Chapter 18
cDNA Libraries
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
lntroduction
Proteins are the work horses of the cell: they comprise the structural and
regulatory molecules, the motors and pumps, enzymes and receptors. Determining the complete sequence of a specific protein, however, or producing it in sufficient quantity to allow its manipulation and characterization
are extremely tedious and expensive procedures if one is limited to the use
of traditional biochemical approaches. Gene cloning and genetic engineering have greatly contributed to the field ofbiochemistry. Determining protein sequences would still be a daunting task, however, if one was restricted
to the use of genomic DNA as the source material since only -2o/o is thought
likely to encode protein. The remainder includes structural and regulatory
elements, introns, untranslated exons, and repetitive, non-coding sequences without known function ("junk DNA"). The effort required to determine a gene product's sequence is greatly reduced if the analysis is restricted to coding sequences. Since one of the principle tenets of molecular
biology isthat mRNA serve as the template for protein synthesis, mRNA
molecules are the ideal substrates for determining the sequence of proteins.
U nfortunately, none of the commonly available cloning vectors accept RN A
molecules as inserts. Therefore, an essential step in the generation of an
expressed sequence library is the conversion of mRNA molecules into double-stranded DNA. DNA copies of mRNA molecules are called cDNA, and a
library comprized ofDNA copies of the mRNA species from a cell or tissue is
called a cDNA library.
Luiz F. Onuchic, Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 970, BalitmoreMarylandUSA, Correspondence to Gregory G.
Germino, Johns Hopkins University, School ofMedicine, Division ofNephrology, 720
Rutland Avenue, Ross 970, BalitmoreMarylandUSA (phone +01-410-614-1650;/a.x +01
410-955-0485; e-mail ggermino@welchlink.welch.jhu.edu)
cDNA Libraries
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
lntroduction
Proteins are the work horses of the cell: they comprise the structural and
regulatory molecules, the motors and pumps, enzymes and receptors. Determining the complete sequence of a specific protein, however, or producing it in sufficient quantity to allow its manipulation and characterization
are extremely tedious and expensive procedures if one is limited to the use
of traditional biochemical approaches. Gene cloning and genetic engineering have greatly contributed to the field ofbiochemistry. Determining protein sequences would still be a daunting task, however, if one was restricted
to the use of genomic DNA as the source material since only -2o/o is thought
likely to encode protein. The remainder includes structural and regulatory
elements, introns, untranslated exons, and repetitive, non-coding sequences without known function ("junk DNA"). The effort required to determine a gene product's sequence is greatly reduced if the analysis is restricted to coding sequences. Since one of the principle tenets of molecular
biology isthat mRNA serve as the template for protein synthesis, mRNA
molecules are the ideal substrates for determining the sequence of proteins.
U nfortunately, none of the commonly available cloning vectors accept RN A
molecules as inserts. Therefore, an essential step in the generation of an
expressed sequence library is the conversion of mRNA molecules into double-stranded DNA. DNA copies of mRNA molecules are called cDNA, and a
library comprized ofDNA copies of the mRNA species from a cell or tissue is
called a cDNA library.
Luiz F. Onuchic, Johns Hopkins University, School ofMedicine, Division ofNephrology,
720 Rutland Avenue, Ross 970, BalitmoreMarylandUSA, Correspondence to Gregory G.
Germino, Johns Hopkins University, School ofMedicine, Division ofNephrology, 720
Rutland Avenue, Ross 970, BalitmoreMarylandUSA (phone +01-410-614-1650;/a.x +01
410-955-0485; e-mail ggermino@welchlink.welch.jhu.edu)