FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1990403938> ?p ?o ?g. }

• W1990403938 endingPage "180" @default.
• W1990403938 startingPage "174" @default.
• W1990403938 abstract "BioTechniquesVol. 39, No. 2 BenchmarksOpen AccessLigation overcomes terminal underrepresentation in multiple displacement amplification of linear DNASimona Panelli, Giuseppe Damiani, Luca Espen & Vittorio SgaramellaSimona Panelli*Address correspondence to: Simona Panelli, CERSA/Fondazione Parco Tecnologico Padano, Lodi, Italy. e-mail: E-mail Address: simona.panelli@unimi.itCERSA/Fondazione Parco Tecnologico Padano, LodSearch for more papers by this author, Giuseppe DamianiIGM-CNR, PaviaSearch for more papers by this author, Luca EspenUniversità di Milano, Milano, ItalySearch for more papers by this author & Vittorio SgaramellaCERSA/Fondazione Parco Tecnologico Padano, LodSearch for more papers by this authorPublished Online:30 May 2018https://doi.org/10.2144/05392BM03AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit The amount of DNA available for informative genomic studies is often limiting. Thus, several methods have been developed to achieve a whole genome amplification (WGA). Particularly promising is a variant, called multiple displacement amplification (MDA) (1,2), which exploits the high processivity of Φ29 phage DNA polymerase and suffers an amplification bias significantly lower than previous, PCR-based WGA (3).Even though MDA-DNA has been successfully used for many applications (1)(4–7), some problems have been reported. For example, sequences near the ends of linear chromosomes appear underrepresented after MDA (6,8,9). Both defective priming events and abortive chain terminations could contribute to this problem, which limits the applications of MDA and jeopardizes its use on short linear DNA (e.g., cDNA and degraded genomes from forensic, archeological, and fetal origin) (10). In these cases, the terminal underrepresentation would cause the loss of substantial information.We studied this problem using λ phage DNA (48.5 kb; Promega Biosciences, San Luis Obispo, CA, USA). MDA was performed using the Genomiphi® kit (Amersham Biosciences, Little Chalfont, England, UK) first on the uncut λ DNA (1 ng) and then on the mixture of its seven HindIII fragments (1 ng). Reactions were incubated overnight at 30°C using the conditions suggested by the manufacturer. We then compared the same amount of amplified samples and unamplified native DNA to assess the representation of the various restriction fragments after MDA. To achieve this aim, we used frequent cutters (RsaI: 113 sites on λ; PstI: 28 sites) in combination with HindIII (Promega Biosciences). Restriction digests with HindIII alone were also performed. Digestions were visualized on either agarose (Eppendorf, Hamburg, Germany) or polyacrylamide gels (Sigma, St. Louis, MO, USA) for discriminating both high and low molecular weight bands, respectively. HindIII and HindIII + PstI digestions were visualized on 1% agarose gels, and HindIII + RsaI digestions were visualized on 1.4% gels. The electrophoresis was run at 80 V for 2 h in 1× TAE buffer (40 mM Tris-base, 20 mM acetic acid, 1 mM EDTA, pH 8.0) and visualized using ethidium bromide (Sigma). Four-percent polyacrylamide gels (acrylamide:bisacrylamide 37.5:1) were run in 0.5× TBE buffer (45 mM Tris-base, 45 mM boric acid, 1 mM EDTA, pH 8.0; Sigma) at 120 V for 30 min and 200 V for 3 h. Patterns were visualized using VistraGreen™ (Amersham Biosciences).The restriction patterns are shown in Figure 1. Dean et al. (1) observed that, in Southern blot experiments, DNA fragments longer than 5 kb appear substantially underrepresented after MDA and their electrophoretic resolution was impaired. The same problem emerges from our electrophoretic patterns, especially with high molecular weight fragments (Figure 1). Dean et al. (1) ascribed this to DNA degradation due to the initial denaturation step required for MDA, the effects of which are supposedly stronger on longer fragments. However, the