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Home / Equine Research Bank / BMC Genomics / A new genomic tool, ultra-frequently cleaving TaqII/sinefung...
BMC genomics2013; 14; 370; doi: 10.1186/1471-2164-14-370

A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries.

Abstract: Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases). Results: We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) - an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5'-GACCGA-3' [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation. Conclusions: In the presence of SIN/DMSO, TaqII REase is transformed from cleaving every 4096 bp on average to cleaving every 58 bp. TaqII SIN/DMSO thus extends the palette of available REase prototype specificities. This phenomenon, employed under partial digestion conditions, was applied to quasi-random DNA fragmentation. Further applications include high sensitivity probe generation and metagenomic DNA amplification.
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Publication Date: 2013-06-01 PubMed ID: 23724933PubMed Central: PMC3681635DOI: 10.1186/1471-2164-14-370Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research paper presents a novel technique, using a bioengineered version of the restriction enzyme TaqII, to produce small and uniform fragments of DNA for genomic studies such as the creation of libraries and amplification of metagenomic DNA.

Restriction Enzymes in Genomics

  • The focus of the study is the field of genomics and metagenomics, two areas where DNA sequencing technologies are developing rapidly, generating huge quantities of sequence data.
  • However, the researchers identify problems related to the assembly of the entire genome (contig assembly) and the production of a representative library due to limitations in the current methods used to break the DNA into smaller pieces – methods such as hydrodynamic shearing, sonication, and DNase I fragmentation.
  • One alternative, and the focus of this research, is the use of restriction enzymes (REases) for DNA breakdown, which could result in higher quality fragments.

Modified TaqII and DNA Fragmentation

  • The team developed a new method to fragment DNA using a bioengineered version of the restriction enzyme TaqII, which originates from a family of bifunctional enzymes found in a bacterium called Thermus sp.
  • Through the use of sinefungin (SIN) and dimethylsulfoxide (DMSO), the traditional 6-base pair (bp) recognition site of TaqII is converted into a theoretical 2.9-bp site. This version of TaqII can recognise shorter DNA sequences and leads to the creation of more and smaller fragments.
  • Using this modified enzyme, they were able to construct a horse genomic library, along with a derivative library made up of DNA in the coding region of the butyrylcholinesterase gene.

Applications of the Study

  • The technique of partial DNA cleavage inherent in the modified TaqII enzyme makes it ideal for generating quasi-random libraries, an important tool in genomic research.
  • The presence of SIN/DMSO allows TaqII to cut DNA at approximately every 58 base pairs instead of every 4,096 base pairs as per the native version of the enzyme. This modifies the specificity of the enzyme and presents a more versatile tool for genomic studies.
  • According to the researchers, this method could also be used for the creation of high-sensitivity probes and amplification of metagenomic DNA, broadening its application in the scientific community.

Cite This Article

APA
Zylicz-Stachula A, Zolnierkiewicz O, Jasiecki J, Skowron PM. (2013). A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries. BMC Genomics, 14, 370. https://doi.org/10.1186/1471-2164-14-370

Publication

BMC genomics
ISSN: 1471-2164
NlmUniqueID: 100965258
Country: England
Language: English
Volume: 14
Pages: 370

Researcher Affiliations

Zylicz-Stachula, Agnieszka
    Zolnierkiewicz, Olga
      Jasiecki, Jacek
        Skowron, Piotr M

          MeSH Terms

          • Adenosine / analogs & derivatives
          • Adenosine / chemistry
          • Animals
          • DNA Cleavage
          • Deoxyribonucleases, Type II Site-Specific / chemistry
          • Genomic Library
          • Genomics / methods
          • Horses / genetics
          • Substrate Specificity
          • Thermus / enzymology

