Recombinant DNATechnology

Deqiao Sheng PhDDept. Of Biochemistry and Mole

cular Biology

Reference Books

1. Hortons’ Principles of Biochemistry 4th edition

2. Molecular Biology, Robert Weaver 2nd edition

3. Harpers’ Biochemistry 26th edition

4. Styers’ Biochemistry

Recombinant DNA and Gene Cloning

Recombinant DNA (rDNA) is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing.

Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future.

Chinese Dragon

龙

The dragon is a mythical creature that can fly and walk. Dragon can change its form and has divine powers to summon wind and rain.

The dragons are said to be made up of many different types of animals of the Earth.

The Nine parts of A Chinese Dragon 

Dragon is an imagination creature, which has deer's antlers, camel's head, hare's eye, snake's neck, carp's scales, eagle's claws, tiger's paws; and ox's ears.

Recombinant DNA:Cloning and Creation of Chimeric Genes

Recombinant DNA technology is one of the recent advances in

biotechnology, which was developed by two scientists named

Boyer and Cohen in 1973.

Stanley N. Cohen (1935–) (top) and Herbert Boyer (1936–) (bottom), who constructed the first recombinant DNA using bacterial DNA and plasmids.

Stanley N. Cohen , who received the Nobel Prize in Medicine in 1986 for his work on discoveries of growth factors.

What is Recombinant DNA Technology?

Recombinant DNA technology is a technology which allows DNA to be produced via artificial means.

The procedure has been used to change DNA in living organisms and may have even more practical uses in the future.

It is an area of medical science that is just beginning to be researched in a concerted effort.

Recombinant DNA technology works by taking DNA from two different sources and combining that DNA into a single molecule. That alone, however, will not do much.

Recombinant DNA technology only becomes useful when that artificially-created DNA is reproduced. This is known as DNA cloning.

Brief Introduction

Recombinant DNA Technology

1. The basic concepts for recombinant DNA technology

2. The basic procedures of recombinant DNA technology

3. Application of recombinant DNA technology

The basic concepts for recombinant DNA technology

In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules composed of nucleotide sequences taken from different sources. The products of these innovations, recombinant DNA molecules, opened exciting new avenues of investigation in molecular biology and genetics, and a new field was born— recombinant DNA technology.

Concept of Recombinant DNA Recombinant DNA is a molecule that combines

DNA from two sources . Also known as gene cloning.

Creates a new combination of genetic material – Human gene for insulin was placed in bacteria– The bacteria are recombinant organisms and

produce insulin in large quantities for diabetics – Genetically engineered drug in 1986

Genetically modified organisms are possible because of the universal nature of the genetic code!

Genetic engineering is the application of this technology to the manipulation of genes. These advances were made possible by methods for amplification of any particular DNA segment( how? ), regardless of source, within bacterial host cells. Or, in the language of recombinant DNA technology, the cloning of virtually any DNA sequence became feasible.

Recombinant technology begins with the isolation of a gene of interest (target gene). The target gene is then inserted into the plasmid or phage (vector) to form replicon.

The replicon is then introduced into host cells to cloned and either express the protein or not.

The cloned replicon is referred to as recombinant DNA. The procedure is called recombinant DNA technology. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells.

Some other terms are also in common use to describe genetic engineering. Gene manipulation Recombinant DNA technology Gene cloning (Molecular cloning) Genetic modification

Cloning——In classical biology, a clone is a population of identical organisms derived from a single parental organism. For example, the members of a colony of bacter

ial cells that arise from a single cell on a petri plate are clones. Molecular biology has borrowed the term to mean a collection of molecules or cells all identical to an original molecule or cell.

Recombinant DNA technology——A series of procedures used to join together (recombine) DNA segments. A recombinant DNA molecule is constructed (recombined) from segments from 2 or more different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate there, autonomously (on its own) or after it has become integrated into a chromosome.

How recombinant technology works

These steps include isolating of the target gene and the vector, specific cutting of DNA at defined sites, joining or splicing of DNA fragments, transforming of replicon to host cell, cloning, selecting of the positive cells containing recombinant DNA, and either express or not in the end.

Six steps of Recombinant DNA

1. Isolating (vector and target gene)

2. Cutting (Cleavage)

3. Joining (Ligation)

4. Transforming

5. Cloning

6. Selecting (Screening)

Recombinant DNA Technology

1. The basic concepts for recombinant DNA technology

2. The basic procedures of recombinant DNA technology

3. Application of recombinant DNA technology

The basic procedures of recombinant DNA technology

DNA molecules that are constructed with DNA from different sources are called recombinant DNA molecules.

