Recombinant DNA (rDNA): DNA made by joining pieces from two or

more different sources

Genetic engineering:
 extraction/syntheses a gene(s) from one organism
 transfer gene(s) into another organism (of same or different species)
 --> gene is expressed in new host

Genetic engineering provides a way of overcoming barriers to gene transfer between species. Indeed
the genes are taken from an organism ina different kingdom, such as a bacterial gene inserted into a
plant or a human gene inserted into a bacterium. Genetic engineering results in the transfer of a single
gene.

Process of genetic engineering

1 The gene that is required is identified. It may be cut from a chromosome, made from mRNA by reverse
transcription or synthesised from nucleotides.

2 Multiple copies of the gene are made using the technique known as the polymerase chain reaction
(PCR).

3 The gene is inserted into a vector which delivers the gene to the cells of the organism. Examples of
vectors are plasmids, viruses and liposomes.

4 The vector takes the gene into the cells.

5 The cells that have the new gene are identified and cloned.

To perform these steps, the genetic engineer needs a ‘toolkit’ consisting of:

■■ enzymes, such as restriction endonucleases, ligase and reverse transcriptase

■■ vectors, including plasmids and viruses

■■ genes coding for easily identifiable substances that can be used as markers.
Enzymes:
Restriction endonuclease: restrict viral infections by recognising and
breaking down DNA of invading viruses.
---> binds to specific target site on DNA (sequence of bases) --> cut sugar-
phosphate backbone:
 straight across = blunt ends
 staggered fashion = sticky ends*
* sticky ends: short lengths of unpaired bases; easily form hydrogen bonds with
complementary base sequences on other fragments of DNA cut with the same
restriction enzyme

Reverse transcriptase:
 uses single-stranded mRNA as the template
 reverses the transcription process: produces single stranded DNA
 single-stranded DNA + DNA polymerase = double-straned complementary
DNA (cDNA)

DNA ligase: links together sugar-phosphate backbones of DNA molecules

and plasmid ---> produces a closed circle of double-stranded DNA containing
the new gene = "recombinant DNA"
Plasmids as vectors in gene cloning
Plasmids are small, circular pieces of double-stranded DNA

 small --> easy to use

 exist naturally in bacteria --> bacteria take up plasmids from
surroundings
 can be produced artificially
 double stranded: can insert genes from prokaryotes and eukaryotes
 replicate independently in bacteria
 can be transferred between different bacterial species

Alternative markers for genetic engineering

A marker is a gene that is deliberately transferred along with the required gene during
the process of genetic engineering. It is easily recognised and used to identify those
cells to which the gene has been successfully transferred. In the production of insulin
in E. coli the original markers were antibiotic resistance genes (Figure 19.7).
Now alternative markers are preferred. This is because of the potential danger of the
antibiotic resistance genes being accidentally transferred to other bacteria, including
eventually, pathogenic strains of E. coli or even pathogens causing other diseases.
Whilst this has not been reported, the possibility must exist. The outcome would be an
‘engineered’ disease-causing microorganism that was resistant to one or more
antibiotic.
Genes for fluorescent (or easily stained) substances are now used as markers
(Figure 19.8) instead of antibiotic resistant genes, alongside the cDNA of the desired
gene. These genes are then linked to a special promoter. The marker gene is
expressed only when the desired gene has been successfully inserted into the genome
of the host. Organisms that have been transformed in this way will glow under UV light
(or can be identified by staining).
The expression of genes, such as those in the lac operon, is controlled by a promoter – the region of
DNA to which RNA polymerase binds as it starts transcription. If we want the gene that we are going to
insert into a bacterium to be expressed, then we also have to insert an appropriate promoter.

The promoter not only allows RNA polymerase to bind to DNA but also ensures that it recognises which
of the two DNA strands is the template strand. Within the sequence of nucleotides in the promoter
region is the transcription start point – the first nucleotide of the gene to be transcribed. In this way, the
promoter can be said to control the expression of a gene and can ensure a high level of gene expression.
In eukaryotes, various proteins known as transcription factors are also required to bind to the promoter
region or to RNA polymerase before transcription can begin.

Process of uptake of recombinant plasmid by host bacteria

It is important to identify which bacteria have been successfully transformed so that they can be used to
make the gene product. This used to be done by spreading the bacteria on agar plates each containing
an antibiotic.