          References

          This article includes 40 references
          1. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM. Genome sequencing in microfabricated high-density picolitre reactors.. Nature 2005 Sep 15;437(7057):376-80.
            pmc: PMC1464427pubmed: 16056220doi: 10.1038/nature03959google scholar: lookup
          2. Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, Carnevali P, Nazarenko I, Nilsen GB, Yeung G, Dahl F, Fernandez A, Staker B, Pant KP, Baccash J, Borcherding AP, Brownley A, Cedeno R, Chen L, Chernikoff D, Cheung A, Chirita R, Curson B, Ebert JC, Hacker CR, Hartlage R, Hauser B, Huang S, Jiang Y, Karpinchyk V, Koenig M, Kong C, Landers T, Le C, Liu J, McBride CE, Morenzoni M, Morey RE, Mutch K, Perazich H, Perry K, Peters BA, Peterson J, Pethiyagoda CL, Pothuraju K, Richter C, Rosenbaum AM, Roy S, Shafto J, Sharanhovich U, Shannon KW, Sheppy CG, Sun M, Thakuria JV, Tran A, Vu D, Zaranek AW, Wu X, Drmanac S, Oliphant AR, Banyai WC, Martin B, Ballinger DG, Church GM, Reid CA. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays.. Science 2010 Jan 1;327(5961):78-81.
            doi: 10.1126/science.1181498pubmed: 19892942google scholar: lookup
          3. Mardis ER. Next-generation DNA sequencing methods.. Annu Rev Genomics Hum Genet 2008;9:387-402.
            doi: 10.1146/annurev.genom.9.081307.164359pubmed: 18576944google scholar: lookup
          4. Valouev A, Ichikawa J, Tonthat T, Stuart J, Ranade S, Peckham H, Zeng K, Malek JA, Costa G, McKernan K, Sidow A, Fire A, Johnson SM. A high-resolution, nucleosome position map of C. elegans reveals a lack of universal sequence-dictated positioning.. Genome Res 2008 Jul;18(7):1051-63.
            doi: 10.1101/gr.076463.108pmc: PMC2493394pubmed: 18477713google scholar: lookup
          5. Rothberg JM, Hinz W, Rearick TM, Schultz J, Mileski W, Davey M, Leamon JH, Johnson K, Milgrew MJ, Edwards M, Hoon J, Simons JF, Marran D, Myers JW, Davidson JF, Branting A, Nobile JR, Puc BP, Light D, Clark TA, Huber M, Branciforte JT, Stoner IB, Cawley SE, Lyons M, Fu Y, Homer N, Sedova M, Miao X, Reed B, Sabina J, Feierstein E, Schorn M, Alanjary M, Dimalanta E, Dressman D, Kasinskas R, Sokolsky T, Fidanza JA, Namsaraev E, McKernan KJ, Williams A, Roth GT, Bustillo J. An integrated semiconductor device enabling non-optical genome sequencing.. Nature 2011 Jul 20;475(7356):348-52.
            doi: 10.1038/nature10242pubmed: 21776081google scholar: lookup
          6. Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Baybayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B, Christians F, Cicero R, Clark S, Dalal R, Dewinter A, Dixon J, Foquet M, Gaertner A, Hardenbol P, Heiner C, Hester K, Holden D, Kearns G, Kong X, Kuse R, Lacroix Y, Lin S, Lundquist P, Ma C, Marks P, Maxham M, Murphy D, Park I, Pham T, Phillips M, Roy J, Sebra R, Shen G, Sorenson J, Tomaney A, Travers K, Trulson M, Vieceli J, Wegener J, Wu D, Yang A, Zaccarin D, Zhao P, Zhong F, Korlach J, Turner S. Real-time DNA sequencing from single polymerase molecules.. Science 2009 Jan 2;323(5910):133-8.
            doi: 10.1126/science.1162986pubmed: 19023044google scholar: lookup
          7. Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM. Accurate multiplex polony sequencing of an evolved bacterial genome.. Science 2005 Sep 9;309(5741):1728-32.
            doi: 10.1126/science.1117389pubmed: 16081699google scholar: lookup
          8. Knierim E, Lucke B, Schwarz JM, Schuelke M, Seelow D. Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.. PLoS One 2011;6(11):e28240.
            doi: 10.1371/journal.pone.0028240pmc: PMC3227650pubmed: 22140562google scholar: lookup
          9. Lam HY, Clark MJ, Chen R, Chen R, Natsoulis G, O'Huallachain M, Dewey FE, Habegger L, Ashley EA, Gerstein MB, Butte AJ, Ji HP, Snyder M. Performance comparison of whole-genome sequencing platforms.. Nat Biotechnol 2011 Dec 18;30(1):78-82.
            doi: 10.1038/nbt.2065pmc: PMC4076012pubmed: 22178993google scholar: lookup