Recombinant DNA molecules are created in nature more often than in the laboratory;– for example, every time a bacteria phage or

eukaryotic virus infects its host cell and integrates its DNA into the host genome, a recombinant is created.

– Occasionally, these viruses pick up a fragment of host DNA when they excise from their host’s genome; these naturally occurring recombinant DNA molecules have been used to study some genes.

Six basic steps are common to most recombinant DNA experiments

1. Isolation and purification of DNA.

Both vector and target DNA molecules can be prepared by a variety of routine methods, which are not discussed here. In some cases, the target DNA is synthesized in vitro.

2. Cleavage of DNA at particular sequences. As we will see, cleaving DNA to generate fragments of defined length, or with specific endpoints, is crucial to recombinant DNA technology. The DNA fragment of interest is called insert DNA. In the laboratory, DNA is usually cleaved by treating it with commercially produced nucleases and restriction endonucleases.

3. Ligation of DNA fragments.

A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and then ligating the insert DNA to vector DNA (recombinant DNA or chimeric DNA). DNA fragments are typically joined using DNA ligase (also commercially produced).– T4 DNA Ligase

4. Introduction of recombinant DNA into compatible host cells. In order to be propagated, the recombinant DNA molecule (insert DNA joined to vector DNA) must be introduced into a compatible host cell where it can replicate. The direct uptake of foreign DNA by a host cell is called genetic transformation (or transformation). Recombinant DNA can also be packaged into virus particles and transferred to host cells by transfection.

5. Replication and expression of recombinant DNA in host cells.

Cloning vectors allow insert DNA to be replicated and, in some cases, expressed in a host cell. The ability to clone and express DNA efficiently depends on the choice of appropriate vectors and hosts.

6. Identification of host cells that contain recombinant DNA of interest. Vectors usually contain easily scored genetic markers, or genes, that allow the selection of host cells that have taken up foreign DNA. The identification of a particular DNA fragment usually involves an additional step—screening a large number of recombinant DNA clones. This is almost always the most difficult step.

DNA cloning in a plasmid vector permits amplification of a DNA fragment.

First step:Isolating DNA

1. Vector

2. Target gene

How to get a target genes?

1. Genomic DNA

2. Artificial synthesis

3. PCR amplification

4. RT-PCR

Polymerase chain reaction (PCR)

A technique called the polymerase chain reaction (PCR) has revolutionized recombinant DNA technology. It can amplify DNA from as little material as a single cell and from very old tissue such as that isolated from Egyptian mummies, a frozen mammoth, and insects trapped in ancient amber.

method is used to amplify DNA sequences

The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube

Number of DNA molecules

InitialDNAsegment

PCR primers

RT-PCR

Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR.

In RT-PCR, however, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional. – Template:RNA

– Products: cDNA

Vectors- Cloning Vehicles

Cloning vectors can be plasmids, bacteriophage, viruses, or even small artificial chromosomes. Most vectors contain sequences that allow them to be replicated autonomously within a compatible host cell, whereas a minority carry sequences that facilitate integration into the host genome.

All cloning vectors have in common at least one unique cloning site, a sequence that can be cut by a restriction endonuclease to allow site-specific insertion of foreign DNA. The most useful vectors have several restriction sites grouped together in a multiple cloning site (MCS) called a polylinker.

Types of vector

1. Plasmid Vectors

2. Bacteriophage Vectors

3. Virus vectors

4. Shuttle Vectors--can replicate in either prokaryotic or eukaryotic cells.

5. Yeast Artificial Chromosomes as Vectors

Plasmid Vectors

Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA.

These extra chromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or symbiotic relationship with their host cell.

Plasmid

Plasmids can replicate autonomously within a host, and they frequently carry genes conferring resistance to antibiotics such as tetracycline, ampicillin, or kanamycin. The expression of these marker genes can be used to distinguish between host cells that carry the vectors and those that do not

pBR322 pBR322 was one of the first versatile plasmid vect

ors developed; it is the ancestor of many of the common plasmid vectors used in biochemistry laboratories.

pBR322 contains an origin of replication (ori) and a gene (rop) that helps regulate the number of copies of plasmid DNA in the cell. There are two marker genes: confers resistance to ampicillin, and confers resistance to tetracycline. pBR322 contains a number of unique restriction sites that are useful for constructing recombinant DNA.

