Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.
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 recently described 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. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that 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.
Diatchenko
L
L
CLONTECH Laboratories, Inc., Palo Alto, CA 94303, USA.
Lau
Y F
YF
Campbell
A P
AP
Chenchik
A
A
Moqadam
F
F
Huang
B
B
Lukyanov
S
S
Lukyanov
K
K
Gurskaya
N
N
Sverdlov
E D
ED
Siebert
P D
PD
D001483:Base Sequence; D002874:Chromosome Mapping; D003360:Cosmids; D015342:DNA Probes; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D014998:Y Chromosome
D015342:DNA Probes; D018076:DNA, Complementary