DNA polymerase in bacteria copies the plasmids; the bacteria then divide by binary fission so that each
daughter cell has several copies of the plasmid. The bacteria transcribe the new gene and may translate
it to give the required gene product, such as insulin.

So if, for example, the insulin gene had been inserted into the plasmid at a point in the gene for
tetracycline resistance in pBR322, then any bacteria which had taken up plasmids with the recombinant
DNA would not be able to grow on agar containing tetracycline

Principle of PCR

The polymerase chain reaction, generally known as PCR, is used in almost every application of gene
technology. It is a method for rapid production of a very large number of copies of a particular fragment
of DNA. Virtually unlimited quantities of a length of DNA can be produced from the smallest quantity of
DNA (even one molecule).

First, the DNA is denatured, usually by heating it.

Th is separates the DNA molecule into its two strands, leaving bases exposed. Th e enzyme DNA
polymerase is then used to build new strands of DNA against the exposed ones. However, DNA
polymerase cannot just begin doing this with no ‘guidance’.

A primer is used to begin the process. Th is is a short length of DNA, oft en about 20 base pairs long, that
has a base sequence complementary to the start of the part of the DNA strand that is to be copied. Th e
primer attaches to the start of the DNA strand, and then the DNA polymerase continues to add
nucleotides all along the rest of the DNA strand.
 Once the DNA has been copied, the mixture is heated again, which once more separates the two
strands in each DNA molecule, leaving them available for copying again. Once more, the primers fi x
themselves to the start of each strand of unpaired nucleotides, and DNA polymerase makes
complementary copies of them.

The three stages in each round of copying need diff erent temperatures.

■■ Denaturing the double-stranded DNA molecules to make single-stranded ones requires a high
temperature, around 95 °C.

■■ Attaching the primers to the ends of the singlestranded DNA molecules (known as annealing)
requires a temperature of about 65 °C.

■■ Building up complete new DNA strands using DNA polymerase (known as elongation) requires a
temperature of around 72 °C. Th e DNA polymerases used for this process come from microorganisms
that have evolved to live in hot environments.

Taq polymerase was the first heat-stable DNA polymerase to be used in PCR. It is valuable for PCR for
two reasons. First, is not destroyed by the denaturation step, so it does not have to be replaced during
each cycle. Second, its high optimum temperature means that the temperature for the elongation step
does not have to be dropped below that of the annealing process, so efficiency is maximised.

Gel electrophoresis

Gel electrophoresis is a technique that is used to separate different molecules. It is used extensively in
the analysis of proteins and DNA. This technique involves placing a mixture of molecules into wells cut
into agarose gel and applying an electric field. The movement of charged molecules within the gel in
response to the electric field depends on a number of factors.

The most important are:

■■ net (overall) charge – negatively charged molecules move towards the anode (+) and positively
charged molecules move towards the cathode (–); highly charged molecules move faster than those
with less overall charge

■■ size – smaller molecules move through the gel faster than larger molecules

■■ composition of the gel – common gels are polyacrylamide for proteins and agarose for DNA; the size
of the ‘pores’ within the gel determines the speed with which proteins and fragments of DNA move.

Electrophoresis of proteins.

The charge on proteins is dependent on the ionisation of the R groups on the amino acid residues.
Whether these R groups are charged or not depends on the pH. When proteins are separated by
electrophoresis, the procedure is carried out at a constant pH using a buffer solution. Usually proteins
have a net negative charge
Gel electrophoresis has been used to separate the polypeptides produced by different alleles of many
genes. For example, allozymes are variant forms of enzymes produced by different alleles of the same
gene. There are also many variants of haemoglobin. Adult haemoglobin is composed of four
polypeptides: 2 α-globins and 2 β-globins .In sickle cell anaemia, a variant of β-globin has an amino acid
with a non-polar R group instead of one with an R group that is charged. These two variants of the β-
globin can be separated by electrophoresis because they have different net charges. This means that
haemoglobin molecules in people who have sickle cell anaemia have a slightly lower negative charge
than normal haemoglobin and so the molecules do not move as far through the gel as molecules of
normal haemoglobin. The test to find out whether someone carries the sickle cell allele makes use of
this difference.