Construction of cDNA Libraries

To obtain a cDNA clone of an mRNA, the mRNA must be copied faithfully into DNA, and the cDNA library must be large enough to represent the abundance class that contains the mRNA of interest. For example, in tobacco, Goldberg (1 ) has shown that it is possible to divide the mRNA population into three classes with most of the mRNAs (11,300) being in the lowest abundance class and making up 39% of the polysomal rnRlVA. To obtain a cDNA library that contains at least one clone for each mRNA of this class will require about 2 � 10⁵ clones (2 ). This can be achieved with a few micrograms of mRNA by using the efficient RNase H method of making double-stranded cDNA (3 ; see Chapter 24 ) and a bacteriophage λ vector that exploits the high efficiency with which in vitro packaged phage can be introduced into Escherichia coli . The choice of λ vector is important because if the DNA to be inserted makes the λ genome > 105% of the wild-type length, the packaged phage will have a low viability.