Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources.
Recombinant DNA and Biotechnology
Recombinant DNA and Biotechnology
Cleaving and Rejoining DNA
Getting New Genes into Cells
Sources of Genes for Cloning
Some Additional Tools for DNA Manipulation
Biotechnology: Applications of DNA Manipulation
Application of Recombinant DNA
Recombinant DNA technology is the manipulation and combination of DNA molecules from different sources.
Humulin & Insulin
Humulin
recombinant human insulin: a form of insulin (trade name Humulin) made from recombinant DNA that is identical to human insulin; used to treat diabetics who are allergic to preparations made from beef or pork insulin - wordnet.princeton.edu/perl/webwn
A polypeptide hormone secreted by the islets of Langerhans and functioning in the regulation of the metabolism of carbohydrates and fats, especially the conversion of glucose to glycogen, which lowers the blood glucose level.
Any of various pharmaceutical preparations containing this hormone that are derived from the pancreas of certain animals or produced through genetic engineering and are used in the medical treatment and management of diabetes mellitus (type I).
- http://www.answers.com/topic/insulin
Tools of Biotechnology
Tools of Biotechnology
Cleaving and Rejoining DNA
Bacteria defend themselves against invasion by viruses by producing restriction enzymes which catalyze the cleavage of DNA into small fragments.
The enzymes cut the bonds between the 3 hydroxyl of one nucleotide, and the 5 phosphate of the next.
There are many such enzymes, each of which recognizes and cuts a specific sequence of bases, called a recognition sequence or restriction site (commonly 4 to 6 base pairs long).
Cleaving and Rejoining DNA
Host DNA is not damaged due to methylation of certain bases at the restriction sites; this is performed by enzymes called methylases.
The enzyme EcoRI cuts
5 ... GAATTC ... 3
3... CTTAAG ... 5
Notice that the sequence is palindromic: It reads the same in the 5-to-3 direction on both strands.
EcoRI
EcoR1 in Action
Sticky End & Blunt Ends
Cleaving and Rejoining DNA
What good are they?
Restriction enzymes serve as “knives” for genetic surgery.
Hundreds of restriction enzymes with different recognition sequences have been purified from various organisms, so the knives come in many forms.
Problem: DNA cut with restriction enzymes creates a collection of fragments all within a microcentrifuge tube. Typically, we want only one of the fragments.
Solution: Gel Electrophoresis
Cleaving and Rejoining DNA
The fragments of DNA can be separated using gel electrophoresis. Because of its phosphate groups, DNA is negatively charged at neutral pH.
When DNA is placed in a semisolid gel and an electric field is applied, the DNA molecules migrate toward the positive pole.
Smaller molecules can migrate more quickly through the porous gel than larger ones.
After a fixed time, the separated molecules can then be stained with a fluorescent dye and examined under ultraviolet light.
Gel Electrophoresis
Electrophoresis gives two types of information:
Size of the DNA fragments can be determined by comparison to DNA fragments of known size added to the gel as a reference.
Southern Hybridization
And…
A specific DNA sequence can be determined by using a complementary labeled single-stranded DNA probe.
Cleaving and Rejoining DNA
SPLICING DNA
Simple techniques give scientists the power to manipulate genetic material; this has revolutionized biological science in the past 30 years.
If two different DNAs are cut so each has sticky ends, fragments with complementary sticky ends can be recombined and sealed with the enzyme DNA ligase.
Gene Splicing
Getting New Genes into Cells
Plasmids are ideal vectors for the introduction of recombinant DNA into bacteria.
Getting New Genes into Cells
A plasmid is small and can divide separately from the host’s chromosome.
They often have just one restriction site, if any, for a given restriction enzyme.
Cutting the plasmid at one site makes it a linear molecule with sticky ends.
If another DNA is cut with the same enzyme, it is possible to insert the DNA into the plasmid.
Plasmids often contain antibiotic resistance genes, which serve as genetic markers.
Inserting a Gene
Getting New Genes into Cells
Only about 10,000 base pairs can be inserted into plasmid DNA, so for most eukaryotic genes a vector that accommodates larger DNA inserts is needed.
For inserting larger DNA sequences, viruses are often used as vectors.
If the genes that cause death and lysis in E. coli are eliminated, the bacteriophage can still infect the host and inject its DNA.
The deleted 20,000 base pairs can be replaced by DNA from another organism, creating recombinant viral DNA.
Getting New Genes into Cells
Bacterial plasmids are not good vectors for yeast hosts because prokaryotic and eukaryotic DNA sequences use different origins of replication.
