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. 2002 Oct;3(5):405–422. doi: 10.1002/cfg.206

A Mathematical Model for Suppression Subtractive Hybridization

Chetan Gadgil 1, Anette Rink 2, Craig Beattie 2, Wei-Shou Hu 1,✉
PMCID: PMC2447336  PMID: 18629052

Abstract

Suppression subtractive hybridization (SSH) is frequently used to unearth differentially expressed genes on a whole-genome scale. Its versatility is based on combining cDNA library subtraction and normalization, which allows the isolation of sequences of varying degrees of abundance and differential expression. SSH is a complex process with many adjustable parameters that affect the outcome of gene isolation.We present a mathematical model of SSH based on DNA hybridization kinetics for assessing the effect of various parameters to facilitate its optimization. We derive an equation for the probability that a particular differentially expressed species is successfully isolated and use this to quantify the effect of the following parameters related to the cDNA sample: (a) mRNA abundance; (b) partial sequence complementarity to other species; and (3) degree of differential expression. We also evaluate the effect of parameters related to the process, including: (a) reaction times; and (b) extent of driver excess used in the two hybridization reactions. The optimum set of process parameters for successful isolation of differentially expressed species depends on transcript abundance. We show that the reaction conditions have a significant effect on the occurrence of false-positives and formulate strategies to isolate specific subsets of differentially expressed genes. We also quantify the effect of non-specific hybridization on the false-positive results and present strategies for spiking cDNA sequences to address this problem.

