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Ligation Based Assembly and Polymerase Chain Reaction-Based Assembly for Extraordinary Adenine/Thymine Rich DNA

  • Chen Yu
  • Li Xu
  • Wenxian Piao
  • Xiao Bao
  • Hairong Wang
  • Meng Xing
  • Jieyu Wu
  • Bang Xu
  • Penghui Yuan
  • Yangxiu Wu
  • Wangyun He
  • Jinhuan Qi
  • Ying Zhang
  • Xiaoqian Ma
  • Qiuyun Liu
Research Article

Abstract

Extraordinary adenine/thymine rich DNA has a low complexity resulting in the occurrence of many short repetitive sequences or longer imperfect repeats, consequently hindering the gene synthesis process. This report describes the rapid synthesis of a DNA fragment with guanine/cytosine content of 11.8% using ligation based assembly and polymerase chain reaction-based assembly respectively, via the use of multiple strategies addressing the adenine/thymine rich nature of the fragment. Sequences can be simply chopped and assembled without Tm optimization. Smaller amount of ligation products as templates in Interference Free polymerase chain reaction yielded markedly more than or equal amount of subassembly products as using larger amount of ligation products, and longer extension time was required for successful subassembly. Longer time at low annealing temperature also had obvious effects on polymerase chain reaction amplifications. No full-length products could be generated without initial oligonucleotide sub-pooling for both approaches. The chemical synthesis of extraordinary adenine/thymine rich DNA could enable researchers to assemble synthetic modules, to study and to access the repetitive DNA such as heterochromatin regions which harbor important functional elements.

Keywords

Extraordinary adenine/thymine rich DNA Low complexity Ligation based assembly Polymerase chain reaction-based assembly 

