Bio-tech Assignment: Recombinant DNA Technology Submitted

It is joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 meters (6 feet) of DNA. Therefore, a small tissue sample will contain many kilometers of DNA. However, recombinant DNA technology has made it possible to isolate one gene or any other segment of DNA, enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a living organism.

Definition: A series of procedures that are used to join together (recombine) DNA segments. A recombinant DNA molecule is constructed from segments of two or more different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate there, either on its own or after it has been integrated into a chromosome.
Requirements to Produce rDNA:

• I. Gene of interest, which is to be cloned.
• II. Molecular scissors to cut out the gene of interest.
• III. Molecular carrier or vector, on which gene of interest could be placed.
• IV. The gene of interest along with the vector is then introduced into an expression system, as a result of which a specific product is made.

How to get a gene?

• I. Isolate it from the chromosome;
• II. Synthesize it chemically; and
• III. Make it from mRNA.

Genes can be isolated from the chromosomes by cutting the chromosomes on the flanking sites of the gene using special enzymes known as restriction endonucleases. (2)

Basics Of rDNA: So What is rDNA?

Before we get to the "r" part of the DNA, we need to understand the DNA. All DNA is made up of the ribose sugar, nitrogen bases, and phosphate. There are four nitrogen bases adenine(A), thymine(T), guanine(G) and cytosine(C). These nitrogen bases are found in pairs, with A&T and G&C are paired together.

These bases can be arranged in an infinite way which gives rise to the formation of the famous "double helix" structure as shown in the figure:

The sugar used in DNA is deoxyribose (oxygen removed from the 2nd carbon of sugar). These all four bases are the same in all organisms but the variety of there arrangement and sequence in DNA leads toward diversity. DNA does not actually make the organism but makes the proteins. The DNA is then transcribed into mRNA and then it is translated into protein which forms the organism. By changing the sequence of DNA, the protein formed will also change. This results in either different protein or inactive protein.

Now we know that what DNA is. Recombinant DNA is combining two strands of different DNA. Thus, the name is the recombinant! which sometimes also called "chimera." By combining different strands of DNA, a scientist can create a different combination of DNA.

How Recombinant DNA is Made?

There are three different ways to make recombinant DNA:

1. Transformation;

2. Phage Introduction; and

3. Non-Bacterial Transformation.

Obtaining DNA:

Step 1: The DNA fragment containing the gene sequence to be cloned (also known as' insert') is isolated.

Step 2: Cutting DNA

Step 3: Joining DNA

Step 2: Insertion of these DNA fragments into a host cell using a "vector" (carrier DNA molecule)

Step 3: The rDNA molecules are generated when the vector self-replicates in the host cell.

Step 4: Transfer of the rDNA molecules into an appropriate host cell.

Step 5: Selection of the host cells carrying the rDNA molecule using a marker

Step 6: Replication of the cells carrying rDNA molecules to get a genetically identical cells population or clone.

The first step in making recombinant DNA is to isolate donor and vector DNA. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA.

Ultracentrifugation:

A protocol for extracting plasmid DNA can be achieved by ultracentrifugation. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube.

Alkaline Lysis:

Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as ? also can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.

Cutting DNA

The restriction enzyme EcoRI cuts a circular DNA molecule bearing one target sequence, resulting in a linear molecule with single-stranded sticky ends.

Joining DNA: Insertion

A vector is any DNA molecule which is capable of multiplying inside the host to which our gene of interest is integrated for cloning. In this process restriction enzyme function as scissors for cutting the DNA molecules. Ligase enzyme is the joining enzyme that joins the vector DNA with the gene of interest. this will produce the recombinant DNA.

Definition:

It is a process for inserting foreign DNA into bacteria, could be used to reliably introduce DNA into bacteria.

Methods

1. Calcium chloride transformation:

In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca2+ (in CaCl2 solution), making the cell become permeable to plasmid DNA. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42 °C for 30–120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/µg or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of the plasmid. Protocols, however, exist for making super-competent cells that may yield a transformation efficiency of over 109. The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.(4)

2. Electroporation:

Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell.[1] In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred cell-penetrating peptides or CellSqueeze could also be used. Electroporation works by passing thousands of volts across a distance of one to two millimeters of suspended cells in an electroporation cuvette (1.0 – 1.5 kV, 250 – 750V/cm). Afterward, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective than chemical transformation.

Selectable markers can be for antibiotic resistance, color changes, or any other characteristic which can distinguish transformed hosts from untransformed hosts. Different vectors have different properties to make them suitable for different applications. Some properties can include symmetrical cloning sites, size, and high copy number.

Antibiotic Selection

It is a technique in which transformed bacterial cells are plated on agar plates with different antibiotics, as a way to identify recombinant bacteria and non transformed cells. Procedure Transformed bacteria are plated on agar plates that contain an antibiotic-ampicillin or any other. Non-transformed bacteria cannot grow in the presence of ampicillin because they lack ampicillin plasmids containing an ampicillin resistance gene(ampR).

Disadvantage

Antibiotic selection alone does not distinguish transformed bacteria with a nonrecombinant plasmid that has recircularized from recombinant plasmids.