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Selected References

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  1. Alizadeh A. A., Staudt L. M. Genomic-scale gene expression profiling of normal and malignant immune cells. Curr Opin Immunol. 2000 Apr;12(2):219–225. doi: 10.1016/s0952-7915(99)00078-3. [DOI] [PubMed] [Google Scholar]
  2. Bittner M., Meltzer P., Chen Y., Jiang Y., Seftor E., Hendrix M., Radmacher M., Simon R., Yakhini Z., Ben-Dor A. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature. 2000 Aug 3;406(6795):536–540. doi: 10.1038/35020115. [DOI] [PubMed] [Google Scholar]
  3. Brenner S., Johnson M., Bridgham J., Golda G., Lloyd D. H., Johnson D., Luo S., McCurdy S., Foy M., Ewan M. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotechnol. 2000 Jun;18(6):630–634. doi: 10.1038/76469. [DOI] [PubMed] [Google Scholar]
  4. Brown P. O., Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet. 1999 Jan;21(1 Suppl):33–37. doi: 10.1038/4462. [DOI] [PubMed] [Google Scholar]
  5. Cho T. J., Park S. S. A simulation of subtractive hybridization. Nucleic Acids Res. 1998 Mar 15;26(6):1440–1448. doi: 10.1093/nar/26.6.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Craig M. E., Crothers D. M., Doty P. Relaxation kinetics of dimer formation by self complementary oligonucleotides. J Mol Biol. 1971 Dec 14;62(2):383–401. doi: 10.1016/0022-2836(71)90434-7. [DOI] [PubMed] [Google Scholar]
  7. Diatchenko L., Lau Y. F., Campbell A. P., Chenchik A., Moqadam F., Huang B., Lukyanov S., Lukyanov K., Gurskaya N., Sverdlov E. D. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):6025–6030. doi: 10.1073/pnas.93.12.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dupressoir A., Barbot W., Loireau M. P., Heidmann T. Characterization of a mammalian gene related to the yeast CCR4 general transcription factor and revealed by transposon insertion. J Biol Chem. 1999 Oct 22;274(43):31068–31075. doi: 10.1074/jbc.274.43.31068. [DOI] [PubMed] [Google Scholar]
  9. Ermolaeva O. D., Lukyanov S. A., Sverdlov E. D. The mathematical model of subtractive hybridization and its practical application. Proc Int Conf Intell Syst Mol Biol. 1996;4:52–58. [PubMed] [Google Scholar]
  10. Ermolaeva O. D., Wagner M. C. SUBTRACT: a computer program for modeling the process of subtractive hydridization. Comput Appl Biosci. 1995 Aug;11(4):457–462. doi: 10.1093/bioinformatics/11.4.457. [DOI] [PubMed] [Google Scholar]
  11. Evans J. D., Wheeler D. E. Differential gene expression between developing queens and workers in the honey bee, Apis mellifera. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5575–5580. doi: 10.1073/pnas.96.10.5575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Glienke J., Schmitt A. O., Pilarsky C., Hinzmann B., Weiss B., Rosenthal A., Thierauch K. H. Differential gene expression by endothelial cells in distinct angiogenic states. Eur J Biochem. 2000 May;267(9):2820–2830. doi: 10.1046/j.1432-1327.2000.01325.x. [DOI] [PubMed] [Google Scholar]
  13. Gotoh M., Hasegawa Y., Shinohara Y., Shimizu M., Tosu M. A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor. DNA Res. 1995 Dec 31;2(6):285–293. doi: 10.1093/dnares/2.6.285. [DOI] [PubMed] [Google Scholar]
  14. Grillari J., Hohenwarter O., Grabherr R. M., Katinger H. Subtractive hybridization of mRNA from early passage and senescent endothelial cells. Exp Gerontol. 2000 Mar;35(2):187–197. doi: 10.1016/s0531-5565(00)00080-2. [DOI] [PubMed] [Google Scholar]
  15. Hastie N. D., Bishop J. O. The expression of three abundance classes of messenger RNA in mouse tissues. Cell. 1976 Dec;9(4 Pt 2):761–774. doi: 10.1016/0092-8674(76)90139-2. [DOI] [PubMed] [Google Scholar]
  16. Hill A. A., Hunter C. P., Tsung B. T., Tucker-Kellogg G., Brown E. L. Genomic analysis of gene expression in C. elegans. Science. 2000 Oct 27;290(5492):809–812. doi: 10.1126/science.290.5492.809. [DOI] [PubMed] [Google Scholar]
  17. Hipfel R., Schittek B., Bodingbauer Y., Garbe C. Specifically regulated genes in malignant melanoma tissues identified by subtractive hybridization. Br J Cancer. 2000 Mar;82(6):1149–1157. doi: 10.1054/bjoc.1999.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hubank M., Schatz D. G. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 1994 Dec 25;22(25):5640–5648. doi: 10.1093/nar/22.25.5640. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ko M. S. An 'equalized cDNA library' by the reassociation of short double-stranded cDNAs. Nucleic Acids Res. 1990 Oct 11;18(19):5705–5711. doi: 10.1093/nar/18.19.5705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee C. K., Klopp R. G., Weindruch R., Prolla T. A. Gene expression profile of aging and its retardation by caloric restriction. Science. 1999 Aug 27;285(5432):1390–1393. doi: 10.1126/science.285.5432.1390. [DOI] [PubMed] [Google Scholar]
  21. Leib-Mösch C., Bachmann M., Brack-Werner R., Werner T., Erfle V., Hehlmann R. Expression and biological significance of human endogenous retroviral sequences. Leukemia. 1992;6 (Suppl 3):72S–75S. [PubMed] [Google Scholar]
  22. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  23. Lipshutz R. J., Fodor S. P., Gingeras T. R., Lockhart D. J. High density synthetic oligonucleotide arrays. Nat Genet. 1999 Jan;21(1 Suppl):20–24. doi: 10.1038/4447. [DOI] [PubMed] [Google Scholar]
