Unit 5 B : Unit 5 B Gene Technology 1
Specification objectives : Specification objectives describe and understand the roles of reverse transcriptase, endonucleases and DNA ligase in the manipulation of DNA
describe the insertion of DNA into a host cell and the multiplication of the host cell
appreciate the use of marker genes to indicate that new genes have been incorporated into host cells
understand how protein synthesis is switched on and the synthesis of a new product by the host cell as illustrated by the introduction of new genes into plants using the bacterium Agrobacterium tumefaciens
The manipulation of DNA : The manipulation of DNA Gene technology involves the manipulation of genetic material so that genes from one organism can be inserted into the genome of another, unrelated organism.
Altered genetic material is referred to as recombinant DNA, and the basis of the technology involves the use of enzymes which enable DNA to be cut, copied and joined.
The basic principles : The basic principles The isolation of the gene required to produce the product
Insertion of this foreign gene into the DNA of a host cell by using a suitable DNA carrier called a vector
Checking to find the host cells which contain the new gene
Multiplying or cloning the organism containing the new gene to produce large numbers of genetically identical cells or organisms for commercial use
The enzymes involved : The enzymes involved Restriction endonucleases
Found naturally in bacteria where they help protect against invasion by viruses by cutting up viral DNA.
They are used to cut a gene out of a chromosome .
Different restriction enzymes cut at different base sequences because only these bases are the right shape to fit into their active site.
Restriction endonucleases : Restriction endonucleases
The enzymes involved : The enzymes involved Ligases
These enzymes are used to join, or anneal, two strands of DNA.
The ligases catalyse the formation of phosphodiester bonds between the pentoses and phosphate groups of two adjacent DNA chains.
The enzymes involved : The enzymes involved Reverse transcriptase
An enzyme first isolated from viruses: it will form DNA from an RNA template, allowing complementary, or copy, DNA (cDNA) to be formed from mRNA.
This allows double-stranded cDNA sequences to be inserted into a suitable plasmid vector, which can then be used to transform bacterial cells.
cDNA : cDNA
Plasmids : Plasmids Plasmids are small, circular loops of DNA, which are present in bacterial cells in addition to their circular chromosomal DNA.
These small loops of DNA contain some genes, such as genes that confer resistance to antibiotics, and replicate independently of the chromosome.
They are used as vectors to introduce genes into a host cell.
Once inside the host cell, the plasmid will replicate so that many copies of the original gene will be produced.
Plasmids can be cut open by restriction endonucleases, the new gene (which has been cut using the same endonuclease) inserted and then DNA ligase used to join the new gene and the plasmid together.
The toolkit : The genetic engineer uses five basic “tools” during the
procedure of Recombinant DNA Technology Isolated genes that code for the desired product The toolkit
Isolating a gene using restriction endonuclease : Restriction enzymes, also known as Restriction Endonucleases, are
a group of enzymes found in bacteria that recognise specific DNA
sequences of four to six nucleotides and make their incision within
that sequence The specific nucleotide sequences, recognised by restriction
enzymes, are called restriction sites and these are usually in the
form of palindromes Palindromes are nucleotide sequences that are symmetrical, about
an axis, and read the same in opposite directions in the two
strands of DNA A restriction enzyme known as
Eco R1, makes double-stranded
cuts between the
A and G nucleotides on
either side of the central axis The cuts from this enzyme
are staggered and produce
single-stranded regions
called ‘sticky ends’ Isolating a gene using restriction endonuclease
Blunt ends : Some restriction enzymes, such as Hpal, cut the DNA strands at positions
directly opposite one another, giving blunt ends to the fragments Hpal recognises the nucleotide sequence GTTAAC and ‘cuts’ between the
T and A nucleotides about the central axis Over seven hundred different restriction enzymes have now been identified and isolated from bacterial cells; each enzyme is named after the bacterial strain from which it was derived Eco R1 is from Escherichia coli,
strain RY13
Bam H1 is from Bacillus
amyloliquefaciens, strain H Blunt ends
The toolkit : Restriction enzymes that generate ‘sticky ends’
are very useful tools to the genetic engineer The same restriction enzyme recognises the same nucleotide sequence in the DNA from different species and creates the same ‘sticky ends’ When the DNA fragments from the
two different species are mixed together, the complementary bases of their ‘sticky ends’ will be attracted to one another and form hydrogen bonds In this way, DNA fragments from different sources can be brought together and joined DNA ligase is the enzyme that
seals fragments of DNA together The toolkit