          10. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, Venter JC. Creation of a bacterial cell controlled by a chemically synthesized genome.. Science 2010 Jul 2;329(5987):52-6.
            doi: 10.1126/science.1190719pubmed: 20488990google scholar: lookup
          11. Schriefer LA, Gebauer BK, Qui LQ, Waterston RH, Wilson RK. Low pressure DNA shearing: a method for random DNA sequence analysis.. Nucleic Acids Res 1990 Dec 25;18(24):7455-6.
            doi: 10.1093/nar/18.24.7455pmc: PMC332897pubmed: 2259642google scholar: lookup
          12. Deininger PL. Approaches to rapid DNA sequence analysis.. Anal Biochem 1983 Dec;135(2):247-63.
            doi: 10.1016/0003-2697(83)90680-2pubmed: 6318598google scholar: lookup
          13. Cavalieri LF, Rosenberg BH. Shear degradation of deoxyribonucleic acid. J Am Chem Soc 1959;81:5136–5139.
            doi: 10.1021/ja01528a029google scholar: lookup
          14. Bodenteich A, Chissoe S, Wang YF, Roe BA. Shotgun cloning as the strategy of choice to generate templates for high throughput dideoxynucleotide sequencing. Automated DNA sequencing and analysis techniques London UK: Academic Press; 1994; pp. 42–50.
          15. Oefner PJ, Hunicke-Smith SP, Chiang L, Dietrich F, Mulligan J, Davis RW. Efficient random subcloning of DNA sheared in a recirculating point-sink flow system.. Nucleic Acids Res 1996 Oct 15;24(20):3879-86.
            doi: 10.1093/nar/24.20.3879pmc: PMC146200pubmed: 8918787google scholar: lookup
          16. Anderson S. Shotgun DNA sequencing using cloned DNase I-generated fragments.. Nucleic Acids Res 1981 Jul 10;9(13):3015-27.
            doi: 10.1093/nar/9.13.3015pmc: PMC327328pubmed: 6269069google scholar: lookup
          17. Sambrook J, Fitsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2. Cold Spring Harbor NY: CSH Press; 1989.
          18. Xia YN, Burbank DE, Uher L, Rabussay D, Van Etten JL. IL-3A virus infection of a Chlorella-like green alga induces a DNA restriction endonuclease with novel sequence specificity.. Nucleic Acids Res 1987 Aug 11;15(15):6075-90.
            doi: 10.1093/nar/15.15.6075pmc: PMC306069pubmed: 2819820google scholar: lookup
          19. Skowron PM, Swaminathan N, McMaster K, George D, Van Etten JL, Mead DA. Cloning and applications of the two/three-base restriction endonuclease R.CviJI from IL-3A virus-infected Chlorella.. Gene 1995 May 19;157(1-2):37-41.
            doi: 10.1016/0378-1119(94)00564-9pubmed: 7607522google scholar: lookup
          20. Mead D, Swaminathan N, Van Etten J, Skowron PM. Recombinant CviJI restriction endonuclease. USA: United States Patent Office no US005472872A; 1995.
          21. Gingrich JC, Boehrer DM, Basu SB. Partial CviJI digestion as an alternative approach to generate cosmid sublibraries for large-scale sequencing projects.. Biotechniques 1996 Jul;21(1):99-104.
            pubmed: 8816243doi: 10.2144/96211st04google scholar: lookup
          22. Swaminathan N, McMaster K, Skowron PM, Mead DA. Thermal cycle labeling: zeptomole detection sensitivity and microgram probe amplification using CviJl* restriction-generated oligonucleotides.. Anal Biochem 1998 Jan 1;255(1):133-41.
            doi: 10.1006/abio.1997.2438pubmed: 9448852google scholar: lookup
          23. Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE--a database for DNA restriction and modification: enzymes, genes and genomes.. Nucleic Acids Res 2010 Jan;38(Database issue):D234-6.
            pmc: PMC2808884pubmed: 19846593doi: 10.1093/nar/gkp874google scholar: lookup
          24. Wu CC, Nimmakayala P, Santos FA, Springman R, Scheuring C, Meksem K, Lightfoot DA, Zhang HB. Construction and characterization of a soybean bacterial artificial chromosome library and use of multiple complementary libraries for genome physical mapping.. Theor Appl Genet 2004 Sep;109(5):1041-50.
            doi: 10.1007/s00122-004-1712-ypubmed: 15164176google scholar: lookup
          25. Zylicz-Stachula A, Harasimowicz-Slowińska RI, Sobolewski I, Skowron PM. TspGWI, a thermophilic class-IIS restriction endonuclease from Thermus sp., recognizes novel asymmetric sequence 5'-ACGGA(N11/9)-3'.. Nucleic Acids Res 2002 Apr 1;30(7):e33.
