Gene Technology Divya

8/2/2019 Gene Technology Divya

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WELCOME

BY

DIVYA. B

I YEAR BIO CHEMISTRY

SACW COLEGE


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GENE TECHNOLOGY


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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 themultiplication of the host cell

appreciate the use of marker genes to indicate that newgenes have been incorporated into host cells

understand how protein synthesis is switched on and thesynthesis of a new product by the host cell as illustrated bythe introduction of new genes into plants using thebacterium Agrobacterium tumefaciens


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THE MANIPULATION OF DNA

Gene technology involves the manipulation ofgenetic material so that genes from one organismcan be inserted into the genome of another,

unrelated organism. Altered genetic material is referred to as

recombinant DNA, and the basis of the technologyinvolves the use of enzymes which enable DNA to

be cut, copied and joined.


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THE BASIC PRINCIPLES

The isolation of the gene required to produce theproduct

Insertion of this foreign gene into the DNA of a hostcell by using a suitable DNA carrier called a vector

Checking to find the host cells which contain thenew gene

Multiplying or cloning the organism containing thenew gene to produce large numbers of genetically

identical cells or organisms for commercial use


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THE ENZYMES INVOLVED

Restriction endonucleases

Found naturally in bacteria where they help protectagainst 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.


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RESTRICTION ENDONUCLEASES


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Ligases

These enzymes are used to join, or anneal,two strands of DNA.

The ligases catalyse the formation ofphosphodiester bonds between the pentosesand phosphate groups of two adjacent DNA

chains.


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Reverse transcriptase

An enzyme first isolated from viruses: it willform DNA from an RNA template, allowing

complementary, or copy, DNA (cDNA) to beformed from mRNA.

This allows double-stranded cDNA

sequences to be inserted into a suitableplasmid vector, which can then be used totransform bacterial cells.


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CDNA


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PLASMIDS

Plasmids are small, circular loops of DNA, which arepresent in bacterial cells in addition to their circularchromosomal DNA.

These small loops of DNA contain some genes, such asgenes 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 sameendonuclease) inserted and then DNA ligase used to jointhe new gene and the plasmid together.


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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 bacteriumE. 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 hostbacterial plasmids are commonlyused as vectors

circularplasmid

An enzyme capable of glueing an isolated gene into a cutvectorDNA 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


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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 sequence

The specific nucleotide sequences, recognised by restrictionenzymes, are called restriction sites and these are usually in the

form ofpalindromes

Palindromes are nucleotide sequences that are symmetrical, aboutan axis, and read the same in opposite directions in the two

strands of DNA

A T T CA

GATT AC

G

portion ofdouble-

strandedDNA

centralaxis

A restriction enzyme known asco 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 regions

called sticky endssticky ends

Restriction Enzymes are the engineersDNAcutting scissors

ISOLATING A GENE USING RESTRICTION ENDONUCLEASE


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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 the

T and A nucleotides about the central axisenzyme cuts

enzyme cuts

T A A CT

GTAA TC

G

blunt endsOver seven hundred differentrestriction enzymes have now been

identified and isolated from bacterialcells; each enzyme is named after the

bacterial strain from which it wasderived

Eco R1 is fromEscherichia coli,strain RY13

Bam H1 is fromBacillusamyloliquefaciens, strain H

BLUNT ENDS


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Restriction enzymes that generate sticky endsare very useful tools to the genetic engineer

The same restriction enzymerecognises the same nucleotide

sequence in the DNA from differentspecies 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 toone another and form hydrogen bonds

In this way, DNA fragments fromdifferent sources can be brought

together and joined

DNA ligase is the enzyme thatseals fragments of DNA together

THE TOOLKIT


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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 endscreated by cutting the DNAfrom each species with the

same restriction enzymeComplementary bases on thesticky 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 backbone

of the DNA moleculeRecombinant DNA isformed


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Vectors are carrier DNA molecules into which DNA fragments containing

specific genes can be insertedVectors 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 (virusesthat infect bacteria) are frequently used as vectors

