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Recombinant DNAand Other Topics in Biotechnology

Recombinant DNAand Other Topics in Biotechnology

Recombinant DNA Technology

Preparing Recombinant DNA

Amplification of DNA by the Polymerase Chain Reaction

Applications of Recombinant DNA Technology

Recombinant DNA Technology

Preparing Recombinant DNA

Amplification of DNA by the Polymerase Chain Reaction

Applications of Recombinant DNA Technology

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The potentialThe potential

The area of recombinant DNA seeks to engineer changes in an organism’s genome to perform useful tasks.

New lab methods have made DNA easy to work with. We can now cut well defined fragments and splice it into the DNA of another species.

Promising potentialPromising potential• Gene replacement therapy• DNA finger printing• Improved agricultural products

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Recombinant DNA technologyRecombinant DNA technology

Gradual increase in DNA knowledge over the last 100 years.

18681868 Discovery of DNA.

19441944 Confirmed as the carrier of genetic information.

19531953 Determination of structure and the methods of replication, transcription, translation and regulation.

1960’s1960’s Deciphering of the genetic code.

mid 1970’smid 1970’s Genetic engineering.

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Recombinant DNARecombinant DNA

DNA recombination or molecular cloningDNA recombination or molecular cloningCovalent insertion of a DNA fragment from one cell or organism into the replicating DNA of another.

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Basic stepsBasic steps

• Select and isolate a DNA molecule to serve as the carrier (vectorvector) for the foreign DNA.

• Cleave the vector DNA strands with a restriction endonuclease.

• Prepare and insert the foreign DNA, producing a recombinantrecombinant or hybrid DNAhybrid DNA molecule.

• Introduce the hybrid into a host organism, often a bacterial cell - transformationtransformation.

• Develop a means to screen and identify host cells that have accepted the hybrid DNA.

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Major breakthroughsMajor breakthroughs

• Isolation of a mutant strain of E. coli that did not have restriction endonucleases - could not degrade foreign DNA.

• Development of bacterial plasmids and bacteriophage DNA as cloning vectors.

• Discovery of restriction endonucleases that permit selective DNA cleavage.

• Discovery of DNA ligase that catalyzes the formation of phosphodiester link for final site closure.

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Cloning vectorsCloning vectors

Bacterial PlasmidsBacterial Plasmids

• Found in most bacterial cells.

• Self-replicating, extra chromosomal DNA.

• Closed, circular, double-stranded.

• Smaller than chromosomal DNA with only 3,000-30,000 base pairs.

• Contain information for translation of specialized and protective proteins.

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Plasmid replicationPlasmid replication

Two possible modesTwo possible modes

StringentStringent - only a few copies are made

RelaxedRelaxed - many, many copies are made.

Some relaxed plasmids may continue to replicate forming 2,000 - 3,000 copies, accounting for 30-40% of all cellular DNA.

A typical plasmid will accept foreign DNA up to 15, 000 base pairs.

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Ideal plasmid cloningvector properties

Ideal plasmid cloningvector properties

It should:It should:• replicate in a relaxed fashion so that many

copies are produced.

• be small so it is easy to separate from chromosomal DNA and easier to handle without damage.

• have only a few sites for attack by restriction enzymes.

• have identifiable markers for screening.

• have a single cleavage site for a given restriction enzyme - within a gene.

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Ideal plasmid cloningvector properties

Ideal plasmid cloningvector properties

Examples:Examples:

E. coliE. coli E1 E1 Bacterial strain that has been shown to be useful for recombinant DNA work.

pBR322pBR322A plasmid of E. coli E. It has 4363 base pairs. It is cleaved at a single site by the restriction endonuclease EcoRI

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Bacteriophage DNABacteriophage DNA

phage is the most widely used.phage is the most widely used.

• Double stranded DNA with ~50,000 base pairs.

• It can produce many DNA copies within a host cell.

• It acts as a package to introduce DNA by infecting host bacteria.

• It is easily screened for.

• DNA fragments up to 23,000 base pairs can be added.

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Preparing recombinant DNAPreparing recombinant DNA

Foreign double stranded DNA must be prepared for insertion.

Methods includeMethods include

Chemical synthesis

Use of restriction endonucleases

Reverse transcription of mRNA

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Preparing recombinant DNAPreparing recombinant DNA

Fragments prepared by restriction endonuclease have either “sticky” or blunt ends.

'5 C G T C

OH

'3 G C A G A A T G

P

5'CAT

OH

3'GTACATT

P

'5 C G T C

OH

'3 G C A G 5'CAT

OH

3'GTA

P

P

Sticky or cohesive ends

Blunt ends

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Preparing recombinant DNAPreparing recombinant DNA

Unpaired cohesive ends, up to 5 nucleotide bases, can be joined to a plasmid that was cleaved by the same endonuclease.

It can lead to regions that may not be stable.

C G T C

G C A G A AT

G CAT

GTACACT

T G

TT

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Preparing recombinant DNAPreparing recombinant DNA

Blunt ends can be attached using DNA ligase.

5’ end must have phosphate groups and the 3’ end must have a free hydroxyl group.

C G T C

G C A G CAT

GTAC G T C

OH

G C A G CAT

OH

GTA

P

PDNA ligase

ATP,

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Use of homopolymer tailsUse of homopolymer tails

This is a more widely used approach.This is a more widely used approach.

• Segments of 50-100 poly-A or poly-G are added to one fragment - typically the plasmid.

