Gene transfer and Recombinant DNA Biotechnology

Tools of recombinant DNA biotechnology

The basic process of recombinant DNA technology revolves around the function of DNA in the synthesis of protein.
By intervening in this process, scientists can change the nature of the DNA and of the gene make-up of an
organism. By inserting genes into the genome of an organism, the scientist can induce the organism to produce a
protein it does not normally produce.
The technology of recombinant DNA has been made possible in part by extensive research on microorganisms
during the last century. One important microorganism in recombinant DNA research is Escherichia coli (E. coli). The
biochemistry and genetics of E. coli are well known, and its DNA has been isolated and made to accept new genes.
The DNA can then be forced into fresh cells of E. coli, and the bacteria will begin to produce the proteins specified
by the foreign genes. Such altered bacteria are said to have been transformed.

Interest in recombinant DNA and biotechnology heightened considerably in the 1960s and 1970s with the
discovery of restriction enzymes. These enzymes catalyze the opening of a DNA molecule at a “restricted” point,
regardless of the DNA's source. Foreign DNA can then be combined with the carrier DNA at this point. An enzyme
called DNA ligase is used to form a permanent link between the dangling ends of the DNA molecules at the point
of union.

Genetic engineering is possible because of the processes genes are capable of, i.e. gene transfer and gene
modification. Genes of interest can also be cloned, i.e. copied

GeneTransfer
Normally, genes and the characteristics they code for are passed down from parent to progeny. This is called
vertical gene transfer. Bacteria and some lower eukaryotes are unique in that they can pass DNA from one cell of
the same generation to another. This is Horizontal Gene Transfer.
Horizontal gene transfer, also known as lateral gene transfer, is a process in which an organism transfers genetic
material to another organism that is not its offspring.
Horizontal gene transfer can occur in any of three ways; Conjugation is the transfer of circular DNA called plasmids
through cell to cell contact. Transformation is the uptake of 'free' DNA from the environment. Transduction is the
transfer of DNA by bacteria-specific viruses called bacteriophage.

Gene modification
This is carried out by gene transfer between species. Because the genetic code is almost universal, an organism can
potentially express a new trait if the appropriate gene is introduced into its genome
The transfer of genes between species is called gene modification, and the new organism created is called
a transgenic

Recombinant DNA
Recombinant DNA (or rDNA) is made by combining DNA from two or more sources. In practice, the process often
involves combining the DNA of different organisms. The process depends on the ability to cut and re-join DNA
molecules at points which are identified by specific sequences of nucleotide bases called restriction sites. DNA
fragments are cut out of their normal position in the chromosome using restriction enzymes (also called restriction
endonucleases) and then inserted into other chromosomes or DNA molecules using enzymes called ligases.

Gene Cloning
Gene cloning is the process in which a gene of interest is located and copied out of DNA extracted from an
organism. The cloned fragments of DNA can then be used for many different purposes, such as creating GM crops,
or finding a cure for disease. There are two types of gene cloning: in vivo, which involves the use of restriction
enzymes and ligases using vectors and cloning the fragments into host cells. The other type is in vitro which is
using the polymerase chain reaction (PCR) method to create copies of fragments of DNA.

For in vivo cloning, a fragment of DNA, containing a single gene or a number of genes, is inserted into a vector that
can be amplified within another host cell. A vector is a section of DNA that can incorporate another DNA fragment
without losing the capacity for self-replication, and a vector containing an additional DNA fragment is known as
a hybrid vector. The fragment of DNA can include one or more genes.

Genetic Modification: Bacteria Producing Human Insulin

Application: Gene transfer to bacteria using plasmids makes use of restriction endonucleases and DNA ligase
The process of gene transfer can be summarised in four key steps:
1. Isolation of gene and vector (by PCR)
2. Digestion of gene and vector (by restriction endonuclease)
3. Ligation of gene and vector (by DNA ligase)
4. Selection and expression of transgenic construct

Step 1: Isolating gene and vector

 DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are
separated
 The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR)
 Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences
(cDNA) lack introns
 A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell
 Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication
and expression
 These plasmids may be modified for further functionality (e.g. selection markers, reporter genes,
inducible expression promoters)
 Other types of vectors include modified viruses and artificial chromosomes

Common Features of a Typical Plasmid Vector

Step 2: Digestion with Restriction Enzymes
 In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at
specific recognition sites
 Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky
ends (complementary overhangs)
 Scientists will often cleave the vector and gene with two different ‘sticky end’ restriction endonucleases
(double digestion) to ensure the gene is inserted in the correct orientation and to prevent the vector from
re-annealing without the desired insert

‘Sticky End’ vs ‘Blunt End’ Restriction Enzymes

Step 3: Ligation of Vector and Insert

 The gene of interest is inserted into a plasmid vector that has been cut with the same
restriction endonucleases
 This occurs because the sticky ends of the gene and vector overlap via complementary base pairing
  The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
 DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent
phosphodiester bond

Formation of a Recombinant Construct

Step 4: Selection and Expression

 The recombinant construct (including the gene of interest) is finally introduced into an appropriate host
cell or organism
 This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or
transformation (for prokaryotes)
 Antibiotic selection is commonly used in order to identify which cells have successfully incorporated the
recombinant construct
 The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will grow in the
presence of antibiotic
 Transgenic cells, once isolated and purified, will hopefully begin expressing the desired trait encoded by
the gene of interest

Isolation of Transgenic Cells via Antibiotic Selection

https://www2.le.ac.uk/projects/vgec/schoolsandcolleges/Microbial%20Sciences/mutation-and-gene-
tranfer

https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/gene-
transfer.html

https://www.britannica.com/science/genetic-engineering