structure proposed for MDA products could suggest another explanation. It was hypothesized (8) that they are characterized by networks of hyperbranched DNA. These structures might compound the electrophoretic problems. In particular, since hyperbranched DNA products are expected to be more frequent in longer amplified fragments, their mobility would be reduced proportionally to their molecular weight. Thus, the quality difference of gels in Figure 1A (high molecular weight bands) and Figure 1B (low molecular weight bands) could be due not only to the different type of gel (agarose versus polyacrylamide) but also to the hyperbranched structure of the products.Figure 1. Restriction patterns resolved on (A) agarose and (B) polyacrylamide gels.In all cases, lanes are the following: 1, native λ DNA; 2, uncut λ DNA + MDA; 3, λ/HindIII + MDA; 4, λ/HindIII + ligase + MDA. (A) Digested unamplified DNA (2 µg) and the same amounts of digested MDA samples were loaded. A band not detectable in the HindIII digestion (lane 2) is signaled with an asterisk to the right of the gel. Lane m, GeneRuler™ 100-bp DNA Ladder Plus (Fermentas Life Sciences, Hanover, MD, USA) (B) Digested unamplified DNA (500 ng) and the same amounts of digested MDA samples were loaded. Bands not detectable in lanes 2 and 3 are signaled with circles and arrowheads, respectively. Lane m, 1-kb Plus DNA Ladder (Invitrogen). MDA, multiple displacement amplification.Figure 1 shows the restriction patterns of the unamplified native λ DNA (lanes 1) and of the MDA products generated from the intact λ genome (lanes 2). Patterns are easily recognizable in amplified samples, suggesting that MDA produces double-stranded DNA that represents nearly the whole λ genome. The disappearance of a terminal band (the 3′ 4.4-kb band in the HindIII digest) may be observed in the MDA sample (Figure 1A, lane 2, signaled with an asterisk to the right of the gel). Other bands present diminished intensity. This is more easily visible in the PstI + HindIII pattern (Figure 1B, bands marked with circles). In accordance with the terminal underrepresentation phenomenon, all of these fragments map in the first 5 kb at the 5′ end of the genome (Figure 2A).Figure 2. Map positions of fragments underrepresented after standard multiple displacement amplification (MDA) as determined through (A)PstI + HindIII or (B)RsaI + HindIII digestion.Numbers in bold above the maps refer to the molecular weight of the lost or underrepresented fragments. Bands not detectable when subjecting the uncut λ DNA to MDA are indicated with circles. Bands lost when amplifying λ/HindIII fragments are signaled with arrowheads. In the first case (circles), PstI fragments confined within 5 kb from the 5′ end present a very diminished intensity after MDA (A). In the second case (arrowheads), the λ/HindIII + MDA sample subjected to PstI + HindIII digestion loses the 704- and 547-bp fragments (A). Both derive from PstI fragments containing internal HindIII sites. The same sample, after RsaI + HindIII digestion, loses several fragments (B), all close to HindIII sites. Other fragments, including those at the termini, could not be unambiguously identified because of co-migration and gel resolution problems. They were thus excluded from our analysis.We then subjected the λ/HindIII fragments to MDA. Their subsequent HindIII digestion did not produce recognizable patterns (Figure 1, lanes 3 of the HindIII panels) as neither intact HindIII fragments nor reconstituted sites are present in this sample. Concerning the other restriction digests, recognizable patterns are still present, but some fragments are no longer detectable (Figure 1B, indicated by arrowheads). These are RsaI or PstI fragments containing, or close to, HindIII sites (Figure 2), again in concordance with terminal underrepresentation.Thus, our analysis shows that after MDA, the sequences in proximity of the template termini are lost or under-represented. We have defined that in the λ genome this phenomenon affects regions within