABI Prism 3100-Avant Genetic Analyzer (both from Applied Biosystems, Foster City, CA, USA), but each requires a fluorescent marker that typically is purchased from a commercial vendor. These markers are often prohibitively expensive. Indeed, the cost of size standards can exceed that of the genotyping assay itself. In response to this dilemma, two recent reports have described novel approaches to the in-house production of fluorescently labeled size standards (1,2). Unfortunately, both of these methods require specific DNA templates that are uncommon in most laboratories, and one of the methods requires multiple labeled primers. Below, we describe a method for marker production that relies upon two of the most common laboratory cloning vectors, plasmids pUC and pBluescript® (pBS).We used the sequences of pUC-19 and pBS II SK(+) to design PCR primers for the amplification of known-length fragments from plasmid mini-preps. The primer sets (Table 1) each include a single labeled forward primer and multiple unlabeled reverse primers that are each used in conjunction with the labeled forward primer. Once the primer sequences were in hand, we used them to amplify DNA fragments from common laboratory plasmids.Table 1. Primer Sequences Complementary to pBS II SK (+) and pUC-19Briefly, plasmid DNA is prepared using conventional miniprep techniques (3), and this DNA is used as a template for subsequent PCR. Plasmid DNA is low complexity and thus makes an excellent template for PCR. We amplified fragments individually as opposed to experimenting with multiplex reactions. Amplifications all utilized the same thermal profile, namely 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. A final extension reaction of 10 min at 72°C ensures that all PCR products are full length. For the pBS size standard, we used Pfu DNA polymerase to avoid the A-tailing inherent to Taq DNA polymerase (4). For the pUC size standard, a naturally occurring or artificial 5′ guanine residue was included in all but four of the unlabeled primer sequences to promote the A-tailing. The pUC fragment sizes reflect this added 5′ guanine and the A-tailing. In our hands, only the 90-bp pUC fragment gave a significant doublet, but fragment analysis parameters can be set such that only the larger molecular weight peak is used for size calculation.Individual amplifications were tested via agarose gel electrophoresis, and PCR products were subsequently combined into a single tube. An aliquot of this mixture was then run on an automated DNA sequencer alongside a known (i.e., commercial) size standard as described earlier (1,2). Our results indicate that plasmid-based size standards are comparable to commercial size standards in their reproducibility, stability, and accuracy (Figure 1). Plasmid-based size standards are also easily customizable for specialized sizing requirements. Furthermore, we estimate their cost to be less than 10% of commercial products.Figure 1. ROX-labeled standards.(A) Gel image from an ABI Prism 377 DNA Sequencer showing ROX-labeled commercial size standards (from Applied Biosystems) and our ROX-labeled pBluescript (pBS) standard. The pBS size standard in the rightmost lanes appears brighter on this gel, but can easily be diluted to the same concentration as the commercial size standard. (B) Chromatogram of pUC size standard run on an ABI Prism 3100-Avant Genetic Analyzer with fragment sizes in bases indicated above each peak.AcknowledgmentsThis project was supported in part by the United States Department of Health and Human Services, award no. U90/CCU923231-01 and by the United States Department of Agriculture National Research Initiative (USDA-NRI) award no. 2003-03616. This manuscript is Agricultural Research Programs (ARP) no. 17390 from Purdue University.Competing Interests StatementThe authors declare that they have no competing interests.References1. Brondani, R.P.V. and D. Grattapaglia. 2001. Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing. BioTechniques 31:793–800.Link, CAS, Google Scholar2. Ueno, S., Y. Tsumura, and I. Washitani. 2003. Cost-effective method to synthesize fluorescently labeled DNA size standards using cloned AFLP fragments. BioTechniques 34:1146–1148.Link, CAS, Google Scholar3. Sambrook, J. and D.W. Russell. 2001. Molecular Cloning: A Laboratory Manual. CSH Laboratory Press, Cold Spring Harbor, NY.Google Scholar4. Brownstein, M.J., J.D. Carpten, and J.R. Smith. 1996. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. 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U90/CCU923231-01 and by the United States Department of Agriculture National Research Initiative (USDA-NRI) award no. 2003-03616. This manuscript is Agricultural Research Programs (ARP) no. 17390 from Purdue University.Competing Interests StatementThe authors declare that they have no competing interests.PDF download
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