Knowledge of a gene or it’s gene product is necessary and essential before a gene can be isolated. This might be knowledge of the a) Gene sequence; b) Protein sequence c) Phenotype As an example, we will look at the isolation of genes which could be used in crop modification:

Complex plant traits such as photosynthesis, flowering, disease resistance, seed development, stress tolerance are coded for by many genes. It is possible to isolate some of the genes coding for these traits but the genes responsible for the complete phenotype are in many cases unknown. It is difficult to engineer organisms to express complex traits because many genes, all interacting in a complex manner, are involved. In all cases of genetic modification currently known, single genes coding for ONE product are transferred .

Example - genetic improvement of legumes to make them nutritionally ideal Legume seeds are an essential food source, but are deficient in cysteine and methionine (essential amino acids) Beneficial to improve seed amino acid content.

To study the protein composition of seeds, a seed protein extract can be electrophoresed on a P.A.G. E. gel Protein band 2 can be sequenced - rich in cysteine.

Key point to note is that only coding DNA is transcribed into mRNA Therefore, a mRNA population represents the expressed, coding part of the genome – non coding DNA is not present in mRNA RNA is transcribed from 3% of the genome mRNA is transcribed from 1.5% of the genome

WHY? If you could transform a mRNA population into DNA, you would have the coding sequences “extracted” from the “junk” DNA. WHY? 1) The coding DNA cannot be extracted from the genome. 2) mRNA is a ss molecule and is susceptible to degradation. DNA is a ds molecule and is RESISTANT to degradation 3) mRNA molecules cannot be cloned as they are ss. DNA molecules can be cloned because they are ds. Remember, cloning is necessary because you can amplify specific DNA molecules.

mRNA can be easily isolated from total RNA (ribosomal, transfer) using magnetic separation.

The enzyme reverse transcriptase is found in retroviruses - viruses which have RNA as the genetic material.

DNA copied from RNA (cDNA) contains genetic info for synthesis of viral proteins