A yeast artificial chromosome, or YAC, has been made that has a yeast origin of replication, a centromere sequence, and telomeres, making it a true eukaryotic chromosome.
YACs have been engineered to include specialized single restriction sites and selectable markers.
YACs can accommodate up to 1.5 million base pairs of inserted DNA.
Getting New Genes into Cells
Plasmid vectors for plants include a plasmid found in the Agrobacterium tumefaciens bacterium, which causes the tumor-producing disease, crown gall, in plants.
Part of the tumor-inducing (Ti) plasmid of A. tumefaciens is T DNA, a transposon, which inserts copies of itself into the host chromosomes.
If T DNA is replaced with the new DNA, the plasmid no longer produces tumors, but the transposon still can be inserted into the host cell’s chromosomes.
The plant cells containing the new DNA can be used to generate transgenic plants.
Getting New Genes into Cells
When a population of host cells is treated to introduce DNA, just a fraction actually incorporate and express it.
In addition, only a few vectors that move into cells actually contain the new DNA sequence.
Therefore, a method for selecting for transfected cells and screening for inserts is needed.
A commonly used approach to this problem is illustrated using E. coli as hosts, and a plasmid vector with genes for resistance to two antibiotics.
Getting New Genes into Cells
Other methods have since been developed for screening.
The gene for luciferase, the enzyme that makes fireflies glow in the dark, has been used as a reporter gene.
Green fluorescent protein, which is the product of a jellyfish gene, glows without any required substrate.
Cells with this gene in the plasmid grow on ampicillin and glow when exposed to ultraviolet light.
Sources of Genes for Cloning
Gene libraries contain fragments of DNA from an organism’s genome.
Restriction enzymes are used to break chromosomes into fragments, which are inserted into vectors and taken up by host cells.
Sources of Genes for Cloning
Using plasmids for insertion of DNA, about one million separate fragments are required for the human genome library.
Phage , which carries four times as much DNA as a plasmid, is used to hold these random fragments.
It takes about 250,000 different phage to ensure a copy of every sequence.
This number seems large, but just one growth plate can hold as many as 80,000 phage colonies.
Sources of Genes for Cloning
A smaller DNA library can be made from complementary DNA (cDNA).
Oligo dT primer is added to mRNA tissue where it hybridizes with the poly A tail of the mRNA molecule.
Reverse transcriptase, an enzyme that uses an RNA template to synthesize a DNA–RNA hybrid, is then added.
The resulting DNA is complementary to the RNA and is called cDNA. DNA polymerase can be used to synthesize a DNA strand that is complementary to the cDNA.
Sources of Genes for Cloning
If the amino acid sequence of a protein is known, it is possible to synthesize a DNA that can code for the protein.
Using the knowledge of the genetic code and known amino acid sequences, the most likely base sequence for the gene may be found.
Often sequences are added to this sequence to promote expression of the protein.
Human insulin has been manufactured using this approach.
Sources of Genes for Cloning
With synthetic DNA, mutations can be created and studied.
Additions, deletions, and base-pair substitutions can be manipulated and tracked.
The functional importance of certain amino acid sequences can be studied.
The signals that mark proteins for passage through the ER membrane were discovered by site-directed mutagenesis.
Some Additional Tools for DNA Manipulation
Homologous recombination is used to study the role of a gene at the level of the organism.
In a knockout experiment, a gene inside a cell is replaced with an inactivated gene to determine the inactivated gene’s effect.
This technique is important in determining the roles of genes during development.
Some Additional Tools for DNA Manipulation
The emerging science of genomics has to contend with two difficulties:
The large number of genes in eukaryotic genomes
The distinctive pattern of gene expression in different tissues at different times
To find these patterns, DNA sequences have to be arranged in an array on some solid support.
DNA chip technology provides these large arrays of sequences for hybridization.
Some Additional Tools for DNA Manipulation
Analysis of cellular mRNA using DNA chips:
In a process called RT-PCR, cellular mRNA is isolated and incubated with reverse transcriptase (RT) to make complementary DNA (cDNA). The cDNA is amplified by PCR prior to hybridization.
The amplified cDNA is coupled to a fluorescent dye and then hybridized to the chip.
A scanner detects glowing spots on the array. The combinations of these spots differ with different types of cells or different physiological states.
Some Additional Tools for DNA Manipulation
DNA chip technology can be used to detect genetic variants and to diagnose human genetic diseases.