Blue-White Selection

It is a technique used to distinguish between recombinant bacteria and nonrecombinant bacteria(containing plasmid without foreign DNA). Procedure In this the agar plates also contain a chromogenic (color-producing)substrate for B-gal called X-gal (5-bromo-4-chloro-3-indolyl-BD-galactopyranoside).X-gal is similar to lactose in structure and turns blue when cleaved by B-gal. As a result, nonrecombinant bacteria -those that contain a plasmid that ligated back to itself without insert DNA-contain a functional LacZ gene, produce B-gal and turn blue. Conversely, recombinant bacteria are identified as white colonies. Because these cells contain the plasmid with foreign DNA inserted into the lacZ gene, B-gal is not produced, and these cells cannot metabolize X-gal. Therefore, through blue-white selection, nontransformed and nonrecombinant bacteria are selected against and white colonies are identified or selected for as the desired colonies containing recombinant plasmids.

Non-Bacterial Transformation

This process is very similar to transformation. The only difference is non-bacterial does not use bacteria such as E.Coli for the host. In microinjection, the DNA is directly injected into the nucleus of the cell being transformed. In biolistics, the host cells are bombarded with high-velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.

Phage Introduction

Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packagings of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods. APPLICATIONS OF RECOMBINANT DNA TECHNOLOGYThe three important applications are (1) Applications in Crop Improvement;(2) Applications in Medicines; and (3) Industrial Applications.

I. Applications in Crop Improvement:

Genetic engineering has several potential applications in crop improvement, such as given below:

1. Distant Hybridization:

With the advancement of genetic engineering, it is now possible to transfer genes between distantly related species. The barriers of gene transfer between species or even genera have been overcome. The desirable genes can be transferred even from lower organisms to higher organisms through recombinant DNA technology.

2. Development of Transgenic Plants:

Genetically transformed plants which contain foreign genes are called transgenic plants. Resistance to diseases, insects and pests, herbicides, drought; metal toxicity tolerance; induction of male sterility for plant breeding purpose; and improvement of quality can be achieved through this recombinant DNA technology. BT-cotton, resistant to bollworms is a glaring example.

3. Development of Root Nodules in Cereal Crops:

Leguminous plants have root-nodules which contain nitrogen-fixing bacteria Rhizobium. This bacteria converts the free atmospheric nitrogen into nitrates in the root nodules. The bacterial genes responsible for this nitrogen fixation can be transferred now to cereal crops like wheat, rice, maize, barley etc. through the techniques of genetic engineering thus making these crops too capable of fixing atmospheric nitrogen.

4. Development of C4 Plants:

Improvement in yield can be achieved by improving the photosynthetic efficiency of crop plants. The photosynthetic rate can be increased by conversion of C3 plants into C4 plants, which can be achieved either through protoplasm fusion or recombinant DNA technology C4 plants have a higher potential rate of biomass production than C3 plants. Most C4 plants (sorghum, sugarcane, maize, some grasses) are grown in tropical and subtropical zones.

Applications in Medicines:

Biotechnology, especially genetic engineering plays an important role in the production of antibiotics, hormones, vaccines and interferon in the field of medicines.

1. Production of Antibiotics:

Penicillium and Streptomyces fungi are used for mass production of famous antibiotics penicillin and streptomycin. Genetically efficient strains of these fungi have been developed to greatly increase the yield of these antibiotics.

2. Production of Hormone Insulin:

Insulin, a hormone, used by diabetics, is usually extracted from the pancreas of cows and pigs. This insulin is slightly different in structure from human insulin. As a result, it leads to allergic reactions in about 5% of patients. Human gene for insulin production has been incorporated into bacterial DNA and such genetically engineered bacteria are used for large-scale production of insulin. This insulin does not cause allergy.

3. Production of Vaccines:

Vaccines are now produced by transfer of antigen-coding genes to disease-causing bacteria. Such antibodies provide protection against the infection by the same bacteria or virus.

4. Production of Interferon:

Interferons are virus-induced proteins produced by virus-infected cells. Interferon is antiviral in action and acts as the first line of defense against viruses causing serious infections, including breast cancer and lymph nodes malignancy. Natural interferon is produced in very small quality from human blood cells. It is thus very costly also. It is now possible to produce interferon by recombinant DNA technology at the much cheaper rate.

5. Production of Enzymes:

Some useful enzymes can also be produced by recombinant DNA technique. For instance, enzyme urokinase, which is used to dissolve blood clots, has been produced by genetically engineered microorganisms.

6. Gene Therapy:

Genetic engineering may one day enable the medical scientists to replace the defective genes responsible for hereditary diseases (e.g., hemophilia, phenylketonuria, alkaptonuria) with normal genes. This new system of therapy is called gene therapy.

7. A solution of Disputed Parentage:

Disputed cases of parentage can now be solved most accurately by recombinant technology than by blood tests.

8. Diagnosis of Disease:

Recombinant DNA technology has provided a broad range of tools to help physicians in the diagnosis of diseases. Most of these involve the construction of probes: short Segments of single-stranded DNA attached to a radioactive or fluorescent marker. Such probes are now used for identification of infectious agents, for instance, food poisoning Salmonella, Pus-forming Staphylococcus, hepatitis virus, HIV, etc. By testing the DNA of prospective genetic disorder carrier parents, their genotype can be determined and their chances of producing an afflicted child can be predicted.

9. Production of Transgenic Animals:

Animals which carry foreign genes are called transgenic animals.

Examples:

Cow, sheep, goat – therapeutic; human proteins in their milk. Fish like common carp, catfish, salmon, and goldfish contain human growth hormone (HGH).

Industrial Applications:

In industries, recombinant DNA technology will help in the production of chemical compounds of commercial importance, improvement of existing fermentation processes and production of proteins from wastes. This can be achieved by developing more efficient strains of microorganisms. Specially developed microorganisms may be used even to clean up the pollutants. Thus, biotechnology, especially recombinant DNA technology has many useful applications in crop improvement, medicines, and industry.