Chapter 3 recombinant dna technology

Recombinant DNA Recombinant DNA TechnologyTechnology

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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 spliced together -- new and spliced together -- new "recombinant ""recombinant " molecules are then molecules are then multiplied in quantity in populations multiplied in quantity in populations of rapidly dividing cells (e.g. bacteria, of rapidly dividing cells (e.g. bacteria, yeast). yeast).


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


Human gene therapy, genetically-Human gene therapy, genetically-engineered crop plants and engineered crop plants and transgenic mice have become transgenic 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 fragments.restriction 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 recognition sitein the DNA called a recognition 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 terminate in a equal length, then they will 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::



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 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.


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.


Transformation and Antibiotic Transformation and Antibiotic SelectionSelection

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

cells. For example, through cells. For example, through electroporationelectroporation..

ElectroporationElectroporation involves applying a involves applying a brief (milliseconds) pulse high voltage brief (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 resistance

PlasmidsPlasmids 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.



The 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.



As 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 DNA

Treatment 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 cells

Production 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

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Chapter 3 recombinant dna technology