GENETIC ENGINEERING

GENETIC ENGINEERING

The method of changing inherited characteristics of an organism by altering its genetic material is called genetic engineering. It is also known as Recombinant DNA technology.
Recombinant DNA technology is the technique, 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 medicine, which is at present in its initial phase of the overall concerted effort.

The cornerstone of most molecular biology technologies is the gene. To facilitate the study of genes, they can be isolated and amplified. One method of isolation and amplification of a gene of interest is to clone the gene by inserting it into another DNA molecule that serves as a vehicle or vector that can be replicated in living cells. When these two DNAs of different origin are combined, the result is a recombinant DNA molecule.

1. TOOLS OF GENETIC ENGINEERING

The tools of genetic engineering are:
1. Complementary DNA
2. Enzymes
3. Vectors

1) Complementary DNA:
Complementary DNA is DNA synthesized from a messenger RNA (mRNA) template in a reaction catalyzed by the enzyme reverse transcriptase and DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists want to express a specific protein in a cell that does not normally express that protein they will transfer the cDNA that codes for the protein to the recipient cell. cDNA is also produced naturally by retroviruses.

2) Enzymes:
Enzymes used in recombinant DNA technology are:
a) Restriction endonucleases
b) DNA ligase
c) Polymerase
d) Alkaline phosphatase
e) Reverse Transcriptase
a) Restriction endonucleases: Restriction endonucleases or restriction enzymes, as they are called popularly, recognize unique base sequence motifs in a DNA strand and cleave the backbone of the molecule at a place within or, at some distance from the recognition site. Recognition site: the site at which restriction endonucleases cuts the DNA.
These are the enzymes that cleave the DNA double strand by breaking phosphodiester bond between nucleic acids that hold the nucleotides together .They belong to the family nucleases. Nucleases are classified into 2 types depending upon the substrate upon which they act :

1. DNAses are those that act on DNA
2. RNAses are those that act on RNA

DNAses are further classified as exonucleases and endonucleases. Exonuclease cut the DNA at the end while endonucleases cut away from the ends.

3 types of endonucleases are of most importance in genetic engineering:
1. Type 1 endonucleases : it cleaves up to 1000 bp from recognition site e.g EcoK1
2. Type 2 endonucleases: cleaves at the recognition site e.g EcoR1
3. Type 3 endonucleases: it cleaves upto 24-26 bp from recognition site e.g EcoP1.

b) DNA ligases:
Recombinant DNA experiments require the joining of two different DNA segments or fragments in vitro. The cohesive ends generated by some RE will anneal themselves by forming hydrogen bonds but the segments annealed thus are weak and do not withstandexperimental conditions. To get stable joining fragments are joined by using an enzyme called ligase. Donor and recipient DNA are incubated at 37 degree centigrade in the presence of DNA ligase for joining.
There are 2 extensively used ligases ,one from E.coli and other form T4 bacteriophage called T4 DNA ligase, join blunt ends of DNA fragments whereas E.coli ligase join cohesive ends which is 2 times faster than blunt ends ligation.

 c) Polymerases
Polymerase enzymes synthesize copies of nucleic acid molecules and are used in many genetic engineering procedures. • When describing a polymerase enzyme, the terms ‘DNA- dependent’ or ‘RNA-dependent’ may be used to indicate the type of nucleic acid template that the enzyme uses. • Thus, a – DNA-dependent DNA polymerase copies DNA into DNA, – an RNA-dependent DNA polymerase copies RNA into DNA, and – a DNA-dependent RNA polymerase transcribes DNA into RNA.

These enzymes synthesize nucleic acids by joining together nucleotides whose bases are complementary to the template strand bases. • The synthesis proceeds in a 5’→3’ direction, as each subsequent nucleotide addition requires a free 3’-OH group for the formation of the phosphodiester bond. • This requirement also means that a short double- stranded region with an exposed 3’-OH (a primer) is necessary for synthesis to begin.

d) Alkaline Phosphatase
The broken fragments of plasmids, instead of joining with foreign DNA, join the cohesive end of the same DNA molecules. The treatment with alkaline phosphatase prevents re-circularization of plasmid vector and increases the frequency of production of recombinant DNA molecule.

e) Reverse Transcriptase:
Retroviruses (possessing RNA) contain RNA dependent DNA polymerase which is called reverse transcriptase. This produces single stranded DNA, which in turn functions as template for complementary long chain of DNA.

