Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes.
A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.
Diatchenko
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CLONTECH Laboratories, Inc., Palo Alto, California 94303-4230, USA.
Lukyanov
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S
Lau
Y F
YF
Siebert
P D
PD
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis*; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping / Q000379:methods; D013737:Testis / Q000378:metabolism; D014998:Y Chromosome
D017931:DNA Primers; D018076:DNA, Complementary; D007202:Indicators and Reagents