pBR322

1. Origin of replication

2. Selectable marker

3. unique restriction sites

Enzymes

Restriction Enzymes and DNA Ligases Allow Insertion of DNA Fragments into Cloning Vectors

1. Restriction endonuclease, RE2. DNA ligase3. Reverse transcriptase4. DNA polymerase, DNA pol5. Nuclease6. Terminal transferase

Restriction enzymes cleave DNA The same sequence of bases is

found on both DNA strands, but in opposite orders. GAATTC

CTTAAG This arrangement is called a

palindrome. Palindromes are words or sentences that read the same forward and backward.

form sticky ends: single stranded ends that have a tendency to join with each other ( the key to recombinant DNA)

Restriction Enzymes Cut DNA Chains at Specific Locations

Restriction enzymes are endonucleases produced by bacteria that typically recognize specific 4 to 8bp sequences, called restriction sites, and then cleave both DNA strands at this site.

Restriction sites commonly are short palindromic sequences; that is, the restriction-site sequence is the same on each DNA strand when read in the 5′ → 3′ direction.

Cut out the gene

Restriction enzymes

Restriction enzymes Restriction enzymes are named after the bact

erium from which they are isolated – For example, Eco RI is from Escherichia coli, a

nd Bam HI is from Bacillus amyloliquefaciens . The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co). These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discovery (eg, EcoRI, EcoRII).

Blunt ends or sticky ends

Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is 4–7 bp long. These DNA cuts result in blunt ends (eg, Hpa I) or overlapping (sticky) ends (eg, BamH I) , depending on the mechanism used by the enzyme.

Sticky ends are particularly useful in constructing hybrid or chimeric DNA molecules .

Results of restriction endonuclease digestion. Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive ends (A) or blunt ends (B). This is an important consideration in devising cloning strategies.

Inserting DNA Fragments into Vectors

DNA fragments with either sticky ends or blunt ends can be inserted into vector DNA with the aid of DNA ligases.

For purposes of DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complementary ends . The vector DNA and restriction fragment are covalently ligated together through the standard 3 → 5 phosphodiester bonds of DNA.

DNA ligase “pastes” the DNA fragments together

Ligation of restriction fragments with complementary sticky ends.

Identification of Host Cells Containing Recombinant DNA

Once a cloning vector and insert DNA have been joined in vitro, the recombinant DNA molecule can be introduced into a host cell, most often a bacterial cell such as E. coli.

In general, transformation is not a very efficient way of getting DNA into a cell because only a very small percentage of cells take up recombinant DNA. Consequently, those cells that have been successfully transformed must be distinguished from the vast majority of untransformed cells.

Identification of host cells containing recombinant DNA requires genetic selection or screening or both.

In a selection, cells are grown under conditions in which only transformed cells can survive; all the other cells die.

In contrast, in a screen, transformed cells have to be individually tested for the presence of the desired recombinant DNA.

Normally, a number of colonies of cells are first selected and then screened for colonies carrying the desired insert.

Selection Strategies Use Marker Genes(Primary screening)

Many selection strategies involve selectable marker genes— genes whose presence can easily be detected or demonstrated. ampR

Selection or screening can also be achieved using insertional inactivation.

A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a piece of DNA is inserted into the unique PstI site. This insertion disrupts the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert.

insertional inactivation

Screening (Strategies)

1. Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments

2. Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method

3. The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture

4. Blotting Techniques Permit Detection of Specific DNA Fragments and mRNAs with DNA Probes

bp—1534

— 994

— 695

— 515

— 377

— 237

A B C M

Gel Electrophoresisnegative charged DNA run to the anode

Sequencing results

Southern blot technique can detect a specific DNA fragment in a complex mixture of restriction fragments.

Radioactive isotope

Hybridization

Types of blotting techniques

Southern blotting Southern blotting techniques is the first nucleic acid

blotting procedure developed in 1975 by Southern. Southern blotting is the techniques for the specific

identification of DNA molecules. Northern blotting

Northern blotting is the techniques for the specific identification of RNA molecules.

Western blotting Western blotting involves the identification of proteins. Antigen + antibody

Expression of Proteins Using Recombinant DNA Technology

Cloned or amplified DNA can be purified and sequenced, used to produce RNA and protein, or introduced into organisms with the goal of changing their phenotype.

One of the reasons recombinant DNA technology has had such a large impact on biochemistry is that it has overcome many of the difficulties inherent in purifying low-abundance proteins and determining their amino acid sequences.

Recombinant DNA technology allows the protein to be purified without further characterization. Purification begins with overproduction of the protein in a cell containing an expression vector.