This enzyme is used to synthesize the copy DNA or complementary DNA (cDNA) by using mRNA as a template. The enzyme is very useful for the synthesis of cDNA and construction of cDNA clone bank and to make short labeled probes

3) Vectors:
Vectors are DNA molecules which carry foreign DNA fragment when inserted into them. They can also be called as vehicle DNA . a vector must have :

 Origin of replication
 Polylinks
 Selectable marker

Some of the common vectors used in genetic engineering are:

 Plasmid
 Phage vectors
 Cosmids
 Viral vectors

Plasmid:
Plasmids are circular DNA molecules that lead an independent existence in the bacterial cells. They are naturally occurring extra chromosomal DNA fragments that are stately inherited from one generation to another in extra chromosomal state. The incorporation of DNA into plasmid vectors not only allow foreign DNA to be replicated in cloned cells for later isolation and identification , but can also be designed so that cells transcribe and translate this DNA into protein. For example PUC19 is obtained from E. coli and have Amp resistance enzyme as selectable marker.

Phage as vectors:
They contain a single nucleic acid usually DNA which carries small genomes enclosed in a protein cover called capsid. Phage vector has a natural advantage over plasmid that they infect the host cell much more efficiently than plasmid transformed cells as the yield of clones with phage vector is usually higher. e.g M13

Cosmid:
Cosimd are the hybrid vectors derived from plasmid and phages. The segment of the phage DNA is inserted into plasmid in E.coli. The points at which bacteriophage lambda is cut are called cos sites. The resulting vector is called a cosmid. They are used to carry larger genes or fragments of DNA.

Viral vectors:

Viral vectors are generally genetically engineered viruses carrying modified viral DNA or RNA that has been rendered noninfectious.

2. TECHNIQUES USE IN BIOTECHNOLOGY

2.1 Polymerase chain reaction

This technique allows the generation of large amount of copies of a specified DNA sequence from a single DNA molecule without the need for cloning. PCR uses single stranded DNA as a template for the synthesis of complementary new strands in a 5’ to 3’ direction.

2.1.1 Denaturation step:

This step is the first regular cycling event and consists of heating the reaction. It causes melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules

2.1.2 Annealing step:

The reaction temperature is lowered allowing annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. DNA polymerase require small fragments of double stranded DNA to initiate DNA synthesis. The polymerase binds to the primer-template hybrid and begins DNA formation

2.1.3 Extension/elongation step:

The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme which reduces the risk of mismatches that occasionally occur at lower

temperatures. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand.

2.1.4 Final elongation:

This single step is occasionally performed after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

2.1.5 Final hold:

This step at 4–15 °C for an indefinite time maybe employed for short-term storage of the reaction.

In PCR both strands of DNA serve as template upon the addition of a pair of primers, one for each strand of DNA. Every PCR cycle is normally repeated up to 30 times. The net result of a PCR at the end of n cycles, will generate maximum of2n double stranded DNA copies of the DNA fragment located between the two primers.

Advantages of PCR:

1. It is highly specific.

2. It is rapid technique.

3. It is versatile,

4. The equipment is inexpensive and allows the analysis of a large number of sequences at one time

5. PCR does not require pure template DNA and can amplify genes from whole cells or tissue samples.

Limitations of PCR:

1. The designing of primers for this technique requires partial knowledge of the DNA sequence to be amplified.

2. The slightest sample contamination can lead to false positive results, which can have detrimental effects when this

technique is used in diagnostics. There is a risk of non specific amplification when the primers bind to closely related sequences, leading

to the amplification of sequence.

2.2 Genes therapy

Genes are found in chromosomes, and are basic physical and functional units of heredity.

Encode how to make a protein. DNA, RNA, proteins. Proteins carry out most of life’s function. When altered causes dysfunction of a protein. Proteins also work to build our numerous cellular structures. When genes are altered so that encoded proteins are unable to carry out their normal functions, genetic disorders can result.

Gene Therapy Is a technique for correcting defective genes that are responsible for disease development.

2.2.1 Approaches for Gene Therapy:

 A normal gene inserted to compensate for a nonfunctional gene.

 An abnormal gene traded for a normal gene.

 An abnormal gene repaired through selective reverse mutation.

2.2.2 Mechanism

 A vector delivers the therapeutic gene into a patient’s target cell

 The target cells become infected with the viral vector

 The vector’s genetic material is inserted into the target cell

 Functional proteins are created from the therapeutic gene causing the cell to return to a normal state.

2.2.3 Type of vector

a) Viruses

b) Non viral vector

a) Viruses

By removing the viral DNA and using the virus as a vehicle to deliver the therapeutic DNA.

A number of viruses have been used for human gene therapy,

• Retrovirus

• Adenovirus

• Adeno-associated virus

• Herpes simplex virus

b) Non-Viral vector

There are several methods for non-viral gene therapy, including the injection of naked DNA, electroporation, inorganic nanoparticles.

2.2.4 Types of Gene Therapy

a) Germ line gene therapy

b) Somatic gene therapy

c) Ex vivo Gene Transfer

d) In vivo Gene Transfer

a) Germ line gene therapy

It involves altering the genetic makeup of a gene of either an egg or a sperm cell before fertilization.

