RECOMBINANT DNA What is genetic recombinant Study about

RECOMBINANT DNA

What is genetic recombinant? • Study about: • Genetic exchange of segment of DNA. • The ability to manipulate the structure of DNA/genome of organism. • Multiple procedure or technique were use to manipulate the genome of organism.

An overview of recombinant DNA technology

The Tools of Recombinant DNA Technology • Tools of recombinant DNA technology include • mutagens, • reverse transcriptase, • synthetic nucleic acids, • restriction enzymes, and • vectors. • To create gene libraries, which are a time saving tool for genetic researchers.

Restriction enzymes (RE) • Function of RE: • cut DNA molecules on specific site • In nature, for protection from phages by cutting phage DNA into nonfunctional pieces. • Namely of RE: • Normally with three/fourth letters • denoting the genus and specific epithet of the source bacterium • Roman numerals : I, III, IV etc (to indicate the order in which enzymes from the same bacterium were discovered). • Example: • Escherichia coli strain R : produces the restriction enzymes Eco. RI and Eco. RII. • Hind. III is the third restriction enzyme isolated from Haemophilus influenzae strain Rd.

Type of RE Type 1: Sticky End Type 2: Blunt End Eco. RI: Cuts of the two strands of DNA, producing fragments that terminate in mortise like sticky ends. Hind. II and Smal: Cuts both strands of DNA at the same point, resulting in blunt ends Sticky end is composed of up to four nucleotides More difficult to make recombinant DNA from blunt ended fragments Bind to complementary sequence of insert/target gene: very specific This enables any blunt ended fragments, even those produced by different restriction enzymes, to be combined easily DNA ligase unites the sugar phosphate backbones of vector and target gene Covalent bond and DNA ligase unites the sugar phosphate backbones of vector and target gene

Type 1: Sticky end

Type 2: Blunt end

• Table 8. 1 identifies several restriction enzymes and their target DNA sequences.

Vector • Vectors are use to deliver a gene into a cell • Vectors are nucleic acid molecules such as viral genomes, transposons and plasmids.

Genetic vectors share several useful properties: • Vectors are small enough to manipulate in a laboratory • Vectors survive inside cells • Vectors contain a recognizable genetic marker • Vectors can ensure genetic expression

Recombinant process used to produce a vector containing a specific gene (insulin).

• After a given restriction enzyme cuts both the DNA molecule containing the gene of interest (in this example, the insulin gene) and the vector DNA (here a plasmid containing a gene for antibiotic resistance (ampr /kanr) • 1) DNA ligase anneals the vector and insert gene to produce a recombinant plasmid. • 2) By using mechanical pressure, recombinant plasmid has been inserted into a bacterial cell • 3) The bacteria are grown on a medium containing the antibiotic • 4) only those cells that contain the recombinant plasmid (and thus insulin gene as well) can grow on the medium: Screening method

Techniques of Recombinant DNA Technology • The tools of recombinant DNA technology were use in a number of basic techniques to • • • Multiply Identify Manipulate Isolate Mapping and sequence the nucleotides of gene? ?

Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction (PCR) • Biotechnology technique to produce a large number of identical molecules of DNA in vitro. • Using PCR, by using a single molecule of DNA and generate billions of exact replicate within hours (2 4 hours). • Such rapid amplification of DNA is critical in a variety of situations.

• PCR is a repetitive process that alternately separates and replicates the two strands of DNA. • Each cycle of PCR consists of the following three steps: • Denaturation. • Priming. • Extension.

A PCR mixture containing: • Recombinant plasmid (vector + target gene) • DNA primers (short nucleotide, 15 18 nucleotide) and this primer are complementary to start codon, Met near the stop codon of the target DNA), • DNA polymerase, • an abundance of the four deoxyribonucleotide triphosphates (A, T, G, and C) • Distilled water , d. H 2 O • 1 st Step: Denaturation. • Exposure to heat (about 94°C) • To open/separates the two strands of the target DNA by breaking the hydrogen bonds between base pairs. • Otherwise leaves the two strands unaltered.

• 2 nd Step: Annealing. • This mixture is then cooled to about 65°C, enabling double stranded DNA to reform. • Because there is an excess of primers, single strands are more likely to bind to a primer than to one another. • The primers provide DNA polymerase with the 3' hydroxyl group it requires for DNA synthesis. • 3 rd Step: Extension. • Raising the temperature to about 72°C increases the rate at which DNA polymerase replicates each strand to produce more DNA

• These steps are repeated over and over, so the number of DNA molecules increases exponentially 10 n. • After only 30 cycles which requires only a few hours to complete • PCR produces over 1 billion identical copies of the original DNA molecule.

