History of

'Genetic Engineering'
- Genetic engineering is the popular term for the more accurate 'Recombinant DNA
Technology'.
- The actual introduction of recombinant DNA technology into real life applications
can not be attributed to a single discovery, however, it is rather an accumulation of
different theories and ideas over a long period of time.
- Of course it returns back to1944 when scientists working in Rockefeller institute
in USA showed that inheritance depends on nucleic acid as a carrier of genetic
information and not protein as previously thought. Then, it was followed by the
breakthrough of Watson and Crick in 1953 who identified the DNA structure as a
double helix based on x-ray diffraction studies. In 1961, scientists start their trials to
know the “rule” controlling information storage in the DNA which was solved later on
by identification of the 'triplet codes' which means that each sequence of 3
nitrogenous bases in the DNA determines the formation of 1 amino acid and by
1966, the triplet codes of the existing 20 amino acids were established.
In 1970,

-Thefirst syntheticgene wasproducedina'test tube' by Khorana andhisgroupby

joiningtogetheritsbasepairs.

- Hamilton Smith discovered a group of enzymes which exist

naturally in microorganisms and are capable of DNA cleavage at
sequence-specific sites. Microorganisms use these enzymes as a
defence tool to attack any invading organism. It was that “precise
specificity” of their cutting that gave them a great role in
recombinant DNA technology with the ability to cut certain 'gene
sequence' out of the whole DNA molecule.
In 1970,
- Temin and Baltimore independently discovered the 'Reverse
Transcriptase Enzyme' which can copy genetic information from RNA to
DNA in certain viruses whose nucleic material is RNA [not DNA] in a
reverse way [hence, the name] to transcription process in which genetic
information is usually transferred from DNA to RNA 'mRNA' which is then
translated by ribosomes into proteins.

- This enzyme allowed the formation of a complementary DNA copy [called

cDNA] of genes encoded by RNA in the laboratory, which then allow the
production of the final gene product 'protein'.
In 1972,
- Success of the first “recombinant” DNA experiment by “joining”
restriction enzyme fragments [from 2 different organisms] together to
constitute a functional molecule.

- Then, plasmids were introduced as carriers for the constructed DNA

fragments 'genes'. Plasmids are capable of independent [of bacterial DNA]
replication in their bacterial hosts allowing the production of a large
quantity of the inserted DNA fragment after growth of their bacterial host in
a suitable medium producing a 'clone of bacterial cells' containing the
same 'gene' [in plasmid].
- The ligation of a specific gene into a plasmid vector represents the
'recombinant' work of the technique.
 In 1973,
- Construction of the first plasmid by Stanley Cohen ' referred to as pSC101'
[p for plasmid, SC for the name of the scientist introduced it] for use in
recombinant DNA experiments.
Restriction Endonucleases

- Exist naturally in bacterial cells which use them to protect themselves against
any invasion by foreign DNA from different strain.

- They are called ‘restriction’ because their activity is 'restricted' to ‘foreign’

DNA & called ‘endo-’ because they cut ‘within’ the DNA molecule.

- The host DNA is not cleaved by its “own” enzyme because the DNA sites
susceptible to be cut by the enzyme are protected by “Methylation” which is
done by another enzyme which is also included in each bacterial cell.

.
*An example: EcoRI enzyme:
E: 1st initial of the genus, Escherichia.

Co: 1st 2 initials of the species, coli.

R: strain designation (if the enzyme is present in a special strain).

I: roman numeral show the order of discovery of the enzyme in this particular
strain.

- Some enzymes, although obtained from different organisms, still can cleave the
same site such as [HindIII & HsuI] or recognise the same nucleotide sequence,
but cut at 2 different sites within that sequence such as (XmaI & SmaI) and are [in
both cases] called 'Isoschizomers'.
- Due to the complementary nature of the base pairing
in DNA molecules, a restriction endonuclease enzyme ---G AATTC---
will cut both strands of the DNA molecules. ---CTTAA G--

*Recognition sequence
- There are 2 types of cut ends; either 'sticky' or 'blunt'
[GATTC] and
according to the type of the enzyme.
*cutting site [between G
& A] of EcoRI enzyme,
- The sticky 'cohesive' ends are so called because the producing 'sticky ends'.

can combine with any complementary sequence [from

another DNA molecule] as that produced in a plasmid - -CCC GGG-
vector by its cutting with the same enzyme to allow -GGG CCC-
recombination of both molecules. Blunt ends produced
by SmaI enzyme,
derived from Serratia
marcescens
- Recognition sequences vary widely.

- Many of them are called Palindromic; that is, the sequence on one strand
reads the same in the same direction [from 5’ to 3’, or 3’ to 5’] on the
complementary strand.

e.g., 5'-GTATAC-3' on one strand is complementary to 3'-CATATG-5', which is

read GTATAC [i.e: the same] from 5' to 3'.

While, 5'-GTAATG-3' is not a palindromic DNA sequence as its complement is

3’-CATTAC-5’ which is read CATTAC from 5’ to 3’ which is different from the
other strand.
Ligation 'Recombination'

- The process of inserting a target DNA fragment into a foreign vector DNA is
called Ligation and is done by using an enzyme called 'DNA ligase'.

- There are several types of DNA ligase as T4 DNA ligase which can link DNA
molecules even those with blunt ends, however, such 'blunt-ended ligation' is less
stable than 'sticky-ended ligation' as the later can occur naturally by hydrogen
bonding between complementary bases 'annealing' before stabilization with the
DNA ligase which catalyses bonds between ends of the DNA molecules.
References:

* Books:

- Alan E.H. Emery and Sue Malcolm (1995) An Introduction to Recombinant DNA
in Medicine, 2nd edition. Wiley : Chichester, England.