– Prokaryotic Expression Vectors

– Eukaryotic Expression Vectors

Prokaryotic Expression Vectors

Expression vectors for bacterial hosts are generally plasmids that have been engineered to contain appropriate regulatory sequences for transcription and translation such as strong promoters, ribosome-binding sites, and transcription terminators.

Eukaryotic proteins can be made in bacteria by inserting a cDNA fragment into an expression vector . Large amounts of a desired protein can be purified from the transformed cells.

In some cases, the proteins can be used to treat patients with genetic disorders. For example, human growth hormone, insulin, and seve

ral blood coagulation factors have been produced using recombinant DNA technology and expression vectors.

Expression of Proteins in Eukaryotes

Prokaryotic cells may be unable to produce functional proteins from eukaryotic genes even when all the signals necessary for gene expression are present because many eukaryotic proteins must be post-translationally modified.

Several expression vectors that function in eukaryotes have been developed.

These vectors contain eukaryotic origins of replication, marker genes for selection in eukaryotes, transcription and translation control regions, and additional features required for efficient translation of eukaryotic mRNA, such as polyadenylation signals and capping sites.

商业化的载体

Recombinant DNA Technology

1. The basic concepts for recombinant DNA technology

2. The basic procedures of recombinant DNA technology

3. Application of recombinant DNA technology

Applications of Recombinant DNA Technology

1. Analysis of Gene Structure and Expression

2. Pharmaceutical Products– Drugs– Vaccines

3. Genetically modified organisms (GMO)

– Transgenic plants– Transgenic animal

4. Application in medicine

5.

Analysis of Gene Structure and Expression

Using specialized recombinant DNA techniques, researchers have determined vast amounts of DNA sequence including the entire genomic sequence of humans and many key experimental organisms. This enormous volume of data, which is growing at a rapid pace, has been stored and organized in two primary data banks: the GenBank at the National Institutes of Health, Bethes

da, Maryland, and the EMBL Sequence Data Base at the European Mo

lecular Biology Laboratory in Heidelberg, Germany.

Pharmaceutical Products

Some pharmaceutical applications of DNA technology: Large-scale production of human hormones a

nd other proteins with therapeutic uses Production of safer vaccines

A number of therapeutic gene products —insulin, the interleukins, interferons, growth hormones, erythropoietin, and coagulation factor VIII—are now produced commercially from cloned genes

Pharmaceutical companies already are producing molecules made by recombinant DNA to treat human diseases.

Recombinant bacteria are used in the production of human growth hormone and human insulin

Use recombinant cells to mass produce proteins– Bacteria

– Yeast

– Mammalian

• Insulin– Hormone required to

properly process sugars and fats

– Treat diabetes – Now easily produced by

bacteria

• Growth hormone deficiency– Faulty pituitary and

regulation– Had to rely on cadaver

source– Now easily produced by

bacteria

Subunit Herpes Vaccine

Not always used for good...

• High doses of HGH can cause permanent side effects– As adults normal growth

has stopped so excessive GH can thicken bones and enlarge organs

Genetically modified organisms (GMO)

Use of recombinant plasmids in agriculture– plants with genetically desirable

traits• herbicide or pesticide resistant corn

& soybean– Decreases chemical insecticide use

– Increases production

• “Golden rice” with beta-carotene– Required to make vitamin A, which in

deficiency causes blindness

Crops have been developed that are better tasting, stay fresh longer, and are protected from disease and insect infestations.

“Golden rice” has been genetically modified to contain beta-carotene

Genetic Engineering of Plants

Plants have been bred for millennia to enhance certain desirable characteristics in important food crops.

Transgenic plants.

The luciferase gene from a firefly is transformed into tobacco plant using the Ti plasmid. Watering the plant with a solution of luciferin (the substrate for firefly luciferase) results in the generation of light by all plant tissues.

Insect-resistant tomato plantsThe plant on the left contains a gene that encodes a bacterial protein that is toxic to certain insects that feed on tomato plants. The plant on the right is a wild-type plant. Only the plant on the left is able to grow when exposed to the insects.

Transgenic animals

Green fluorescence Red fluorescence

Transgenic animals

A transgenic mouse

Mouse on right is normal; mouse on left is transgenic animal expressingrat growth hormone

Trangenic plants and animals have genes from other organisms.