• The process can be automated using a thermocycler, • a device that automatically performs PCR by continuously cycling all the necessary reagents • DNA polymerase, • primers, and • triphosphate deoxynucleotides • through the three temperature regimes. • A thermocycler uses DNA polymerase derived from hyperthermophilic archaea such as Thermus aquaticus. • This enzyme is not denatured at 94°C, so the machine need not be replenished with DNA polymerase after each cycle.

Genetic recombination and transfer • Horizontal gene transfer • A donor cell contributes part of its genom to a recipient cell which may be of a different species or even a different genus from the donor. • 3 types • Transformation • Transduction • Bacterial conjugation (animation)

Separating DNA Molecules: Gel Electrophoresis and the Southern Blot • Electrophoresis: • technique for separating molecules based on their electrical charge, • • • size, and shape. Use to isolate fragments of DNA molecules that can then be inserted into vectors, multiplied by PCR, or preserved in a gene library. DNA molecules, which have an overall negative charge, are drawn through a semisolid gel by an electric current toward the positive electrode within an electrophoresis chamber. The gel is typically composed of a purified sugar component of agar, called agarose. Agarose acts as a molecular sieve that retards the movement of DNA fragments down the chamber and separates the fragments by size. Smaller DNA fragments move faster and farther than larger ones. The size of a fragment can determine by comparing the distance it travels to the distances traveled by standard DNA fragments of known sizes, Marker.

• Southern Blot: • DNA probes allow us to find specific DNA sequences such as genes in a cell. • Use probes to localize specific sequences in electrophoresis gels, but because gels are flimsy, easily broken, and deform as they dry, it is difficult to probe gels.

• In 1975, Ed Southern (1938) devised a method, called the Southern blot, to transfer DNA from agarose gels to nitrocellulose membranes(PDMF), which are less delicate. • The Southern blot technique begins with the procedures of gel electrophoresis just described. • Once the DNA fragments have been separated by size, the liquid in the electrophoresis gel is blotted out, the DNA is denatured with Na. OH, and its single strands are transferred and bonded to a nitrocellulose membrane. • Radioactive probes are used to localize DNA sequences of inter est in the membrane. • Southern blots were use for a variety of purposes, including genetic ‘fingerprinting’ and diagnosis of infectious diseases. For example, we can detect the presence of genetic sequences unique to hepatitis B virus in a blood sample of an infected patient even before the patient shows symptoms or an immune response. • Southern blotting also were use to demonstrate the incidence and prevalence in an environmental sample of archaea, bacteria, and viruses, particularly those that cannot be cultured. Most microorganisms have never been grown in a laboratory; indeed, scientists know them only by unique DNA patterns in electrophoresis gels and Southern blot membranes, called their DNA fingerprints or ‘signatures’. • A northern blot is a similar technique used to detect specific RNA molecules, whereas western blot used to detect specific protein.

DNA Microarrays • A recent advance in biotechnology is the development of DNA micro arrays (DNA chips). An array consists of molecules of single stranded DNA either genetic DNA or c. DNA immobilized on glass slides, silicon chips, or nylon membranes. • Robots, similar to those that construct computer chips, deposit PCR derived copies of hundreds of thousands of different DNA sequences in precise locations on the array. • An array may consist of DNA from a single species (for example, DNA micro arrays containing sequences from all the genes of E. coli are available commercially), or a DNA array may contain sequences from numerous species. In any case, single strands of fluorescently labeled DNA in a sample washed over an array adhere only to locations on the array where there are complementary DNA sequences.

DNA micro arrays were used in a number of ways, including: • Monitoring gene expression • Diagnosis of infection. • Identification of organisms in an environmental sample.

Inserting DNA into Cells • A goal of recombinant DNA technology is the insertion of a gene into a cell, a process also known as transformation. • In addition to using vectors and the natural methods of trans formation of competent cells, transduction, and conjuga tion, there also several artificial methods that have been developed to introduce DNA into cells, including: • Electroporation • Protoplast fusion • Injection

Electroporation • Electroporation involves using an electrical current to puncture microscopic holes through a cell's membrane so that DNA can enter the cell from the environment. • Electroporation can be used on all types of cells, though the thick walled cells of fungi and algae must first be converted to protoplasts, which are cells whose cell walls have been enzymatically removed. • Cells treated by electroporation repair their membranes and cell walls after a time.

Protoplast fusion • When protoplasts encounter one another, their cytoplasmic membranes may fuse to form a single cell that contains the genomes of both "parent" cells. • Exposure to polyethylene glycol, PG 5000/PG 8000 increases the rate of fusion. The DNA from the two fused cells recombines to form a recombinant molecule. • Scientists often use protoplast fusion for the genetic modification of plants.

Injection • Two types of injection are commonly used with larger eukaryotic cells. • Researchers use a gene gun powered by a blank. 22 caliber cartridge or compressed gas to fire tiny tungsten or gold beads coated with DNA into a target cell. • The cell eventually eliminates the inert metal beads. • In microinjection, a geneticist inserts DNA into a target cell with a glass micropipette having a tip diameter smaller than that of the cell or nucleus. • Unlike electroporation and protoplast fusion, injection can be used on intact tissues such as in plant seeds.

