2 recombinant dna technology

Recombinant DNA Recombinant DNA TechnologyTechnology


Recombinant DNA technology Recombinant DNA technology procedures by which DNA from procedures by which DNA from different species can be isolated, cut different species can be isolated, cut and and spliced together spliced together -- new -- new "recombinant ""recombinant " molecules are then molecules are then multiplied (cloning) in quantity in multiplied (cloning) in quantity in populations of rapidly dividing cells populations of rapidly dividing cells (e.g. bacteria, yeast). (e.g. bacteria, yeast).


The term The term gene cloning, recombinant gene cloning, recombinant DNA DNA technology and genetic engineering technology and genetic engineering may seems similar, however they are may seems similar, however they are different techniques in Biotechnology different techniques in Biotechnology and and they are interrelatedthey are interrelated

Clone: a collection of molecules or cells, Clone: a collection of molecules or cells, all all identicalidentical to an original molecule or to an original molecule or cellcell


Three goals Three goals - Eliminate undesirable Eliminate undesirable phenotypic phenotypic

traits (observable characteristics)traits (observable characteristics)- Combine beneficial traits of two or Combine beneficial traits of two or

more organismsmore organisms- Create organisms that synthesize Create organisms that synthesize

products humans needproducts humans need


Human Human gene therapygene therapy, genetically-, genetically-engineered engineered cropcrop plants and plants and transgenictransgenic mice have become mice have become possible because of the powerful possible because of the powerful techniques developed to manipulate techniques developed to manipulate nucleic acids and proteins.nucleic acids and proteins.


In the early 1970s it became possible In the early 1970s it became possible to isolate a specific piece of DNA out to isolate a specific piece of DNA out of the millions of base pairs in a of the millions of base pairs in a typical genome.typical genome.


Currently it is relatively easy to cut Currently it is relatively easy to cut out a specific piece of DNA, produce out a specific piece of DNA, produce a large number of copies , determine a large number of copies , determine its nucleotide sequence, slightly alter its nucleotide sequence, slightly alter it and then as a final step transfer it it and then as a final step transfer it back into cell in.back into cell in.


Recombinant DNA technology is based Recombinant DNA technology is based on a number of important things:on a number of important things:

Bacteria contain extrachromosomal Bacteria contain extrachromosomal molecules of DNA called molecules of DNA called plasmidsplasmids which are circular.which are circular.


Bacteria also produce enzymes Bacteria also produce enzymes called called restriction endonucleasesrestriction endonucleases that cut DNA molecules at specific that cut DNA molecules at specific places into many smaller fragments places into many smaller fragments called called restriction fragmentsrestriction fragments..


Restriction Enzymes and Restriction Enzymes and plasmidplasmid

There are many different kinds of There are many different kinds of restriction endonucleases restriction endonucleases

Each nuclei cuts DNA at a specific Each nuclei cuts DNA at a specific site defined by a sequence of bases site defined by a sequence of bases in the DNA called a in the DNA called a recognition siterecognition site



A restriction enzyme cuts only A restriction enzyme cuts only double-helical segments that contain double-helical segments that contain a particular sequence, and it makes a particular sequence, and it makes its incisions only within that its incisions only within that sequence--known as a "recognition sequence--known as a "recognition sequence".sequence".



Sticky endSticky end and and blunt endblunt end are the are the two possible configurations resulting two possible configurations resulting from the breaking of double-stranded from the breaking of double-stranded DNA DNA


Restriction Enzymes and plasmidRestriction Enzymes and plasmid If two complementary strands of DNA are of If two complementary strands of DNA are of

equal length, then they will equal length, then they will terminate in a terminate in a blunt endblunt end, as in the following example:, as in the following example:

5'-5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpTCpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3'-3'

3'-3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpAGpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-5'-5'



However, if one strand extends beyond However, if one strand extends beyond the complementary region, then the the complementary region, then the DNA is said to possess an DNA is said to possess an overhangoverhang: :

5'-5'-ApTpCpTpGpApCpTApTpCpTpGpApCpT-3'-3' 3'-3'-TpApGpApCpTpGpApCpTpApCpGTpApGpApCpTpGpApCpTpApCpG-5'-5'



If another DNA fragment exists with a If another DNA fragment exists with a complementary overhang, then these complementary overhang, then these two overhangs will tend to associate two overhangs will tend to associate with each other and each strand is said with each other and each strand is said to possess a to possess a sticky endsticky end::