Plasmids are small, circular, self-replicating double-stranded DNA moleculesfound in bacterial cells,which are separate from the main bacterial chromosome

mainchromosome

plasmid

VECTORS


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Courtesy ofProf. Stanley Cohen

Science Photo Library

This electronmicrograph showsa single bacterialplasmid extracted

from the bacteriumE. coli

BACTERIAL PLASMID


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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

PLASMIDS


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

Plasmid

The two DNA moleculesare attracted to one anotherand, in the presence of DNA

ligase, form a recombinantDNA 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 complementaryto those on the cut plasmid

Recombinant plasmid

MAKING RECOMBINANT PLASMIDS

When host bacterial cells are mixed with these recombinant plasmids they may take them


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When host bacterial cells are mixed with these recombinant plasmids, they may take themup and become transformed; these bacterial cells are now described as transgenicorganisms as they contain and express the genetic material from a different species

When this transformedbacterial cell divides, the

recombinant plasmidreplicates and copies of the

plasmid (containing theforeign DNA) are passed to

the daughter cells

The foreign DNA has been cloned


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MANUFACTURING INSULIN


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MARKER GENES

Only a few bacteria will take up the plasmidcontaining the required gene (the recombinantplasmid). These bacteria are said to be modified ortransformed.

Other bacteria will take up non-recombinantplasmids without the gene.

The modified bacteria have to be identified andseparated (screened) from the others.

This involves genetic markers such as genes forantibiotic resistance.


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MARKER GENES

Plasmids with a gene forantibiotic resistance are usedas the vectors

Cutting the plasmid and

inserting the human DNA isinserted within the bases ofthe antibiotic gene

If the gene has been insertedsuccessfully, the antibiotic

cannot function properlybecause it has beendamaged

Before

After

Antibioticresistantgene

Cut here

Human DNAadded withinthe gene

REPLICA PLATING


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Replica plating is a technique that allows molecular biologists to transfersamples 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

Replica Plating Tool

handle

sterilised felt orvelvet surface

The felt or velvet-coveredtool is pressed gently

onto the surface of thefirst agar plate containing

colonies of bacteria

Cells from each of thebacterial colonies stickto the velvet and canbe transferred to the

replica plate in thesame positions relative

to one another

REPLICA PLATING


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REPLICA PLATING

Antibiotic plate transgenicbacteria dont grow

Master platedoesnt containantibiotic - all bacteria grow

These colonies aremissing and show

where, on themaster plate,

bacterial coloniescontaining

recombinantplasmids

are growing

These coloniescontain

recombinantplasmids; thecolonies are

removed andgrown on anew agar plate


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SWITCHING ON GENES

Genes are expressed when their base sequence is beingtranscribed into mRNA for protein synthesis

Many genes have to be activated (switched on) before theycan be expressed.

This prevents a protein being produced (and wasted) at thewrong 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 areknown as an operon


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SWITCHING ON GENES


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THE USE OF THE BACTERIUMAGROBACTERIUM

TUMEFACIENS AS A VECTOR WITH PLANTS

This is a bacterium that naturallyinfects plants causing swellingscalled galls.

To insert a gene into a plant, therequired gene is inserted into aplasmid and then the plasmid isinserted into Agrobacterium.

Agrobacteruim is then allowed toinfect plant cells grown in culturewhere it transfers plasmids intothe plant cells

Plants grown from these GM plantcells will contain the inserted gene

This was used in 1993 to produceGM oilseed rape plants.

The transgenic plants produced adifferent type of oil which could beextracted and used commerciallyto make detergents


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THE USE OF THE BACTERIUMAGROBACTERIUM

TUMEFACIENS AS A VECTOR WITH PLANTS


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BY

DIVYA. B

I YEAR BIO CHEMISTRY

SACW COLEGE

Gene Technology Divya

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