• Equal length segments of poly-T or poly-C are added to the other fragment.

• Both are accomplished using deoxynucleotidyl transferase.

• They are then allowed to incubate together to allow hydrogen bonding to bring them together.

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Use of homopolymer tailsUse of homopolymer tails

P

C G T T

OH

G C

OH

A A

P

P

C G T T

G C A A

P

Tn

Tn

HO

OH

OH P

HOP

An P

AnP

OH

HO

n-dATP

n-dTTP

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Use of homopolymer tailsUse of homopolymer tails

P

P

An

P

C G T T

G C A A

P

Tn

Tn

OH

OH

HO

AnOH

Foreign DNA withpoly T tails

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Linearized plasmidwith poly A tails

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Getting the hybrid into the hostGetting the hybrid into the host

TransformationTransformation• Introduction of the hybrid DNA into host cell.• Current methods are inefficient with only 1

molecule in 10,000 being successfully replicated.

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Transformation of E. coli cellsTransformation of E. coli cells

One can promote the introduction of hybrid plasmid using CaCl2

• Wash cells with CaCl2 solution.

• Incubate with a solution of hybrid DNA.

• If DNA is incorporated, it is replicated.

• Selective markers can be tested for.

Cloning of bacteriophage DNA is done by infecting the host bacteria.

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Screening and separationScreening and separation

pBR322 example.pBR322 example.

• This plasmid has resistance for two antibiotics - ampicillin and tetracycline.

• The foreign sequence is added within the gene that imparts tetracycline resistance.

• The resistance is destroyed.

• Sequential testing using both antibiotics can identify hybrid plasmids.

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Screening and separationScreening and separation

Yellow = Ampicillin treatedGreen = Tetracycline treated

identical transfer

incubate

Circled colonies areampicillin immuneand tetracyclinesensitive. Theymust be coloniesof cells containingthe hybrid plasmid.

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Restriction enzyme mapsRestriction enzyme maps

Mapping is valuable in the selection of plasmids for cloning and characterization of DNA fragments.

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Isolation and cloningof a single gene

Isolation and cloningof a single gene

A human gene can contain 40,000 - 1,000,000 base pairs.

• That represents only about 0.03% of the entire genome.

• We have the goal of identifying all human genes - Human Genome Project.

• The first step is to construct a genomic library - brute force, hit or miss approach.

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Constructing a genomic libraryConstructing a genomic library

• Cut DNA into thousands of fragments using restriction enzymes yielding random, overlapping pieces.

• Separate fragments based on molecular size using methods like gel electrophoresis.

• Add homopolymer tails and insert into vectors and then into host cells.

• Clone the cells. This results in large population of cells, each containing a different DNA fragment.

• Hope that all original genomic DNA is represented.

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Constructing a genomic libraryConstructing a genomic library

Now one must screen to find which cells contain a gene of interest.

• Grow each cell on an agar plate.

• Each colony has a different recombinant DNA.

• Sample cells by blotting to identify which colonies have the sequence of interest.

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BlottingBlotting

• Apply nitrocellulose paper to the plate to produce an imprint.

• Treat the paper with dilute NaOH to lyse the cells, releasing the DNA which stays on the paper.

• Add a radiolabeled hybridization probe - complementary DNA or RNA molecule.

• Labeled spots on the paper identify which colonies have the sequence of interest.

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Amplification of DNAAmplification of DNA

Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)

• Method is used to make multiple copies of DNA without cloning.

• It requires that at least part of the sequence for a DNA fragment be known.

• The advantage is that the process can be repeated many times by altering temperature.

• The amount of DNA increases exponentially.

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Amplification of DNAAmplification of DNA

Requirements for PCRRequirements for PCR

• Two synthetic oligonucleotide primers of approximately 20 base pairs. They must be complementary to the ‘flanking sequences.”

• Heat stable DNA polymerase.

• All four deoxyribonucleotides as triphosphates.

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Polymerase chain reactionPolymerase chain reaction

I II III IV V

I II III IV V

Targetsequence

Separate strands byheating to 95oC.

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Polymerase chain reactionPolymerase chain reaction

I II III IV V

I II III IV V

Hybridize primerscooling to 54oC.

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Polymerase chain reactionPolymerase chain reaction

I II III IV V

I II III IV V

Synthesize DNA byextending primers at 72oC.

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Application of recombinantDNA technology

Application of recombinantDNA technology

Recombinant protein productsRecombinant protein productsBacteria can serve as factories for various proteins like human insulin.

Bacteria can’t be used for many eukaryotic genes since they are unable to remove introns.

Genetically altered organismsGenetically altered organismsAlter DNA to remove defects or improve quality/quantity of normal products.

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ExamplesExamples

BacteriaBacteriaModified Pseudomonas are being constructed to

degrade hazardous waste.

PlantsPlants Flavr Savr tomato developed by Calgene. It was

altered to inhibit rotting and allow it to ripen longer on the vine.

Animals Animals The rat growth hormone, somatotropin, was added

to a plasmid and added to mouse eggs. Resulted in mice twice normal size. This was transgenic since it was transferred to subsequent generations.

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ExamplesExamples

Human gene therapyHuman gene therapyThere are approximately 4,000 known genetic

diseases.There are several clinical trials ongoing or

planned. They involve:

• Removal of somatic cells from a patient.

• Insertion of a normal gene

• Reintroduction of the cells

Bone marrow, skin or liver cells are typically used.