Restriction Enzymes and plasmidRestriction Enzymes and plasmid 5'-5'-ApTpCpTpGpApCpT ApTpCpTpGpApCpT

pGpApTpGpCpGpTpApTpGpCpTpGpApTpGpCpGpTpApTpGpCpT-3'-3'

3'-3'-TpApGpApCpTpGpApCpTpApCpGpTpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpACpApTpApCpGpA-5'-5'

BecomesBecomes 5'-5'-ApTpCpTpGpApCpTApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpTpGpApTpGpCpGpTpApTpGpCpT-3'-3'

3'-3'-TpApGpApCpTpGpApCpTpApCpGpTpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpACpApTpApCpGpA-5'-5'


Restriction Enzymes and plasmidRestriction Enzymes and plasmidRestriction Enzymes are primarily found Restriction Enzymes are primarily found

in bacteria and are given abbreviations in bacteria and are given abbreviations based on genus and species of the based on genus and species of the bacteria.bacteria.

One of the first restriction enzymes to One of the first restriction enzymes to be isolated was from EcoRIbe isolated was from EcoRI

EcoRI is so named because it was EcoRI is so named because it was isolated from isolated from Escherichia Escherichia coli strain coli strain called RY13. called RY13.


Digestion of DNA by EcoRI to Digestion of DNA by EcoRI to produce cohesive ends ( Fig. produce cohesive ends ( Fig.

3.1):3.1):


Creating recombinant DNA :Creating recombinant DNA :

The first Recombinant DNA The first Recombinant DNA molecules were made by Paul Berg molecules were made by Paul Berg at Stanford University in 1972. at Stanford University in 1972.

In 1973 Herbert Boyer and Stanley In 1973 Herbert Boyer and Stanley Cohen created the first recombinant Cohen created the first recombinant DNA organisms. DNA organisms.

Overview of recombinant DNA technologyOverview of recombinant DNA technology

Bacterial cell

Bacterialchromosome

Plasmid

Gene of interest

DNA containinggene of interest

Isolate plasmid.

Enzymatically cleaveDNA into fragments.

Isolate fragmentwith the gene ofinterest.

Insert gene into plasmid.

Insert plasmid and gene intobacterium.

Culture bacteria.

Harvest copies ofgene to insert intoplants or animals

Harvest proteinscoded by gene

Eliminateundesirablephenotypictraits

Produce vaccines,antibiotics,hormones, orenzymes

Createbeneficialcombinationof traits

Ligase

E. Coli

E. Coli


Creating Recombinant DNA (Fig Creating Recombinant DNA (Fig 3.2):3.2):


Reading materials :Summary of Reading materials :Summary of Recombinant DNA technology process:Recombinant DNA technology process:

Recombinant DNA technology requires DNA Recombinant DNA technology requires DNA extraction, purification, and fragmentation. extraction, purification, and fragmentation.

Fragmentation of DNA is done by specific Fragmentation of DNA is done by specific 'restriction' enzymes and is followed by 'restriction' enzymes and is followed by sorting and isolation of fragments sorting and isolation of fragments containing a particular gene. containing a particular gene.


Summary of Recombinant DNA Summary of Recombinant DNA technology process:technology process:

This portion of the DNA is then This portion of the DNA is then coupled to a carrier molecule.coupled to a carrier molecule.

The hybrid DNA is introduced into a The hybrid DNA is introduced into a chosen cell for reproduction and chosen cell for reproduction and synthesis.synthesis.


Transformation and Antibiotic Transformation and Antibiotic SelectionSelection

Transformation is the genetic Transformation is the genetic alteration of a cell resulting from the alteration of a cell resulting from the introduction, uptake and expression introduction, uptake and expression of foreign DNA. of foreign DNA.

Recombinant DNA Recombinant DNA TechnologyTechnologyTransformation and Antibiotic Transformation and Antibiotic

SelectionSelection

There are more aggressive techniques for There are more aggressive techniques for inserting foreign DNA into eukaryotic cells. inserting foreign DNA into eukaryotic cells.

For example, through For example, through electroporationelectroporation.. ElectroporationElectroporation involves applying a brief involves applying a brief

(milliseconds) pulse high voltage (milliseconds) pulse high voltage electricity to create tiny holes in the electricity to create tiny holes in the bacterial cell wall that allows DNA to bacterial cell wall that allows DNA to

enter.enter.