about 5 kb of the ends. The behavior of the HindIII + PstI 5′ terminal fragments (Figure 1B and 2A, circles) makes it likely that premature detachment of the growing chain contributes to the terminal under-representation. The relevant bands are much less intense when amplifying the entire λ genome, while their intensities become comparable to those of the unamplified native λ after MDA of the HindIII fragments.In order to overcome the termini problem, we developed a simple protocol based on ligation of linear templates before MDA to obtain a random permutation of the original fragments and possibly the circularization of the resulting products. T4 DNA ligase (3 Weiss units; Invitrogen, Carlsbad, CA, USA) was used to ligate the cohesive ends of λ/HindIII fragments (7.5 ng/µL). Other restriction enzymes have been tried with comparable results (PstI and BglII, not shown). Reactions were incubated overnight at 30°C and were analyzed on 1% agarose gels (11). The ligated sample (750 pg) was subjected to amplification. For all MDA reactions, we obtained 10–20 µg product.The product was then compared with the unamplified native DNA as described above. Lanes 4 (Figure 1) show the ligated samples. Each digestion was repeated at least three times using different λ/HindIII + ligase and MDA preparations. We always observed the same patterns, and the resultant bands were reproducible. The patterns shown were obtained from representative experiments. The restriction patterns were the most similar to those of the unamplified DNA. Nearly all the bands not detectable in Figure 1, lanes 2–3, were recovered, even if their intensities did not always perfectly match those of the unamplified DNA, indicating slight differences in the ligation and/or amplification efficiency of different fragments.Special consideration was given to the terminal sequences of λ DNA, cosL and cosR, which after HindIII digestion are borne respectively by the 23- and 4.4-kb fragments. Once ligated, they are resolvable only by specialized enzymes. The λ/HindIII 3′ band (4.4 kb) is not rescued after ligation (Figure 1A). In this sample, the 4.4-kb fragment, after cosL/R joining, forms a 27.4-kb band, indistinguishable from the 23-kb band, as they both lie in the nonresolving region of the gel.In both gels of Figure 1, A and B, there are some faint bands that do not correspond to the expected restriction patterns. The clearest example is a band of about 4.0 kb, in lanes 2 and 4 of the HindIII digestion (Figure 1A). The unexpected bands are not produced by the ligase treatment, since they are also present in lane 2. Moreover, they generally disappear in lane 3 (i.e., when digesting the unligated λ/HindIII fragments). In the light of data on the enzymatic properties of the Φ29 DNA polymerase (12), a reasonable hypothesis is that the unexpected bands are due to the presence of hairpins in the DNA template. These structures are known to cause polymerase detachment from the template, template switching, or specific endonucleolytic activity (13,14). The result is the accumulation of discrete small DNA molecules. We found a palindromic sequence, GGTTGATATCAACC, starting at nucleotide 41269 (GenBank® accession no. NC_001416) and located 3810 bp downstream of the 37459 λ/HindIII site. It is related to the sequence TGTTTCACGTGGAACA, which Murthy et al. (12) call a dyad symmetry element (DSE) and show to be specifically responsible for the replication block of the Φ29 DNA polymerase. This hairpin may thus be responsible for the production of the cited band in lanes 2 and 4 of the HindIII digestion (Figure 1A). This hypothesis also explains why this band disappears in lane 3. In fact, when subjecting the unligated λ/HindIII fragments to MDA, the relevant region is underrepresented in the MDA product since it is located near a terminus.In conclusion, the ligation of templates helps to overcome under-representation of the termini and expands the potential for the application of MDA. It is useful to obtain faithful amplifications of