Advantages - Germ line gene therapy is done before the organism has grown or developed; therefore, the cure is inherited by future generations of that organism.

Disadvantages - Germ line gene therapy also holds numerous risks, such as a margin for possible error during the gene 'transplant'

b) Somatic gene therapy

It involves altering the genetic code or chromosomes of a person's somatic cells, or body cells. It is mostly performed in fully grown organisms.

Advantages - Somatic gene therapy is relatively effective as a procedure. Somatic gene therapy is significantly less controversial than germ line gene therapies.

Disadvantages - it will not be passed on to the patient/organism's offspring. In addition, the methodology of somatic gene therapy, such as the use of viral vectors, is difficult.

2.2.4 Current status of Gene Therapy

The food and drug administration (FDA) has not yet approved any human gene therapy product for sale. Current gene therapy is experimental and has not proven very successfully in clinical trials. FDA’s Centre for Biologics Evaluation & Research (CBER) works with researcher and industry during all stages of product development.

2.2.5 Factors effecting gene therapy

Following are the factor due to which gene therapy is less effective for the treatment of genetic diseases.

a) 1-Short -lived nature of gene therapy

b) 2-Immune Response

c) 3-Problems with viral vectors.

a) Short -lived nature of gene therapy

Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long term benefits.

Patients have to undergo multiple round of gene therapy.

b) Immune Response

The natural immune system is designed to invade any foreign object. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk.

c) Problems with viral vectors

Viruses cause toxicity immune disorders inflammatory responses etc. so these vectors may cause severe diseases.

3. Nanotechnology for Genetic engineering

Bioballistics:

Projectile method that used metal slivers to deliver genetic material to the cell. Genetic material is coated on the slivers. Once genetic material is transported to cell, it is incorporated in host gene.

Microinjection:

It is a direct gene transfer method with no use of vector. It is done with tipped glass needle. Injected gene will find the host cel gene and incorporate themselves among them.

For example,

Most common method of making genetically altered mice.

Electro and chemical poration:

This process creat pores in the cell membrane to allow direct entry of new gene. It is done by bathing cells in solution of special chemicals or weak electric current.

4. Applications of Genetic Engineering:

Pharmaceutical application:

Use of genetic engineering in pharmaceutical involves use of genetically modified organisms to produce pharmaceutical products.Genetic engineering has many applications to medicine that include manufacturing of drugs, creations of model animals that mimic human conditions and gene therapy. One of earliest use was to mass production of human insuline in E.Coli. This application is now applied to

 Recombinant human insulin (humulin R by Lilly company).

 Recombinant somatotropin (human growth hormone, Genotropin by Pfizer)

 Recombinant hepatitis B vaccine (hepagam B)

 Tissue plasminogen activator (dissolve blood clot, TNKase)

 Adenosine deaminase (ADA) (for treatment of severe combined immunodeficiency SCID, Nipent)

Genetic engineering is used to create animal model. Genetically modified mice is most common model to study cancer, obesity,anxiety,heart disease and parkinsons disease.

Genetically modified pigs have been bred with aim of increasing success of pig to human organ transplantion. Pig embryo is injected with human Hb gene that synthesize human Hb.

Since 1990, gene therapy has been used to treat diseases like AIDs, cancer, cystic fibrosis etc.

Applications in research field:

Organism are geneticaly modfied to discover the function of certain gene.Gene and other genatic information from an organism can be transferred to bacteria for storage and modification.Creating geneticaly modified bacteria are cheap,easy to grow,multiply quickly.

 Loss of function experiment: Also called gene knockout experiment.Organism is geneticaly modified to lack activity of one or more gene.

 Gain of function experiment: It assigned the increase in function of gene usualy providing extra copies of gene.

 Expression studies: It discover when and where specific proteins are prepared.DNA seqence has reporter gene or enzyme that catalyze dye so time and pace where gene is produce is expressed.

Industrial applications:

 Genetically modified virus is use in labs for environmental friendly lithium free battery.

 They also use in making biofuels,cleaning up oil spoils,carbon and other toxic waste.

Agricultural applications:

 Golden rice(increase level of vitamin A)

 Howell(enzyme in fieflies is injected in tobacco plant so when there is light the gene show expression and plant glow)

 BT corn(soil bacterium that resist insecticides s injected in corn to make it insect esistant)

 Calgene tomato(stay fresh longer because enzyme to breakdown pectin are reduced by genetic engineering)

Bio art:

 some bacteria are geneticaly engineered to create black and white photograph.

 Genetic engineering has also been use to create novelty items like blue roses ,glowing fish.

Tab. 1. examples of drugs produced by recombinant technology