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Gene Technology or Recombinant DNA Technology is about the manipulation of genes

Recombinant DNA Technology involves the isolation of DNA sequences from one organism and combining them with the DNA from a different organism

This technique allows organisms to express genes and thus manufactureproteins that they would not normally produce – their DNA has been engineered

to manufacture a product that is foreign to their natural genetic make-up

The procedure of Recombinant DNA Technology involves introducingsegments of DNA (genes) into a HOST by means of a carrier (VECTOR) system

The foreign DNA (gene) becomes a permanent feature of the host such that, during replication of the host cell, the gene is also passed on to its daughter cells

along with the rest of its own DNA – the foreign gene is therefore cloned

The manufacture of human insulinand growth hormone are

examples of theapplication of Recombinant

DNA Technology

GrowthHormone

HumanInsulin

Gene TechnologyGene Technology

Obtain the gene that codes for the desiredprotein product

Select a suitable vector (carrier) for transferring the gene to the host cells;the most commonly used vectors are bacterial plasmids and viruses

Insert the selected gene into the chosen vector

Allow the vector to carry the gene into the host cells; commonly used host cellsare bacteria and yeast cells – they are chosen for their rapid rates

of reproduction and allow the gene to be CLONED

Locate the host cells that have successfully taken up the desired gene

Culture the transformed cells on a commercial scale using large fermenters

Isolate and purify the desired ‘gene product’ – DownstreamProcessing

The Procedure of Recombinant DNA TechnologyThe Procedure of Recombinant DNA Technology

The genetic engineer uses five basic ‘tools’ during theprocedure of Recombinant DNA Technology

• A host organism that will take up the vectorcontaining the gene and reproduce rapidly

in order to supply many copies of thegene (gene cloning) – the bacterium E. coli

is commonly used as a host

• Vectors (carriers) into which the desired gene may beinserted and which are capable of carrying the

gene into a suitable host – bacterial plasmids are commonlyused as vectors

circularplasmid

• An enzyme capable of ‘glueing’ an isolated gene into a ‘cut’vector – DNA ligase is responsible for re-forming the DNA

backbone following insertion of the gene

plasmidwith

insertedgene

• Isolated genes that code for the desired product

• Enzymes capable of ‘cutting’ DNA at specific sites – theseenzymes are known as Restriction Enzymes

The ‘Toolkit’The ‘Toolkit’

Restriction enzymes, also known as Restriction Endonucleases, area group of enzymes found in bacteria that recognise specific DNAsequences of four to six nucleotides and make their incision within

that sequenceThe specific nucleotide sequences, recognised by restriction

enzymes, are called restriction sites and these are usually in theform 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 T T CA

GATT AC

Gportion of

double-stranded

DNA

centralaxis

A restriction enzyme known asEco R1, makes double-stranded

cuts between theA and G nucleotides on

either side of the central axis

enzyme cuts

enzyme cuts

ATT AC

G A T TA C

G

The cuts from this enzymeare staggered and produce

single-stranded regionscalled ‘sticky ends’

‘sticky ends’

Restriction Enzymes are the engineer’s DNA – cutting scissors

The ‘Toolkit’The ‘Toolkit’

T A A CT

GTAA TC

G

Some restriction enzymes, such as Hpal, cut the DNA strands at positionsdirectly opposite one another, giving blunt ends to the fragments

Hpal recognises the nucleotide sequence GTTAAC and ‘cuts’ between theT and A nucleotides about the central axis

enzyme cuts

enzyme cuts

T A A CT

GTAA TC

G

blunt endsOver 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

The ‘Toolkit’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 fromdifferent species and creates

the same ‘sticky ends’

When the DNA fragments from thetwo 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 thatseals fragments of DNA together

The ‘Toolkit’The ‘Toolkit’

A T T CA

GATT AC

G

ATT AC

G A T TA C

G

ATT AC

G

Fragment of DNA from species X

A T TA C

G

Fragment of DNA from species Y

Complementary sticky ends created by cutting the DNA from each species with the

same restriction enzymeComplementary bases on the sticky ends of the DNA from

the different species are attracted to one another

Hydrogen bondsform between the

bases and theenzyme DNA ligase

seals the sugar-phosphate backboneof the DNA molecule

Recombinant DNA is formed

VECTORSVectors 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 cellswhere the desired gene is then cloned

The isolated plasmids of bacterial cells and the DNA of bacteriophages (virusesthat 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

mainchromosome

plasmid

The ‘Toolkit’The ‘Toolkit’

Genes coding for ‘desirable products’ can be spliced into plasmids to formRECOMBINANT PLASMIDS

When these plasmids are taken up by bacterial host cells, they replicate alongwith the host cell and clone the desired gene

Plasmids are obtained from cultures of bacterial cells; bacterial cells arebroken open and the plasmids are separated out by centrifugation

Homogenised bacterial cells, when subjected to centrifugation, provide theplasmids into which foreign genes can be inserted

The ‘Toolkit’The ‘Toolkit’

Total human DNA extracted from human cells- known as genomic DNA

Plasmid

The two DNA moleculesare attracted to one another and, in the presence of DNAligase, form a recombinant

DNA molecule

Both the plasmid and the human DNA are treatedwith the SAME restriction enzyme so that the DNA from

both sources will have complementary ‘sticky ends’

DNA fragment with sticky ends complementary to those on the ‘cut’ plasmid

Recombinant plasmid

Making Recombinant PlasmidsMaking Recombinant Plasmids

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