small, linear templates that otherwise could not be successfully amplified. Both cohesive and blunt ends can be satisfactorily ligated before MDA. For a wider range of applications, and especially for the study of degraded genomic DNA, one could consider blunt-ending (mediated, for example, by T4 DNA polymerase) and ligation, mediated by adaptors containing restriction recognition sequences. Our protocol provides a simpler alternative to the OmniPlex® technology (Rubicon Genomics, Ann Arbor, MI, USA) (15) for performing WGA on such samples.Recently, a method has been described for isothermal in vitro amplification of specific regions (16). It exploits DNA helicase to generate a single-stranded template for primer hybridization and subsequent extension. However, its application could be problematic if the template is limited or degraded. Our procedure, through the fill-in and blunt-end ligation of linear DNA before MDA, could substitute DNA extraction as the first step of a totally isothermal process for amplifying specific DNA sequences regardless of the template quantity and quality.AcknowledgmentsWe thank Claudio Bandi for hospitality and the Cariplo Foundation for a grant to V.S.Competing Interests StatementThe authors declare no competing interests.References1. Dean, F.B., S. Hosono, L. Fang, X. Wu, F. Faruqi, P. Bray-Ward, Z. Sun, Q. Zong, et al.. 2002. Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. USA 99:5261–5266.Crossref, Medline, CAS, Google Scholar2. Dean, F.B., J.R. Nelson, T.L. Giesler, and R.S. Lasken. 2001. Rapid amplification of plasmid and phage DNA using phi 29 DNA polymerase and multiply-primed rolling-circle amplification. Genome Res. 11:1095–1099.Crossref, Medline, CAS, Google Scholar3. Cheung, V.G. and S.F. Nelson. 1996. Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. Proc. Natl. Acad. Sci. USA 93:14676–14679.Crossref, Medline, CAS, Google Scholar4. Detter, J.C., J.M. Jett, S.M. Lucas, E. Dalin, A.R. Arellano, M. Wang, J.R. Nelson, J. Chapman, et al.. 2002. Isothermal strand-displacement amplification applications for high-throughput genomics. Genomics 80:691–698.Crossref, Medline, CAS, Google Scholar5. Hellani, A., S. Coskun, M. Benkhalifa, A. Tbakhi, N. Sakati, A. Al-Odaib, and P. Ozand. 2004. Multiple displacement amplification on single cell and possible PGD applications. Mol. Hum. Reprod. 10:847–852.Crossref, Medline, CAS, Google Scholar6. Hosono, S., A.F. Faruqi, F.B. Dean, D. Yuefen, Z. Sun, X. Wu, J. Du, S.F. Kingsmore, et al.. 2003. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13:954–964.Crossref, Medline, CAS, Google Scholar7. Wong, K.K., Y.T.M. Tsang, J. Shen, R.S. Cheng, Y.M. Chang, T.K. Man, and C.C. Lau. 2004. Allelic imbalance analysis by high-density single nucleotide polymorphic allele (SNP) array with whole genome amplified DNA. Nucleic Acids Res. 32:e69.Crossref, Medline, Google Scholar8. Lage, J.M., J.H. Leamon, T. Pejovic, S. Hamann, M. Lacey, D. Dillon, R. Segraves, B. Vossbrinck, et al.. 2003. Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH. Genome Res. 13:294–307.Crossref, Medline, CAS, Google Scholar9. Tzvetkov, M.V., C. Becker, B. Kulle, P. Nurnberg, J. Brockmoller, and L. Wojonowski. 2005. Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification. Electrophoresis 26:710–715.Crossref, Medline, CAS, Google Scholar10. Li, Y., E. Di Naro, A. Vitucci, B. Zimmermann, W. Holzgreve, and S. Hahn. 2005. Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma. JAMA 293:843–849.Crossref, Medline, CAS, Google Scholar11. Sambrook, J. and D.W. Russell. 1996. Molecular Cloning: A Laboratory Manual, 3rd ed. CSH Laboratory Press, Cold Spring Harbor.Google Scholar12. Murthy, V., W.J.J. Meijer, L. Blanco, and M. Salas. 1998. DNA polymerase template switching at specific sites on the Φ29 genome causes the in vivo accumulation of subgenomic Φ29 DNA molecules. Mol. Microbiol. 29:787–798.Crossref, Medline, CAS, Google Scholar13. Hacker, K.J. and B.M. Alberts. 