Notes

Acknowledgements

This work was supported by the Guangdong Natural Science Foundation (S2011010004264), Guangdong Science and Technology Program (No. 2008B020100001, 2016B020204001), Open Fund of MOE Key Laboratory of Aquatic Product Safety, Foreign Expert Program at Sun Yat-sen University, Open Fund of Laboratory (20160215) and 2016 Key Project Budget at Sun Yat-sen University, and The National Natural Science Foundation of China (J1310025, 21601209, 30370799). The authors thank Dr. Yuchuan Wang, Ms Jing Li, Mr. Xiang Zhu, Ms Fan Yang, Mr. Xingqiang Lai and Ms Cui Yang for technical help. The authors are grateful to Dr. Weiguo Cao and Ms Yan Shi for editorial assistance, and anonymous reviewer for valuable suggestions.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Roth TL, Milenkovic L, Scott MP (2014) A rapid and simple method for DNA engineering using cycled ligation assembly. PLoS ONE 9(9):e107329CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Guo YY, Shi ZY, Fu XZ, Chen JC, Wu Q, Chen GQ (2015) A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation. Microb Cell Fact 14:18CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Tsuge K, Sato Y, Kobayashi Y, Gondo M, Hasebe M, Togashi T, Tomita M, Itaya M (2015) Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments. Sci Rep 5:10655CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Gibson DG, Smith HO, Hutchison CA 3rd, Venter JC, Merryman C (2010) Chemical synthesis of the mouse mitochondrial genome. Nat Methods 7(11):901–903CrossRefPubMedGoogle Scholar
  5. 5.
    Torella JP, Boehm CR, Lienert F, Chen JH, Way JC, Silver PA (2014) Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Res 42(1):681–689CrossRefPubMedGoogle Scholar
  6. 6.
    Kahl LJ, Endy D (2013) A survey of enabling technologies in synthetic biology. J Biol Eng 7(1):13CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bartholdy B, Mukhopadhyay R, Lajugie J, Aladjem MI, Bouhassira EE (2015) Allele-specific analysis of DNA replication origins in mammalian cells. Nat Commun 6:7051CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Agier N, Romano OM, Touzain F, Cosentino Lagomarsino M, Fischer G (2013) The spatiotemporal program of replication in the genome of Lachancea kluyveri. Genome Biol Evol 5(2):370–388CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Shalem O, Sharon E, Lubliner S, Regev I, Lotan-Pompan M, Yakhini Z, Segal E (2015) Systematic dissection of the sequence determinants of gene 3′ end mediated expression control. PLoS Genet 11(4):e1005147CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Rouillard JM, Lee W, Truan G, Gao X, Zhou X, Gulari E (2004) Gene2Oligo: oligonucleotide design for in vitro gene synthesis. Nucleic Acids Res 32:W176–W180CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Smith HO, Hutchison CA 3rd, Pfannkoch C, Venter JC (2003) Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc Natl Acad Sci USA 100(26):15440–15445CrossRefPubMedGoogle Scholar
  12. 12.
    Chakrabarti R, Schutt CE (2001) The enhancement of PCR amplification by low molecular weight amides. Nucleic Acids Res 29(11):2377–2381CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gyllensten UB, Erlich HA (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 85(20):7652–7656CrossRefPubMedGoogle Scholar
  14. 14.
    Wang S, Luo Y, Yi X et al (2007) A highly efficient and highly reliable protocol for transformation of Escherichia coli by electroporation. J Rapid Methods Autom Microbiol 15(2007):253–258CrossRefGoogle Scholar
  15. 15.
    Michael RG, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  16. 16.
    Arratia R, Martin D, Reinert G, Waterman MS (1996) Poisson process approximation for sequence repeats, and sequencing by hybridization. J Comput Biol 3(3):425–463CrossRefPubMedGoogle Scholar
  17. 17.
    Arratia R, Goldstein L, Gordon L (1990) Poisson approximation and the Chen–Stein method. Stat Sci 5(4):403–424CrossRefGoogle Scholar
  18. 18.
    Lewis AP, Sims MJ, Gewert DR, Peakman TC, Spence H, Crowe JS (1994) Taq DNA polymerase extension of internal primers blocks polymerase chain reactions allowing differential amplification of molecules with identical 5′ and 3′ ends. Nucleic Acids Res 22(14):2859–2861CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Su XZ, Wu Y, Sifri CD, Wellems TE (1996) Reduced extension temperatures required for PCR amplification of extremely A + T-rich DNA. Nucleic Acids Res 24(8):1574–1575CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ye HY, Huang MC, Li MH, Ying JY (2009) Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis. Nucleic Acids Res 37(7):e51CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Gibson DG, Benders GA, Pfannkoch CA et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319(5867):1215–1220CrossRefPubMedGoogle Scholar
  22. 22.
    Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345CrossRefPubMedGoogle Scholar
  23. 23.
    Bang D, Church GM (2008) Gene synthesis by circular assembly amplification. Nat Methods 5(1):37–39CrossRefPubMedGoogle Scholar
  24. 24.
    Sorek R, Zhu Y, Creevey CJ, Francino MP, Bork P, Rubin EM (2007) Genome-wide experimental determination of barriers to horizontal gene transfer. Science 318(5855):1449–1452CrossRefPubMedGoogle Scholar

Copyright information

© The National Academy of Sciences, India 2017

Authors and Affiliations

  • Chen Yu
    • 1
  • Li Xu
    • 1
  • Wenxian Piao
    • 1
  • Xiao Bao
    • 1
  • Hairong Wang
    • 2
  • Meng Xing
    • 1
  • Jieyu Wu
    • 1
  • Bang Xu
    • 1
  • Penghui Yuan
    • 1
  • Yangxiu Wu
    • 1
  • Wangyun He
    • 1
  • Jinhuan Qi
    • 1
  • Ying Zhang
    • 3
  • Xiaoqian Ma
    • 4
  • Qiuyun Liu
    • 1
  1. 1.MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, School of Life SciencesSun Yat-sen UniversityGuangzhouChina
  2. 2.School of BiotechnologySouthern Medical UniversityGuangzhouChina
  3. 3.Guangzhou Center for Disease Control and PreventionGuangzhouChina
  4. 4.Cell Transplantation and Gene Therapy InstituteThe Third Xiang Ya Hospital of Central South UniversityChangshaChina

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