Instead of sequencing the entire gene, it is possible to make a chip with 20-nucleotide fragments including every possible mutant sequence.
Hybridizing that sequence with a person’s DNA may reveal whether any of the DNA hybridized to a mutant sequence on the chip.
Some Additional Tools for DNA Manipulation
Base-pairing rules can also be used to stop mRNA translation.
Antisense RNA is complementary to a sequence of mRNA.
The antisense RNA forms a double-stranded hybrid with an mRNA, which inhibits translation.
These hybrids are broken down rapidly in the cytoplasm, so translation does not occur.
In the laboratory, antisense RNA can be made and added to cells to block translation.
Some Additional Tools for DNA Manipulation
A related technique uses interference RNA (RNAi) which inhibits mRNA translation in the inactive X chromosome of mammals.
Scientists can synthesize a small interfering RNA (siRNA) to inhibit translation of any known gene.
Some Additional Tools for DNA Manipulation
The two-hybrid system allows scientists to test for protein interactions within a living cell.
A two-hybrid system uses a transcription factor that activates the transcription of an easily detectable reporter gene.
This transcription factor has two domains: one that binds to DNA at the promoter, and another that binds to the transcription complex to activate transcription.
An example is the yeast two-hybrid system.
Biotechnology: Applications of DNA Manipulation
Biotechnology is the use of microbial, plant, and animal cells to produce materials—such as foods, medicines, and chemicals—that are useful to people.
The use of yeast to create beer and wine and bacterial cultures to make yogurt and cheese are examples of centuries-old biotechnology.
Gene cloning techniques of modern molecular biology have vastly increased the number of these products beyond those that are naturally made by microbes.
Biotechnology: Applications of DNA Manipulation
Expression vectors are typical vectors, but they also have extra sequences needed for the foreign gene to be expressed in the host cell.
An expression vector might have an inducible promoter, which can be stimulated into expression by responding to a specific signal such as a hormone.
A tissue-specific promoter is expressed only in a certain tissue at a certain time.
Targeting sequences are sometimes added to direct the protein product to an appropriate destination.
Biotechnology: Applications of DNA Manipulation
Many medical products have been made using recombinant DNA technology.
For example, tissue plasminogen activator (TPA), is currently being produced in E. coli by recombinant DNA techniques.
TPA is an enzyme that converts blood plasminogen into plasmin, a protein that dissolves clots.
Recombinant DNA technology has made it possible to produce the naturally occurring protein in quantities large enough to be medically useful.
Biotechnology: Applications of DNA Manipulation
Selective breeding has been used for centuries to improve plant and animal species to meet human needs.
Molecular biology is accelerating progress in these applications.
There are three major advantages over traditional techniques:
Specific genes can be affected.
Genes can be introduced from other organisms.
Plants can be regenerated much more quickly by cloning than by traditional breeding.
Biotechnology: Applications of DNA Manipulation
Insecticides tend to be nonspecific, killing both pest and beneficial insects. They can also be blown or washed away to contaminate and pollute non-target sites.
Bacillus thuringiensis bacteria produce a protein toxin that kills insect larvae pests and is 80,000 times more toxic than the typical chemical insecticide.
Transgenic tomato, corn, potato, and cotton plants have been made that produce a toxin from B. thuringiensis.
Biotechnology: Applications of DNA Manipulation
The process of producing pharmaceuticals using agriculture is nicknamed “pharming.”
Transgenic sheep are being used to produce human a-1-antitrypsin (a-1-AT) in their milk; this protein inhibits the enzyme elastase, which breaks down connective tissue in the lungs. Treatment with a-1-AT alleviates symptoms in people suffering from emphysema.
Other products of “pharming” include blood clotting factors and antibodies for treating colon cancer.
Biotechnology: Applications of DNA Manipulation
Crops that are resistant to herbicides:
Glyphosate (“Roundup”) is a broad-spectrum herbicide that inhibits an enzyme system in chloroplasts that is involved in the synthesis of amino acids.
A bacterial gene, which confers resistance to glyphosate, is inserted into useful food crops (corn, cotton, soybeans) to protect them from the herbicide, which otherwise would kill them along with the weeds.
Biotechnology: Applications of DNA Manipulation
Grains with improved nutritional characteristics:
Genes from bacteria and daffodil plants are transferred to rice using the vector Agrobacterium tumefaciens.
Now a genetically modified strain of rice produces -carotene, a molecule that is converted to vitamin A in animals.