1994. The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for the polymerase release following the termination of each Okazaki fragment. J. Biol. Chem. 269:24221–24228.Crossref, Medline, CAS, Google Scholar14. Tombline, G., D. Bellizzi, and V. Sgaramella. 1996. Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endo-nuclease activity of DNA polymerase and to premature stops. Proc. Natl. Acad. Sci. USA 93:2724–2728.Crossref, Medline, CAS, Google Scholar15. Barker, D.L., M.S.T. Hansen, A.F. Faruqi, D. Giannola, O.R. Irsula, R.S. Lasken, M. Latterich, V. Makarov, et al.. 2004. Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genome Res. 14:901–907.Crossref, Medline, CAS, Google Scholar16. Vincent, M., Y. Xu, and H. Kong. 2004. Helicase-dependent isothermal DNA amplification. EMBO Rep. 5:1–6.Crossref, Medline, Google ScholarFiguresReferencesRelatedDetailsCited ByA lab-in-a-foil microfluidic reactor based on phaseguidingMicroelectronic Engineering, Vol. 187-188Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome30 January 2014 | Microbiome, Vol. 2, No. 1Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose5 February 2014 | PLoS ONE, Vol. 9, No. 2The future is now: single-cell genomics of bacteria and archaea1 May 2013 | FEMS Microbiology Reviews, Vol. 37, No. 3Amplification of viral RNA from drinking water using TransPlex™ whole-transcriptome amplification5 May 2011 | Journal of Applied Microbiology, Vol. 111, No. 1Single Virus Genomics: A New Tool for Virus Discovery23 March 2011 | PLoS ONE, Vol. 6, No. 3Somatic genome variations interact with environment, genome and epigenome in the determination of the phenotype: A paradigm shift in genomics?DNA Repair, Vol. 9, No. 4Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primersHirokazu Takahashi, Kimiko Yamamoto, Toshio Ohtani & Shigeru Sugiyama25 April 2018 | BioTechniques, Vol. 47, No. 1In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule levelAnalytical Biochemistry, Vol. 383, No. 2Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotypingAnalytical Biochemistry, Vol. 368, No. 2Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samplesForensic Science International, Vol. 166, No. 1Single molecule transcription profiling with AFM21 December 2006 | Nanotechnology, Vol. 18, No. 4Phi29-based amplification of small genomesAnalytical Biochemistry, Vol. 354, No. 1Sequencing genomes from single cells by polymerase cloning28 May 2006 | Nature Biotechnology, Vol. 24, No. 6Gene paucity, genome instability, clonal development: Has an individual genome the potential to encode for more than one brain?DNA Repair, Vol. 5, No. 5Towards the analysis of the genomes of single cells: Further characterisation of the multiple displacement amplificationGene, Vol. 372 Vol. 39, No. 2 Follow us on social media for the latest updates Metrics History Received 12 January 2005 Accepted 27 May 2005 Published online 30 May 2018 Published in print August 2005 Information© 2005 Author(s)AcknowledgmentsWe thank Claudio Bandi for hospitality and the Cariplo Foundation for a grant to V.S.Competing Interests StatementThe authors declare no competing interests.PDF download" @default.
• W1990403938 created "2016-06-24" @default.
• W1990403938 creator A5039525663 @default.
• W1990403938 creator A5046167967 @default.
• W1990403938 creator A5048568924 @default.
• W1990403938 creator A5052660394 @default.
• W1990403938 date "2005-08-01" @default.
• W1990403938 modified "2023-09-27" @default.
• W1990403938 title "Ligation overcomes terminal underrepresentation in multiple displacement amplification of linear DNA" @default.
• W1990403938 cites W1560122944 @default.
• W1990403938 cites W1968762427 @default.
• W1990403938 cites W2009978569 @default.
• W1990403938 cites W2019342009 @default.
• W1990403938 cites W2022008291 @default.
• W1990403938 cites W2037325598 @default.
• W1990403938 cites W2040041836 @default.
• W1990403938 cites W2063513406 @default.
• W1990403938 cites W2080944438 @default.
• W1990403938 cites W2086814185 @default.
• W1990403938 cites W2097097085 @default.
• W1990403938 cites W2097348296 @default.