            doi: 10.1093/nar/30.7.e33pmc: PMC101857pubmed: 11917039google scholar: lookup
          26. Skowron PM, Majewski J, Zylicz-Stachula A, Rutkowska SM, Jaworowska I, Harasimowicz-Słowińska RI. A new Thermus sp. class-IIS enzyme sub-family: isolation of a 'twin' endonuclease TspDTI with a novel specificity 5'-ATGAA(N(11/9))-3', related to TspGWI, TaqII and Tth111II.. Nucleic Acids Res 2003 Jul 15;31(14):e74.
            doi: 10.1093/nar/gng074pmc: PMC167652pubmed: 12853651google scholar: lookup
          27. Zylicz-Stachula A, Bujnicki JM, Skowron PM. Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family.. BMC Mol Biol 2009 May 29;10:52.
            doi: 10.1186/1471-2199-10-52pmc: PMC2700111pubmed: 19480701google scholar: lookup
          28. Barker D, Hoff M, Oliphant A, White R. A second type II restriction endonuclease from Thermus aquaticus with an unusual sequence specificity.. Nucleic Acids Res 1984 Jul 25;12(14):5567-81.
            doi: 10.1093/nar/12.14.5567pmc: PMC320015pubmed: 6087291google scholar: lookup
          29. Zylicz-Stachula A, Zołnierkiewicz O, Sliwińska K, Jeżewska-Frąckowiak J, Skowron PM. Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving.. BMC Biochem 2011 Dec 5;12:62.
            doi: 10.1186/1471-2091-12-62pmc: PMC3280180pubmed: 22141927google scholar: lookup
          30. Zylicz-Stachula A, Zolnierkiewicz O, Lubys A, Ramanauskaite D, Mitkaite G, Bujnicki JM, Skowron PM. Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities.. BMC Mol Biol 2012 Apr 10;13:13.
            doi: 10.1186/1471-2199-13-13pmc: PMC3384240pubmed: 22489904google scholar: lookup
          31. Shinomiya T, Kobayashi M, Sato S. A second site specific endonuclease from Thermus thermophilus 111, Tth111II.. Nucleic Acids Res 1980 Aug 11;8(15):3275-85.
            doi: 10.1093/nar/8.15.3275pmc: PMC324152pubmed: 6255411google scholar: lookup
          32. Zylicz-Stachula A, Zołnierkiewicz O, Jeżewska-Frąckowiak J, Skowron PM. Chemically-induced affinity star restriction specificity: a novel TspGWI/sinefungin endonuclease with theoretical 3-bp cleavage frequency.. Biotechniques 2011 Jun;50(6):397-406.
            pubmed: 21781040doi: 10.2144/000113685google scholar: lookup
          33. Wei H, Therrien C, Blanchard A, Guan S, Zhu Z. The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.. Nucleic Acids Res 2008 May;36(9):e50.
            doi: 10.1093/nar/gkn182pmc: PMC2396408pubmed: 18413342google scholar: lookup
          34. Madhusoodanan UK, Rao DN. Diversity of DNA methyltransferases that recognize asymmetric target sequences.. Crit Rev Biochem Mol Biol 2010 Apr;45(2):125-45.
            doi: 10.3109/10409231003628007pubmed: 20184512google scholar: lookup
          35. Swaminathan N, Mead DA, McMaster K, George D, Van Etten JL, Skowron PM. Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.. Nucleic Acids Res 1996 Jul 1;24(13):2463-9.
            doi: 10.1093/nar/24.13.2463pmc: PMC145972pubmed: 8692682google scholar: lookup
          36. Swaminathan N, George D, McMaster K, Szablewski J, Van Etten JL, Mead DA. Restriction generated oligonucleotides utilizing the two base recognition endonuclease CviJI*.. Nucleic Acids Res 1994 Apr 25;22(8):1470-5.
            doi: 10.1093/nar/22.8.1470pmc: PMC308007pubmed: 8190639google scholar: lookup
          37. Fitzgerald MC, Skowron P, Van Etten JL, Smith LM, Mead DA. Rapid shotgun cloning utilizing the two base recognition endonuclease CviJI.. Nucleic Acids Res 1992 Jul 25;20(14):3753-62.
            doi: 10.1093/nar/20.14.3753pmc: PMC334028pubmed: 1322530google scholar: lookup
          38. GenBank. http://www.ncbi.nlm.nih.gov/genome/461?project_id=58033.
          39. Molecular station. http://www.molecularstation.com/dna/genomic-dna-isolation.
          40. Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.. Proc Natl Acad Sci U S A 1992 Sep 15;89(18):8794-7.
            doi: 10.1073/pnas.89.18.8794pmc: PMC50007pubmed: 1528894google scholar: lookup