Biotechnology: Applications of DNA Manipulation
Crops that adapt to the environment:
A gene was recently discovered in the thale cress (Arabidopsis thaliana) that allows it to thrive in salty soils.
When this gene is added to tomato plants, they can grow in soils four times as salty as the normal lethal level.
This finding raises the prospect of growing useful crops on previously unproductive soils with high salt concentration.
Biotechnology may allow us to adapt plants to different environments.
Biotechnology: Applications of DNA Manipulation
There is public concern about biotechnology:
Genetically modified E. coli might share their genes with the E. coli bacteria that live normally in the human intestines.
Researchers now take precautions against this. For example, the strains of E. coli used in the lab have a number of mutations that make their survival in the human intestine impossible.
Biotechnology: Applications of DNA Manipulation
There are concerns that genetic manipulation interferes with nature, that genetically altered foods are unsafe, and that genetically altered plants might allow transgenes to escape to other species and thus threaten the environment.
Regarding safety for human consumption, advocates of genetic engineering note that typically only single genes specific for plant function are added.
As plant biotechnology moves from adding genes to improve plant growth to adding genes that affect human nutrition, such concerns will become more pressing.
Biotechnology: Applications of DNA Manipulation
The risks to the environment are more difficult to assess.
Transgenic plants undergo extensive field testing before they are approved for use, but the complexity of the biological world makes it impossible to predict all potential environmental effects of transgenic organisms.
Because of the potential benefits of agricultural biotechnology, most scientists believe we should proceed, but with caution.
Biotechnology: Applications of DNA Manipulation
With the exception of identical twins, each human being is genetically distinct from all other human beings.
Characterization of an individual by DNA base sequences is called DNA fingerprinting.
Biotechnology: Applications of DNA Manipulation
Scientists look for DNA sequences that are highly polymorphic.
Sequences called VNTRs (variable number of tandem repeats) are easily detectable if they are between two restriction enzyme recognition sites.
Different individuals have different numbers of repeats. Each gets two sequences of repeats, one from the mother and one from the father.
Using PCR and gel electrophoresis, patterns for each individual can be determined.
Biotechnology: Applications of DNA Manipulation
The many applications of DNA fingerprinting include forensics and cases of contested paternity.
DNA from a single cell is sufficient to determine the DNA fingerprint because PCR can amplify a tiny amount of DNA in a few hours.
PCR is used in diagnosing infections in which the infectious agent is present in small amounts.
Genetic diseases such as sickle-cell anemia are now diagnosable before they manifest themselves.
Getting New Genes into Cells
The goal of recombinant DNA work is to produce many copies (clones) of a particular gene.
To make protein, the genes must be introduced, or transfected, into a host cell.
The host cells or organisms, referred to as transgenic, are transfected with DNA under special conditions.
The cells that get the DNA are distinguished from those that do not by means of genetic markers, called reporter genes.
Getting New Genes into Cells
Bacteria have been useful as hosts for recombinant DNA.
Bacteria are easy to manipulate, and they grow and divide quickly.
They have genetic markers that make it easy to select or screen for insertion.
They have been intensely studied and much of their molecular biology is known.
Getting New Genes into Cells
Bacteria have some disadvantages as well.
Bacteria lack splicing machinery to excise introns.
Protein modifications, such as glycosylation and phosphorylation, fail to occur as they would in a eukaryotic cell.
In some applications, the expression of the new gene in a eukaryote (the creation of a transgenic organism) is the desired outcome.
Getting New Genes into Cells
Saccharomyces, baker’s and brewer’s yeast, are commonly used eukaryotic hosts for recombinant DNA studies.
In comparison to many other eukaryotic cells, yeasts divide quickly, they are easy to grow, and have relatively small genomes (about 20 million base pairs).
Getting New Genes into Cells
Plants are also used as hosts if the goal is to make a transgenic plant.
It is relatively easy to regenerate an entire plant from differentiated plant cells because of plant cell totipotency.
The transgenic plant can then reproduce naturally in the field and will carry and express the gene on the recombinant DNA.
Getting New Genes into Cells
New DNA can be introduced into the cell’s genome by integration into a chromosome of the host cell.
If the new DNA is to be replicated, it must become part of a segment of DNA that contains an origin of replication called a replicon, or replication unit.
Getting New Genes into Cells
New DNA can be incorporated into the host cell by a vector, which should have four characteristics:
The ability to replicate independently in the host cell
A recognition sequence for a restriction enzyme, permitting it to form recombinant DNA
A reporter gene that will announce its presence in the host cell