• In every case, foreign DNA that enters a cell remains in a cell's progeny only if the DNA is self replicating, as in the case of plasmid and viral vectors, or if the DNA integrates into a cellular chromosome by recombination.

Applications of Recombinant DNA Technology • Genomics is the sequencing (genetic mapping), analysis, and comparison of genomes. Genetic sequencing has been speeded up by an automated machine that distinguishes among fluorescent dyes attached to each type of nucleotide base. • Scientists synthesize subunit vaccines by introducing genes for a pathogen's polypeptides into cells or viruses. When the cells, the viruses, or the polypeptides they produce are injected into a human, the body's immune system is exposed to and reacts against relatively harmless antigens instead of the potentially harmful pathogen. • Genetic screening can detect infections and inherited diseases before a patient shows any sign of disease. • Genetic fingerprinting (DNA fingerprinting), which identifies unique sequences of DNA, is used in paternity investigations, crime scene forensics, diagnostic microbiology, and epidemiology.

• Gene therapy cures various diseases by replacing defective genes with normal genes. • In xenotransplants involving recombinant DNA technology, human genes would be inserted into animals to produce cells, tissues, or organs for introduction into the human body. • Transgenic plants and animals have been genetically altered by the inclusion of genes from other organisms. • Agricultural uses of recombinant DNA technology include advances in herbicide resistance, salt tolerance, freeze resistance, and pest resistance, as well as improvements in nutritional value and yield.

The Ethics and Safety of Recombinant DNA Technology • The procedures of recombinant DNA technology provide the opportunity to transfer genes among unrelated organisms, even among organisms in different kingdoms, but how safe and ethical are they? "Super broccoli, " "franken food, " "biological Russian roulette, " and" designer humans" are some of the terms opponents use to denigrate gene therapy and transgenic agricultural products. • Some opponents question the ethics of raising genetically altered animals solely for creating products for human use. They contend that this exemplifies a supremacist view the view that humans are of greater intrinsic value than animals. • Other critics of transgenic crops and animals correctly state that the long term effects of transgenic manipulations are unknown, and that unforeseen problems arise from every new technology and procedure. Recombinant DNA technology may burden society with complex and as yet unforeseen regulatory, administrative, financial, legal, social, and environmental problems.

• Critics also argue that natural genetic transfer through sexual reproduction and processes such as transformation and transduction could deliver genes from transgenic plants and animals into other organisms. • For example, if a herbicide resistant plant cross pollinates with a related weed species, we might be cursed with a weed that is nearly impossible to kill. • Opponents further express concern that transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic. • Therefore, some opponents of recombinant DNA technology desire a ban on all genetically modified agricultural products.

• The U. S. National Academy of Sciences, the U. S. National Research Council, and 81 research projects conducted between 1985 and 2001 by the European Union have not revealed any risks to human health or the environment from genetically modified agricultural products beyond the usual uncertainties inherent in conventional plant breeding. • In fact, the European Union concluded in 2001 that "the use of more precise technology and the greater regulatory scrutiny probably make them [genetically modified foods] even safer than conventional plants and foods. "

• As the debate continues, governments continue to impose standards on laboratories involved in recombinant DNA technology. These are intended to prevent the accidental release of altered organisms or exposure of laboratory workers to potential dangers. • Additionally, genetic researchers often design organisms to lack a vital gene so that they cannot survive for long outside of a laboratory.

• Unfortunately, biologists can apply the procedures used to create beneficial crops and animals to create biological weapons that are more infective and more resistant to treat ment than their natural counterparts are, though international treaties prohibit the development of biological weapons. • Nevertheless, B. anthracis spores were used in bioterrorist attacks in the United States in 2001, though, thankfully, the strain utilized was not genetically altered to realize its deadliest potential.

• Emergent recombinant DNA technologies raise numer ous other ethical issues. Should people be routinely screened for diseases that are untreatable or fatal? Who should pay for these procedures: individuals, employers, prospective employers, insurance companies, HMOs, government agencies? What rights do individuals have to genetic privacy? • If entities other than individuals pay the costs involved in genetic screening, should those entities have access to all the genetic information that results? Should businesses be allowed to have patents on and make profits from any living organisms they have genetically altered? Should governments be allowed to require genetic screening and then force genetic manipulations on individuals to correct so called genetic abnormalities that some claim are the bases of criminality, manic depression, risk taking behavior, and alcoholism? Should HMOs, physicians, or the government demand ge netic screening and then refuse to provide services related to the birth or care of supposedly" defective" children? • We as a society will have to confront these and other ethical considerations as the genomic revolution continues to affect people's lives in many unpredictable ways.