How were the first genes isolated from genomic or cDNA libraries without already knowing a hybridization probe?
The following is based on a chapter on Cloning DNA in the 10th edition of The Biochemistry of the Nucleic Acids (RLP Adams et al.) published by Chapman &amp; Hall in 1986. (This is probably more pertinent than the 11th and final edition, which was published in 1992 and had a more contemporary treatment.) Apologies for the fact that it will be difficult to find.  It should be available from some university libraries and through inter-library loan systems in certain countries. Section A.7.3 gives a taste of the methodology used at that time. 

Protein properties that could be used as the basis of a cloning strategy

In attempting to clone DNA for a particular protein for which there were no pre-existing clones to use as hybridization probes, one had to consider the properties of the protein one is able to exploit. These might include:


Amino acid sequence 
Antibodies to the protein
Biological activity
Particular physical properties, such as size


(Clearly if one had no such ‘handle’ to the protein one was not in a position to try to clone its cDNA or gene.)

Three general approaches

There seemed to be three general approaches:


No expression 
Indirect expression
Direct expression


The approach that one might take was also governed by whether the protein is particularly abundant in a specific tissue, when one might expect less sophisticated more labour-intensive approaches to stand a chance of success.

Another point to emphasize is that, at least with eukaryotes, one would look for a cDNA clone first, and only try for a genomic clone after one had this to employ as a hybridization probe.

1. ‘No expression’ approaches

In the no expression approaches one relied on the amino acid sequence to identify the cloned DNA. One such approach was to use hybridization probes against stretches of amino acids, the codons of which had limited redundancy (Met and Trp have a single codon, and some other amino acids only two) using low stringency hybridization conditions. This required luck. Alternatively one could sequence random clones and hope to find one that correlated with the amino acid sequence. At that time DNA sequencing was slow and labour intensive, so that this was only applicable to proteins that were relatively abundant in the tissue from which the libarary was prepared.

2. Direct expression approaches

Direct expression generally entailed using a bacterial expression vector which produced a fusion of a bacterial protein with a part of the eukaryotic protein. (Statistically only one in six of clones would have the correct reading frame in a fusion protein.). This approach was favoured if one had an antibody, which did not require a complete protein.

3. Indirect expression approaches

By indirect expression was meant using immobilized cloned cDNA to select corresponding mRNAs (‘hybrid selection’) which could be expressed in a cell-free system. This was appropriate if the distinguishing characteristic required a full-length protein, as in the case of biological activity or size.