• W1990403938 cites W2117240806 @default.
• W1990403938 cites W2120147786 @default.
• W1990403938 cites W2153125376 @default.
• W1990403938 doi "https://doi.org/10.2144/05392bm03" @default.
• W1990403938 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16116788" @default.
• W1990403938 hasPublicationYear "2005" @default.
• W1990403938 type Work @default.
• W1990403938 sameAs 1990403938 @default.
• W1990403938 citedByCount "17" @default.
• W1990403938 countsByYear W19904039382013 @default.
• W1990403938 countsByYear W19904039382014 @default.
• W1990403938 countsByYear W19904039382018 @default.
• W1990403938 countsByYear W19904039382021 @default.
• W1990403938 crossrefType "journal-article" @default.
• W1990403938 hasAuthorship W1990403938A5039525663 @default.
• W1990403938 hasAuthorship W1990403938A5046167967 @default.
• W1990403938 hasAuthorship W1990403938A5048568924 @default.
• W1990403938 hasAuthorship W1990403938A5052660394 @default.
• W1990403938 hasBestOaLocation W19904039381 @default.
• W1990403938 hasConcept C104317684 @default.
• W1990403938 hasConcept C153911025 @default.
• W1990403938 hasConcept C156385826 @default.
• W1990403938 hasConcept C179437574 @default.
• W1990403938 hasConcept C2779664074 @default.
• W1990403938 hasConcept C41008148 @default.
• W1990403938 hasConcept C49105822 @default.
• W1990403938 hasConcept C54355233 @default.
• W1990403938 hasConcept C552990157 @default.
• W1990403938 hasConcept C70721500 @default.
• W1990403938 hasConcept C76155785 @default.
• W1990403938 hasConcept C78063203 @default.
• W1990403938 hasConcept C86803240 @default.
• W1990403938 hasConceptScore W1990403938C104317684 @default.
• W1990403938 hasConceptScore W1990403938C153911025 @default.
• W1990403938 hasConceptScore W1990403938C156385826 @default.
• W1990403938 hasConceptScore W1990403938C179437574 @default.
• W1990403938 hasConceptScore W1990403938C2779664074 @default.
• W1990403938 hasConceptScore W1990403938C41008148 @default.
• W1990403938 hasConceptScore W1990403938C49105822 @default.
• W1990403938 hasConceptScore W1990403938C54355233 @default.
• W1990403938 hasConceptScore W1990403938C552990157 @default.
• W1990403938 hasConceptScore W1990403938C70721500 @default.
• W1990403938 hasConceptScore W1990403938C76155785 @default.
• W1990403938 hasConceptScore W1990403938C78063203 @default.
• W1990403938 hasConceptScore W1990403938C86803240 @default.
• W1990403938 hasIssue "2" @default.
• W1990403938 hasLocation W19904039381 @default.
• W1990403938 hasLocation W19904039382 @default.
• W1990403938 hasLocation W19904039383 @default.
• W1990403938 hasLocation W19904039384 @default.
• W1990403938 hasOpenAccess W1990403938 @default.
• W1990403938 hasPrimaryLocation W19904039381 @default.
• W1990403938 hasRelatedWork W1988958686 @default.
• W1990403938 hasRelatedWork W1995477860 @default.
• W1990403938 hasRelatedWork W2002128513 @default.
• W1990403938 hasRelatedWork W2080534576 @default.
• W1990403938 hasRelatedWork W2089817402 @default.
• W1990403938 hasRelatedWork W2097272551 @default.
• W1990403938 hasRelatedWork W2185219645 @default.
• W1990403938 hasRelatedWork W2323003408 @default.
• W1990403938 hasRelatedWork W2404044552 @default.
• W1990403938 hasRelatedWork W2092874662 @default.
• W1990403938 hasVolume "39" @default.
• W1990403938 isParatext "false" @default.
• W1990403938 isRetracted "false" @default.
• W1990403938 magId "1990403938" @default.
• W1990403938 workType "article" @default.