I am having difficulty understanding the theory behind cloning used back in 80's when we didn't have access to the full human genome. I was wondering if somebody could explain to me the methodology used at that time to search for amino acids coded by a gene and the full translated information of it's DNA. I found this paper (Identification of cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1072, 1989) as an example of method used but these papers are way too brief and difficult to comprehend. Can any expert in this field help me better understand these initial methods used to find genes and their translated protein sequences.
Well, I was still an undergrad working on biochemistry in the late 80s, but my 1993 I was a full blown graduate student in molecular genetics, so I know a few things. In general, there was a lot of cloning and Sanger Sequencing using known primers in the vector backbone to start then then primers internally as you made progress across a cDNA. I actually worked on a project that was a bit more like WGS. We had two almost identical Cosmids (large DNA clones propagated in bacterial cells) that we sequenced using shotgun sequencing (shear it up, clone it, sequence the clones, assemble de novo). It worked!!! Now everything in the sequencing world is too easy, so we stopped thinking about the methods as much.
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DNA sequencing was commonly used for genes and regions (but not genomes) beginning in the late 1970's. So it was possible to know the sequence of a transcript. As to how a gene was identified, it depended on the organism in use. In some cases genetic methods were used, in other cases DNA:DNA or DNA:RNA hybridization methods were used to identify cloned DNA that was similar to something you already knew about.
Essentially, you prepared libraries of cDNA clones (obtained by reverse transcription with random primers or, more commonly, oligo dT) or fragmented genomic DNA into plasmid or bacteriophage vectors. These libraries would then be screened by immunodectection (using e.g. a polyclonal antiserum against the naturally purified protein) or hybridization with oligonucleotides designed from part of the aminoacid sequence of that protein (obtained e.g. by Edman sequencing) or simply with cloned genes from a genetically related organism. Positive clones would then be sequenced, and sequence data would then be used for further experimentation (Northern blotting, recombinant expression in bacterial/yeast/mammalian hosts, etc).
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