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LUIZ F. ONUCHIC AND GREGORY G. GERMINO
An ideal cDNA library is a faithful and complete representation of all
mRNA molecules present in the tissue from which it was prepared.
Many of the principles reviewed in the preceding sections are also applicable to cDNA libraries:
• the choice of vector and host are determined by the ultimate use of the
library;
• the strategy used to generate inserts depends on the optimal size of insert
desired;
• some expressed sequences confer significant growth advantages/disadvantages and thereby distort their representation within the library;
• chimerk clones are common; and
• one must consider the relative cost and effort of constructing one's own
library versus the availability of previously prepared libraries. There are a
number of features unique to expressed sequence libraries, however,
which are reviewed in the following sections.
mRNA purification
The first step in constructing a cDNA library consists of isolating the RNA
molecules that are expressed within a given tissue or cell type. mRNAs comprise only 1-5% of the total RNA present within a cell, so additional purification techniques are required to enrich for mRNA. Selection of mRNA
from total cellular RNA is based on the observation that virtually all eukaryotic mRNAs have a long stretch (>20 bp) of adenosines at their 3'
ends. This sequence, called the poly(A) tail, is unique to mRNA molecules
and is absent in the other major RNA species, ribosomal RNAs (rRNAs) and
transfer RNAs (tRNAs). The poly(A) tail usually is long enough to hybridize
to its complement, a synthetic oligonucleotide made of deoxythymidines
[oligo(dT)], and it is this property that allows selection of mRNAs from
a mixture ofRNA molecules. Although there are numerous methods available for selecting mRNA molecules, they primarily differ in how the oligo(dT)-mRNA complex is separated from the total RNA mixture. The protocols fall basically into three groups:
• Oligo(dT) affinity chromatography: The original protocol by Aviv and
Leder (1972) used affinity chromatography with an oligo(dT) (12-18
mer) linked to cellulose. In this procedure, the total RNA mixture is
loaded onto the top of a column of the matrix, and mRNAs anneal to
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
An ideal cDNA library is a faithful and complete representation of all
mRNA molecules present in the tissue from which it was prepared.
Many of the principles reviewed in the preceding sections are also applicable to cDNA libraries:
• the choice of vector and host are determined by the ultimate use of the
library;
• the strategy used to generate inserts depends on the optimal size of insert
desired;
• some expressed sequences confer significant growth advantages/disadvantages and thereby distort their representation within the library;
• chimerk clones are common; and
• one must consider the relative cost and effort of constructing one's own
library versus the availability of previously prepared libraries. There are a
number of features unique to expressed sequence libraries, however,
which are reviewed in the following sections.
mRNA purification
The first step in constructing a cDNA library consists of isolating the RNA
molecules that are expressed within a given tissue or cell type. mRNAs comprise only 1-5% of the total RNA present within a cell, so additional purification techniques are required to enrich for mRNA. Selection of mRNA
from total cellular RNA is based on the observation that virtually all eukaryotic mRNAs have a long stretch (>20 bp) of adenosines at their 3'
ends. This sequence, called the poly(A) tail, is unique to mRNA molecules
and is absent in the other major RNA species, ribosomal RNAs (rRNAs) and
transfer RNAs (tRNAs). The poly(A) tail usually is long enough to hybridize
to its complement, a synthetic oligonucleotide made of deoxythymidines
[oligo(dT)], and it is this property that allows selection of mRNAs from
a mixture ofRNA molecules. Although there are numerous methods available for selecting mRNA molecules, they primarily differ in how the oligo(dT)-mRNA complex is separated from the total RNA mixture. The protocols fall basically into three groups:
• Oligo(dT) affinity chromatography: The original protocol by Aviv and
Leder (1972) used affinity chromatography with an oligo(dT) (12-18
mer) linked to cellulose. In this procedure, the total RNA mixture is
loaded onto the top of a column of the matrix, and mRNAs anneal to