Natural Computing

, Volume 8, Issue 2, pp 333–347 | Cite as

Successful preparation and analysis of a 5-site 2-variable DNA library

Article

Abstract

DNA code sequences generated by the program SynDCode were used to construct a 5-site, 2-variable computational DNA library by parallel overlap self-assembly. The final library was amplified using the polymerase chain reaction (PCR) to obtain a fragment of the expected size. Twelve library sequences randomly selected from the library by cloning were sequenced and found to be distinct and correctly assembled library strands. A Birthday Problem-like analysis suggests that we have all 32 different molecules in our library mixture. We then developed new protocols using DNA hybridization to successfully identify single members of this library. We have also used this protocol to analyze mixtures of clones from the library. This approach shows the experimental validation of the ability to distinguish different sequences generated from the SynDCode program. We are in the process of working out protocols to separate out specific library members and to expand this library.

Keywords

Bayesian approach Birthday Problem analysis DNA code design DNA hybridization Parallel overlap assembly 

Abbreviations

EDTA

Ethylenedinitrilotetraacetic acid

CH

Cross-hybridized

LMW

Low molecular weight marker

PCR

Polymerase chain reaction

SDS

Sodium dodecyl sulfate

SSC

Sodium citrate buffer

TBE

Tris-borate-EDTA buffer

TBS

Tris buffered saline

WC

Watson–Crick

References

  1. Adleman L (1994) Molecular computation of solutions of combinatorial problems. Science 266:1021–1024. doi:10.1126/science.7973651 CrossRefGoogle Scholar
  2. Andronescu M, Aguirre-Hernandez R, Condon A et al (2003) RNAsoft: a suite of RNA secondary structure prediction and design software tools. Nucleic Acids Res 31:3416–3422. doi:10.1093/nar/gkg612 CrossRefGoogle Scholar
  3. Bishop M, Macula A, Renz T (2006) SynDCode suite, http://syndcode.geneseo.edu/, which has a link to run the SynDCode program as well as a manuscript describing the program and the parameters used; cited 19 Mar 2007
  4. Bishop M, D’yachkov A, Macula A et al (2007) Free Energy Gap and Statistical Thermodynamic. J Comput Biol (in press)Google Scholar
  5. Bracho MA, Moy A, Barrio E (1998) Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity. J Gen Virol 79:2921–2928Google Scholar
  6. Braich S, Chelyapov N, Johnson C et al (2002) Solution of a 20-variable 3-SAT problem on a DNA computer. Science 296:499–502. doi:10.1126/science.1069528 CrossRefGoogle Scholar
  7. Cai H, White P, Torney D et al (2000) Flow cytometry-based minisequencing: a new platform for high throughput single nucleotide polymorphism scoring. Genomics 66:135–143. doi:10.1006/geno.2000.6218 CrossRefGoogle Scholar
  8. Chen J, Deaton R, Garzon M (2006) Characterization of non-crosshybridizing DNA oligonucleotides manufactured in vitro. Nat Comput 5:65–181. doi:10.1007/s11047-005-4460-2 CrossRefMathSciNetGoogle Scholar
  9. D’yachkov A, Erdös P, Macula AJ et al (2003) Exordium for DNA codes. J Comb Optim 7(4):369–380. doi:10.1023/B:JOCO.0000017385.39168.0d MATHCrossRefMathSciNetGoogle Scholar
  10. D’yachkov AG, Vilenkin PA, Ismagilov IK et al (2005) On DNA codes. Prob Inf Trans 41(4):349–367. doi:10.1007/s11122-006-0004-3 MATHCrossRefMathSciNetGoogle Scholar
  11. Eason R, Pourmand N, Tongprasit W et al (2004) Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains. Proc Natl Acad Sci USA 101:11046–11051. doi:10.1073/pnas.0403672101 CrossRefGoogle Scholar
  12. Fish D, Horne M, Searles R et al (2007) Multiplex SNP discrimination. Biophys J 92:L89–L92. doi:10.1529/biophysj.107.105320 CrossRefGoogle Scholar
  13. Hardenbol P, Baner J, Jain M et al (2003) Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat Biotechnol 6:673–678. doi:10.1038/nbt821 CrossRefGoogle Scholar
  14. Kaderali L, Deshpande A, Nolan J et al (2003) Primer-design for multiplexed genotyping. Nucleic Acids Res 31:1796–1802. doi:10.1093/nar/gkg267 CrossRefGoogle Scholar
  15. Kaplan PD, Ouyang Q, Thaler DS et al (1997) Parallel overlap assembly for the construction of computational DNA libraries. J Theor Biol 188:333–4116. doi:10.1006/jtbi.1997.0475 CrossRefGoogle Scholar
  16. Ouyang Q, Kaplan PD, Liu S et al (1997) DNA solution of the maximal clique problem. Science 278:446–449. doi:10.1126/science.278.5337.446 CrossRefGoogle Scholar
  17. Pagano A, Gal S (2007) An approach to using modified nucleotides in aqueous DNA computing. Lect Notes Comput Sci 4848:161–169CrossRefGoogle Scholar
  18. Penchovsky R, Ackermann J (2003) DNA library design for molecular computation. J Comput Biol 10:215–229. doi:10.1089/106652703321825973 CrossRefGoogle Scholar
  19. Pogozelski WK, Bernard MP, Priore SF et al (2006) Experimental validation of DNA sequences for DNA computing: Use of a SYBR green assay. Lect Notes Comput Sci 3892:322–331Google Scholar
  20. Ritter T (1994) Estimating population from repetitions in accumulated random samples. Cryptologia 18(2):155–190. doi:10.1080/0161-119491882847 MATHCrossRefGoogle Scholar
  21. Rose J, Deaton R, Suyama A (2004) Statistical thermodynamic analysis and design of DNA-based computers. Nat Comput 3:443–359. doi:10.1007/s11047-004-2641-z CrossRefMathSciNetGoogle Scholar
  22. Shortreed M, Chang S, Hong D (2005) A thermodynamic approach to designing structure-free combinatorial DNA word sets. Nucleic Acids Res 33:4965–4977. doi:10.1093/nar/gki812 CrossRefGoogle Scholar
  23. Tulpan D, Andronescu M, Chang S et al (2005) Thermodynamically based DNA strand design. Nucleic Acids Res 33:4951–4964. doi:10.1093/nar/gki773 CrossRefGoogle Scholar
  24. Valignat M, Theodoly O, Crocker J et al (2005) Reversible self-assembly and directed assemblyof DNA-linked micrometer-sized colloids. Proc Natl Acad Sci USA 102:4225–4229. doi:10.1073/pnas.0500507102 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Susannah Gal
    • 1
  • Nancy Monteith
    • 1
  • Anthony J. Macula
    • 2
  1. 1.Department of Biological SciencesBinghamton UniversityBinghamtonUSA
  2. 2.Biomathematics GroupSUNY-GeneseoGeneseoUSA

Personalised recommendations