1. 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:

2. 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 .

3. 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.

4. 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.

6. 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

7. 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.

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

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

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