Slide 15 : Complementary bases on the sticky ends of the DNA from the different species are attracted to one another Hydrogen bonds
form between the
bases and the
enzyme DNA ligase
seals the sugar-phosphate backbone
of the DNA molecule Recombinant DNA is formed
Vectors : Vectors are carrier DNA molecules into which DNA fragments containing
specific genes can be inserted Vectors are the means by which selected genes are carried into host cells
where the desired gene is then cloned The isolated plasmids of bacterial cells and the DNA of bacteriophages (viruses
that infect bacteria) are frequently used as vectors Plasmids are small, circular, self-replicating double-stranded DNA molecules
found in bacterial cells,which are separate from the main bacterial chromosome Vectors
Bacterial plasmid : Courtesy of
Prof. Stanley Cohen
Science Photo Library This electron
micrograph shows
a single bacterial
plasmid extracted
from the bacterium
E. coli Bacterial plasmid
Plasmids : Genes coding for ‘desirable products’ can be spliced into plasmids to form
RECOMBINANT PLASMIDS When these plasmids are taken up by bacterial host cells, they replicate along
with the host cell and clone the desired gene Plasmids are obtained from cultures of bacterial cells; bacterial cells are
broken open and the plasmids are separated out by centrifugation Homogenised bacterial cells, when subjected to centrifugation, provide the
plasmids into which foreign genes can be inserted Plasmids
Making recombinant plasmids : The two DNA molecules
are attracted to one another
and, in the presence of DNA
ligase, form a recombinant
DNA molecule Both the plasmid and the human DNA are treated
with the SAME restriction enzyme so that the DNA from
both sources will have complementary ‘sticky ends’ Making recombinant plasmids
Slide 20 : When host bacterial cells are mixed with these recombinant plasmids, they may take them
up and become transformed; these bacterial cells are now described as transgenic
organisms as they contain and express the genetic material from a different species When this transformed
bacterial cell divides, the
recombinant plasmid
replicates and copies of the
plasmid (containing the
foreign DNA) are passed to
the daughter cells The foreign DNA has been cloned
Manufacturing insulin : Manufacturing insulin
Marker genes : Marker genes Only a few bacteria will take up the plasmid containing the required gene (the recombinant plasmid). These bacteria are said to be modified or transformed.
Other bacteria will take up non-recombinant plasmids without the gene.
The modified bacteria have to be identified and separated (screened) from the others.
This involves genetic markers such as genes for antibiotic resistance.
Marker genes : Marker genes Plasmids with a gene for antibiotic resistance are used as the vectors
Cutting the plasmid and inserting the human DNA is inserted within the bases of the antibiotic gene
If the gene has been inserted successfully, the antibiotic cannot function properly because it has been damaged Before After Antibiotic resistant gene Cut here Human DNA added within the gene
Replica plating : Replica plating is a technique that allows molecular biologists to transfer
samples of bacterial colonies from one nutrient agar plate to another Using this method, duplicate bacterial samples can be grown on a second agar
plate in exactly the same position that they were growing on the first, master plate The felt or velvet-covered
tool is pressed gently
onto the surface of the
first agar plate containing
colonies of bacteria Cells from each of the
bacterial colonies stick
to the velvet and can
be transferred to the
replica plate in the
same positions relative
to one another Replica plating
Replica plating : Replica plating Antibiotic plate – transgenic bacteria don’t grow Master plate – doesn’t contain antibiotic - all bacteria grow
Switching on genes : Switching on genes Genes are expressed when their base sequence is being transcribed into mRNA for protein synthesis
Many genes have to be activated (switched on) before they can be expressed.
This prevents a protein being produced (and wasted) at the wrong time or in the wrong place.
Other sections of DNA are involved in this switching on, including promoters, regulators and operators
One structural gene (codes for actual functional protein) together with the regulatory genes which control it are known as an operon
Switching on genes : Switching on genes
The use of the bacterium Agrobacterium tumefaciens as a vector with plants : The use of the bacterium Agrobacterium tumefaciens as a vector with plants This is a bacterium that naturally infects plants causing swellings called galls.
To insert a gene into a plant, the required gene is inserted into a plasmid and then the plasmid is inserted into Agrobacterium.
Agrobacteruim is then allowed to infect plant cells grown in culture where it transfers plasmids into the plant cells
Plants grown from these GM plant cells will contain the inserted gene
This was used in 1993 to produce GM oilseed rape plants.
The transgenic plants produced a different type of oil which could be extracted and used commercially to make detergents
The use of the bacterium Agrobacterium tumefaciens as a vector with plants : The use of the bacterium Agrobacterium tumefaciens as a vector with plants