Plasmids and Antibiotic resistancePlasmids and Antibiotic resistancePlasmidsPlasmids were discovered in the were discovered in the

late sixties, and it was quickly late sixties, and it was quickly realized that they could be used to realized that they could be used to amplify a gene of interest.amplify a gene of interest.

A plasmid containing resistance to an A plasmid containing resistance to an antibiotic (usually ampicillin) or antibiotic (usually ampicillin) or Tetracycline, is used as a vector. Tetracycline, is used as a vector.


The gene of interest (resistant to The gene of interest (resistant to Ampicillin) is inserted into the vector Ampicillin) is inserted into the vector plasmid and this newly constructed plasmid and this newly constructed plasmid is then put into E. coli that is plasmid is then put into E. coli that is sensitive to ampicillin.( Text bk:Pg sensitive to ampicillin.( Text bk:Pg 58)58)

The bacteria are then spread over a The bacteria are then spread over a plate that contains ampicillin. plate that contains ampicillin.


Plasmids and Antibiotic resistancePlasmids and Antibiotic resistanceThe ampicillin provides a selective The ampicillin provides a selective

pressure because only bacteria that pressure because only bacteria that have acquired the plasmid can grow have acquired the plasmid can grow on the plate.on the plate.

Those bacteria which do not acquire Those bacteria which do not acquire the plasmid with the inserted gene of the plasmid with the inserted gene of interest will die.interest will die.


Plasmids and Antibiotic resistancePlasmids and Antibiotic resistanceAs long as the bacteria grow in As long as the bacteria grow in

ampicillin, it will need the plasmid to ampicillin, it will need the plasmid to survive and it will continually survive and it will continually replicate it, along with the gene of replicate it, along with the gene of interest that has been inserted to the interest that has been inserted to the plasmid .plasmid .


Fig 3.3 Fig 3.3 (a).(a).

Selecting Selecting a Gene in a Gene in a plasmid a plasmid

and and Antibiotic Antibiotic selection.selection.


Assignment: For above Assignment: For above procedure,procedure,

Read Transformation of Read Transformation of Bacterial cells and Antibiotic Bacterial cells and Antibiotic

selection pg 61.selection pg 61.


Human Gene cloningHuman Gene cloning

Once inside a bacterium, the plasmid Once inside a bacterium, the plasmid containing the human cDNA can containing the human cDNA can multiply to yield several dozen multiply to yield several dozen replicas. replicas.


Reading materials:Reading materials:Summary of Recombinant DNA and Summary of Recombinant DNA and

Cloning (Fig. below):Cloning (Fig. below): Isolation of two kinds of DNAIsolation of two kinds of DNATreatment of plasmid and foreign DNA Treatment of plasmid and foreign DNA

with the same restriction enzyme with the same restriction enzyme Mixture of foreign DNA with plasmidsMixture of foreign DNA with plasmids


Addition of DNA ligaseAddition of DNA ligase Introduction of recombinant plasmid Introduction of recombinant plasmid

into bacterial cellsinto bacterial cellsProduction of multiple gene copies by Production of multiple gene copies by

gene cloninggene cloning


Summary of Recombinant DNA Summary of Recombinant DNA and Cloning (Fig.):and Cloning (Fig.):


This segment is "glued" into place This segment is "glued" into place using an enzyme called DNA ligase. using an enzyme called DNA ligase.

The result is an edited, or The result is an edited, or recombinant, DNA molecule.recombinant, DNA molecule.


When this recombinant plasmid DNA When this recombinant plasmid DNA is inserted into is inserted into E. coliE. coli, the cell will be , the cell will be able to process the instructions to able to process the instructions to assemble the amino acids for insulin assemble the amino acids for insulin production. production.


More importantly, the new More importantly, the new instructions are passed along to the instructions are passed along to the next generation of next generation of E. coliE. coli cells in the cells in the process known as gene cloning. process known as gene cloning.

Assignment: Human gene cloning pg Assignment: Human gene cloning pg 6363


Fig: Fig: Inserting Inserting

a DNA a DNA sample sample into a into a

PlasmidPlasmid


ReferencesReferences http://en.wikipedia.org/wiki/Restriction_enzymehttp://en.wikipedia.org/wiki/Restriction_enzyme http://web.mit.edu/esgbio/www/rdna/cloning.htmlhttp://web.mit.edu/esgbio/www/rdna/cloning.html http://faculty.plattsburgh.edu/donald.slish/http://faculty.plattsburgh.edu/donald.slish/

Transformation.htmlTransformation.html

2 recombinant dna technology

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