  24. Martin K. J., Pardee A. B. Identifying expressed genes. Proc Natl Acad Sci U S A. 2000 Apr 11;97(8):3789–3791. doi: 10.1073/pnas.97.8.3789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Milner J. J., Cecchini E., Dominy P. J. A kinetic model for subtractive hybridization. Nucleic Acids Res. 1995 Jan 11;23(1):176–187. doi: 10.1093/nar/23.1.176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nemeth E., Millar L. K., Bryant-Greenwood G. Fetal membrane distention: II. Differentially expressed genes regulated by acute distention in vitro. Am J Obstet Gynecol. 2000 Jan;182(1 Pt 1):60–67. doi: 10.1016/s0002-9378(00)70491-1. [DOI] [PubMed] [Google Scholar]
  27. Nemeth E., Tashima L. S., Yu Z., Bryant-Greenwood G. D. Fetal membrane distention: I. Differentially expressed genes regulated by acute distention in amniotic epithelial (WISH) cells. Am J Obstet Gynecol. 2000 Jan;182(1 Pt 1):50–59. doi: 10.1016/s0002-9378(00)70490-x. [DOI] [PubMed] [Google Scholar]
  28. Oh M. K., Liao J. C. DNA microarray detection of metabolic responses to protein overproduction in Escherichia coli. Metab Eng. 2000 Jul;2(3):201–209. doi: 10.1006/mben.2000.0149. [DOI] [PubMed] [Google Scholar]
  29. Pardinas J. R., Combates N. J., Prouty S. M., Stenn K. S., Parimoo S. Differential subtraction display: a unified approach for isolation of cDNAs from differentially expressed genes. Anal Biochem. 1998 Mar 15;257(2):161–168. doi: 10.1006/abio.1997.2550. [DOI] [PubMed] [Google Scholar]
  30. Patanjali S. R., Parimoo S., Weissman S. M. Construction of a uniform-abundance (normalized) cDNA library. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1943–1947. doi: 10.1073/pnas.88.5.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rynditch A. V., Zoubak S., Tsyba L., Tryapitsina-Guley N., Bernardi G. The regional integration of retroviral sequences into the mosaic genomes of mammals. Gene. 1998 Nov 5;222(1):1–16. doi: 10.1016/s0378-1119(98)00451-x. [DOI] [PubMed] [Google Scholar]
  32. Sagerström C. G., Sun B. I., Sive H. L. Subtractive cloning: past, present, and future. Annu Rev Biochem. 1997;66:751–783. doi: 10.1146/annurev.biochem.66.1.751. [DOI] [PubMed] [Google Scholar]
  33. Sandhu H., Dehnen W., Roller M., Abel J., Unfried K. mRNA expression patterns in different stages of asbestos-induced carcinogenesis in rats. Carcinogenesis. 2000 May;21(5):1023–1029. doi: 10.1093/carcin/21.5.1023. [DOI] [PubMed] [Google Scholar]
  34. Sasaki Y. F., Ayusawa D., Oishi M. Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system. Nucleic Acids Res. 1994 Mar 25;22(6):987–992. doi: 10.1093/nar/22.6.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shen J., Gudas L. J. Molecular cloning of a novel retinoic acid-responsive gene, HA1R-62, which is also up-regulated in Hoxa-1-overexpressing cells. Cell Growth Differ. 2000 Jan;11(1):11–17. [PubMed] [Google Scholar]
  36. Simpson K. S., Adams M. H., Behrendt-Adam C. Y., Baker C. B., McDowell K. J. Identification and initial characterization of calcyclin and phospholipase A2 in equine conceptuses. Mol Reprod Dev. 1999 Jun;53(2):179–187. doi: 10.1002/(SICI)1098-2795(199906)53:2<179::AID-MRD7>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  37. Spiegelman G. B., Haber J. E., Halvorson H. O. Kinetics of ribonucleic acid-deoxyribonucleic acid membrane filter hybridization. Biochemistry. 1973 Mar 13;12(6):1234–1242. doi: 10.1021/bi00730a034. [DOI] [PubMed] [Google Scholar]
  38. Stuart R. O., Bush K. T., Nigam S. K. Changes in global gene expression patterns during development and maturation of the rat kidney. Proc Natl Acad Sci U S A. 2001 May 1;98(10):5649–5654. doi: 10.1073/pnas.091110798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Velculescu V. E., Zhang L., Vogelstein B., Kinzler K. W. Serial analysis of gene expression. Science. 1995 Oct 20;270(5235):484–487. doi: 10.1126/science.270.5235.484. [DOI] [PubMed] [Google Scholar]
  40. Vernier P., Mastrippolito R., Helin C., Bendali M., Mallet J., Tricoire H. Radioimager quantification of oligonucleotide hybridization with DNA immobilized on transfer membrane: application to the identification of related sequences. Anal Biochem. 1996 Mar 1;235(1):11–19. doi: 10.1006/abio.1996.0085. [DOI] [PubMed] [Google Scholar]
  41. Wang X., Li X., Yaish-Ohad S., Sarau H. M., Barone F. C., Feuerstein G. Z. Molecular cloning and expression of the rat monocyte chemotactic protein-3 gene: a possible role in stroke. Brain Res Mol Brain Res. 1999 Aug 25;71(2):304–312. doi: 10.1016/s0169-328x(99)00203-x. [DOI] [PubMed] [Google Scholar]
  42. Wetmur J. G., Davidson N. Kinetics of renaturation of DNA. J Mol Biol. 1968 Feb 14;31(3):349–370. doi: 10.1016/0022-2836(68)90414-2. [DOI] [PubMed] [Google Scholar]
  43. Wetmur J. G. Hybridization and renaturation kinetics of nucleic acids. Annu Rev Biophys Bioeng. 1976;5:337–361. doi: 10.1146/annurev.bb.05.060176.002005. [DOI] [PubMed] [Google Scholar]
  44. White K. P., Rifkin S. A., Hurban P., Hogness D. S. Microarray analysis of Drosophila development during metamorphosis. Science. 1999 Dec 10;286(5447):2179–2184. doi: 10.1126/science.286.5447.2179. [DOI] [PubMed] [Google Scholar]
  45. Yang G. P., Ross D. T., Kuang W. W., Brown P. O., Weigel R. J. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res. 1999 Mar 15;27(6):1517–1523. doi: 10.1093/nar/27.6.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhang J., Underwood L. E., D'Ercole A. J. Formation of chimeric cDNAs during suppression subtractive hybridization and subsequent polymerase chain reaction. Anal Biochem. 2000 Jul 1;282(2):259–262. doi: 10.1006/abio.2000.4613. [DOI] [PubMed] [Google Scholar]
  47. Zhang L., Zhou W., Velculescu V. E., Kern S. E., Hruban R. H., Hamilton S. R., Vogelstein B., Kinzler K. W. Gene expression profiles in normal and cancer cells. Science. 1997 May 23;276(5316):1268–1272. doi: 10.1126/science.276.5316.1268. [DOI] [PubMed] [Google Scholar]

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