Abstract
Suppression subtractive hybridization (SSH) is a widely used method for separating DNA molecules that distinguish two closely related DNA samples. Two of the main SSH applications are cDNA subtraction and genomic DNA subtraction. In fact, SSH is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. The SSH method is based on a suppression PCR effect and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of DNA fragments within the target population, and the subtraction step excludes sequences that are common to the populations being compared. This dramatically increases the probability of obtaining low-abundance differentially expressed cDNA or genomic DNA fragments, and simplifies analysis of the subtracted library. In our hands, the SSH technique has enriched over 1000-fold for rare sequences in a single round of subtractive hybridization.
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References
Luk’ianov, S. A., Gurskaya, N. G., Luk’ianov, K. A., Tarabykin, V. S., and Sverdlov, E. D. (1994) Highly efficient subtractive hybridization of cDNA. J. Bioorgan. Chem. 20, 386–388.
Gurskaya, N. G., Diatchenko, L., Chenchik, A., et al. (1996) Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal. Biochem. 240, 90–97.
Akopyants, N. S., Fradkov, A., Diatchenko, L., et al. (1998) PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc. Natl. Acad. Sci. USA 95, 13108–13113.
Diatchenko, L., Lau, Y. F. C., Campbell, A. P., et al. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025–6030.
Lukyanov, K. A., Launer, G. A., Tarabykin, V. S., Zaraisky, A. G., and Lukyanov, S. A. (1995) Inverted terminal repeats permit the average length of amplified DNA fragments to be regulated during preparation of cDNA libraries by polymerase chain reaction. Anal. Biochem. 229, 198–202.
Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A., and Lukyanov, S. A. (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23, 1087–1088.
Jin, H., Cheng, X., Diatchenko, L., Siebert, P. D., and Huang, C. C. (1997) Differential screening of a subtracted cDNA library: a method to search for genes preferentially expressed in multiple tissues. BioTechniques 23, 1084–1086.
Desai, S., Hill, J., Trelogan, S., Diatchenko, L., and Siebert, P. (2000) Identification of differentially expressed genes by suppression subtractive hybridization, in Functional Genomics (Hunt, S. P. and Livesey, F. J., eds.), Oxford University Press, 81–111.
Rebrikov, D. V., Britanova, O. V., Gurskaya, N. G., Lukyanov, K. A., Tarabykin, V. S., and Lukyanov, S. A. (2000) Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization. Nucleic Acids Res. 28, e90.
Gubler, U. and Hoffman, B. J. (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263–269.
Chenchik, A., Zhu, Y. Y., Diatchenko, L., Li, R., Hill, J., and Siebert, P. D. (1998) Generation and Use of High-Quality cDNA from Small Amounts of Total RNAby SMART PCR in Gene Cloning and Analysis by RT-PCR (Siebert, P. D. and Larrick, J. W., eds.), Molecular Laboratory Methods Number 1, 305–319.
Britten, R. J. and Davidson, E. H. (1985) In Nucleic Acid Hybridization-A Practical Approach (Hames, B. D. and Higgins, S., eds.), IRL Press, Oxford, 3–15.
Kellogg, D. E., Rybalkin, I., Chen, S., et al. (1994) TaqStart Antibody: “hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16, 1134–1137.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor.
Ausubel, F. M., Brent, R., Kingston, R. E., et al. (1994) Current Protocols in Molecular Biology. Greene Publishing Associates and John Wiley & Sons, Inc., NY. 1, Ch. 2.4.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chlorophorm extraction. Anal. Biochem. 162, 156–159.
Farrell, R. E., Jr. (1993) RNA Methodologies: A Guide for Isolation and Characterization. Academic, San Diego, CA.
Garcia-Fernandez, J., Marfany, G., Baguna, J., and Salo, E. (1993) Infiltration of mariner elements. Nature 364, 109–110.
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Rebrikov, D.V., Desai, S.M., Siebert, P.D., Lukyanov, S.A. (2004). Suppression Subtractive Hybridization. In: Shimkets, R.A. (eds) Gene Expression Profiling. Methods in Molecular Biology, vol 258. Humana Press. https://doi.org/10.1385/1-59259-751-3:107
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DOI: https://doi.org/10.1385/1-59259-751-3:107
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