          Citations

          This article has been cited 6 times.
          1. Zebrowska J, Jezewska-Frackowiak J, Wieczerzak E, Kasprzykowski F, Zylicz-Stachula A, Skowron PM. Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change. Appl Microbiol Biotechnol 2019 Apr;103(8):3439-3451.
            doi: 10.1007/s00253-019-09731-0pubmed: 30879089google scholar: lookup
          2. Krefft D, Papkov A, Prusinowski M, Zylicz-Stachula A, Skowron PM. Randomized DNA libraries construction tool: a new 3-bp 'frequent cutter' TthHB27I/sinefungin endonuclease with chemically-induced specificity. BMC Genomics 2018 May 11;19(1):361.
            doi: 10.1186/s12864-018-4748-0pubmed: 29751745google scholar: lookup
          3. Skowron PM, Anton BP, Czajkowska E, Zebrowska J, Sulecka E, Krefft D, Jezewska-Frackowiak J, Zolnierkiewicz O, Witkowska M, Morgan RD, Wilson GG, Fomenkov A, Roberts RJ, Zylicz-Stachula A. The third restriction-modification system from Thermus aquaticus YT-1: solving the riddle of two TaqII specificities. Nucleic Acids Res 2017 Sep 6;45(15):9005-9018.
            doi: 10.1093/nar/gkx599pubmed: 28911108google scholar: lookup
          4. Zylicz-Stachula A, Zebrowska J, Czajkowska E, Wrese W, Sulecka E, Skowron PM. Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site. Mol Biol Rep 2016 Apr;43(4):269-82.
            doi: 10.1007/s11033-016-3949-3pubmed: 26886214google scholar: lookup
          5. Zylicz-Stachula A, Jeżewska-Frąckowiak J, Skowron PM. Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N₄₇₃A variant in the NPPY motif. Mol Biol Rep 2014;41(4):2313-23.
            doi: 10.1007/s11033-014-3085-xpubmed: 24442320google scholar: lookup
          6. Zylicz-Stachula A, Zolnierkiewicz O, Sliwinska K, Jezewska-Frackowiak J, Skowron PM. Modified 'one amino acid-one codon' engineering of high GC content TaqII-coding gene from thermophilic Thermus aquaticus results in radical expression increase. Microb Cell Fact 2014 Jan 11;13:7.
            doi: 10.1186/1475-2859-13-7pubmed: 24410856google scholar: lookup
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