Farm Animals and “Pharm” Animals

These transgenic sheep carry a gene for a human blood protein– This protein may help in

the treatment of cystic fibrosis

just a joke

Other benefits of GMOs

Disease resistance There are many viruses, fungi, bacteria that cause plant

diseases “Super-shrimp”

Cold tolerance Antifreeze gene from cold water fish introduced to

tobacco and potato plants

Drought tolerance & Salinity tolerance As populations expand, potential to grow crops in

otherwise inhospitable environments

Where in the world?

Downsides???

Introduce allergens? Pass trans-genes to

wild populations?– Pollinator transfer

R&D is costly– Patents to insure

profits• Patent infringements• Lawsuits• potential for capitalism

to overshadow humanitarian efforts

Application in medicine

Human Gene Therapy Diagnosis of genetic disorders Forensic Evidence

Human Gene Therapy

Human gene therapy seeks to repair the damage caused by a genetic deficiency through introduction of a functional version of the defective gene. To achieve this end, a cloned variant of the gene must be incorporated into the organism in such a manner that it is expressed only at the proper time and only in appropriate cell types. At this time, these conditions impose serious technical and clinical difficulties.

Gene therapy is the alteration of an afflicted individual’s genes

Gene therapy holds great potential for treating disorders traceable to a single defective gene

Vectors are used for delivery of genes into cells Gene therapy raises ethical questions, such as

whether human germ-line cells should be treated to correct the defect in future generations

Many gene therapies have received approval from the National Institutes of Health for trials in human patients, including the introduction of gene constructs into patients. Among these are constructs designed to cure ADA- SCID (severe combined immunodeficiency due to adenosine deaminase [ADA] deficiency), neuroblastoma, or cystic fibrosis, or to treat cancer through expression of the E1A and p53 tumor suppressor genes.

Cloned gene

Retroviruscapsid

Bonemarrowcell frompatient

Inject engineeredcells into patient.

Insert RNA version of normal alleleinto retrovirus.

Viral RNA

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Bonemarrow

Somatic cells Only!

Not for reproductive cells !!

However, there are some challenging issues that need to be considered:

1. In mammalian cells, mRNA is processed before it is translated into a protein:

– Introns are cut out and exons are spliced together

– Bacteria can not process mRNA

2. Post-translational modifications– Enzymatic modifications of protein molecul

es after they are synthesized in cells– Post-translational modifications include:

• Disulfide bond formation (catalyzed by disulfide isomerases) and protein folding

• Glycosylation (addition of sugar molecules to protein backbone, catalyzed by glycosyl transferases)

• Proteolysis (clipping of protein molecule, e.g., processing of proinsulin to insulin)

• Sulfation, phosphorylation (addition of sulfate, phosphate groups)

3. Recombinant proteins are particularly susceptible to proteolytic degradation in bacteria

4. Recombinant protein may accumulate in bacteria as refractile inclusion bodies

So how can these problems be tackled?

Problem:

1. mRNA processing in mammalian cells but not in bacteria

Solution:• Synthesize chemically gene containing only exon

s and insert that into vector; • or, Make cDNA by reverse-transcription of proc

essed mRNA (using the enzyme reverse transcriptase)

Problem:2. Bacteria cannot perform post-translationa

l modificationsSolution:• This is a tough one! Only proteins that do

not undergo extensive post-translational processing can be synthesized in bacteria

Problem:3. Recombinant proteins particularly susceptible to

proteolysisSolution:• Design fusion protein consisting of an

endogenous bacterial protein connected to the recombinant protein through a specific amino acid sequence. Fusion protein is then specifically cleaved at the fusion site

The Hope

Summary

1. Recombinant DNA technology builds on a few basic techniques: isolation of DNA, cleavage of DNA at particular sequences, ligation of DNA fragments, introduction of DNA into host cells, replication and expression of DNA, and identification of host cells that contain recombinants.

2. DNA Fragments generated by restriction endonucleases can be ligated into a wide range of cloning vectors, including: plasmids, bacteriophage, viruses, or artificial chromosomes.

3. Cells containing recombinant DNA molecules can be selected, often by the activity of a marker gene. Cells containing the desired recombinant are identified by screening.

4. The product of a gene that has been incorporated into an appropriate expression vector can be generated in prokaryotic or eukaryotic cells. Foreign genes can also be stably incorporated into the genomes of animals and plants.

5. Recombinant DNA methods allow the production of proteins for therapeutic use and the identification of individuals with genetic defects.

REVIEW QUESTIONS

Choose the ONE BEST answer or completion.

Plasmids used as cloning vectorsA. are circular molecules.

B. have an origin of replication.

C. carry antibiotic resistance genes.

D. have unique restriction endonuclease cutting sites.

E. all of the above.