12th Class Biology Genetics Genetic Engineering

Genetic Engineering

Category : 12th Class

Recombinant DNA technology

Genetic engineering, a kind of biotechnology, is the latest branch in applied genetics dealing the alteration of the genetic make up of cells by deliberate and artificial means. Genetic engineering involves transfer or replacement of genes, so also known as recombination DNA technology or gene splicing.

Tools of genetic engineering : Two enzymes used in genetic engineering are restriction endonuclease and ligases. R.E. is used to cut the plasmid as well as the foreign DNA molecules of specific points while ligase is used to seal gaps or to join bits of DNA.

Steps of recombinant DNA technology

(1) Isolating a useful DNA segment from the donor organism.

(2) Splicing it into a suitable vector under conditions to ensure that each vector receives no more than one DNA fragment.

(3) Producing of multiple copies of his recombinant DNA.

(4) Inserting this altered DNA into a recipient organism.

(5) Screening of the transformed cells.

Vectors : Vector in genetic engineering is usually a DNA segment used as a carrier for transferring selected DNA into living cells. Which are as follows :

(1) Plasmid : Plasmid are extrachromosomal, closed circular double stranded molecules of DNA present in most eukaryotes. All plasmid carry replicons pieces of DNA that have the genetic information required to replicate. Plasmid pBR 322 was one of the first widely used cloning vectors, it contain both ampicillin and tetracycline resistance genes.

(2) Phage : It is constructed from the phage l chromosomes and acts as bacteriophage cloning vectors.

(3) Cosmid : The hybrids between plasmid and the phage l chromosome give rise to cosmid vectors.

Beside all these there are artificial chromosomes like

BACs (Bacterial Artificial chromosomes)

YACs (Yeast Artificial chromosomes)

MACs (Mammalian Artificial chromosomes) are very efficient vectors for eukaryotic gene transfers.

Natural genetic engineer : When as gene transfer occurs without human effort, the bacterium is known as "natural genetic engineer"  of plants. e.g., A soil inhabiting, plant pathogenic bacterium, Agrobacterium tumefaciens.

Application of recombinant DNA technology : The technique of recombinant DNA can be employed in the following ways :

(1) It can be used to elucidate molecular events in the biological process such as cellular differentiation and ageing. The same can be used for making gene maps with precision.

(2) In biochemical and pharmaceutical industry, by engineering genes, useful chemical compounds can be produced cheaply and efficiently which is shown in table.

(3) Production of transgenic plants.

(4) Production of genetically modified microorganisms.

 

Applications of recombinant DNA products

Medically useful recombinant products

Applications

Human insulin

Treatment of insulin-dependent diabetes

Human growth hormone

Replacement of missing hormone in short stature people

Calcitonin

Treatment of rickets

Chronic gonadotropin

Treatment of infertility

Blood clotting factor VIII/IX

Replacement of clotting factor missing in patients with Haemophilia A/B

Tissue plasminogen activator

Dissolving blood clots after heart attacks and strokes

Erythropoitin

Stimulation of the formation of erythrocytes (RBCs) for patients suffering from anaemia during kidney dialysis or side effects of AIDS patients treated by drugs

Platelet derived growth factor

Stimulation of wound healing

Interferon

Treatment of pathogenic viral infections, cancer

Interleukins

Enhancement of action of immune system

Vaccines

Prevention of infectious diseases such as hepatitis B, herpes, influenza, pertussis, meningitis, etc.

 

Genetic engineering in plants

The main steps in plant genetic engineering are given below:

(1) Agronomically important gene is identified and isolated.

(2) Plasmid is isolated from the bacterium, Agrobacterium tumefaciens.

(3) Plant DNA containing the gene of interest in integrated into the T DNA of the plasmid by using restriction endonuclease and ligase enzymes.

(4) Recombinant plasmid is introduced into the cultured plant cells.

(5) T DNA integrates into the plant cells chromosomes DNA.

(6) As the plant cells divide, each daughter cell receives a copy of T DNA and the gene of interest it carries.

(7) The cells give rise to a plantlet, which, when transferred into soil, grows into a new plant that may express the new gene.   

Cloning

Cloning is the process of producing many identical organisms or clones. In this process nucleus of ovum (n) is removed and replaced by nucleus of diploid cell of same organism. Now the egg with 2n nucleus is transferred to the uterus of mother to have normal pregnancy and delivers clone of itself.

Examples of organism cloning

(1) Cloning of sheep was done by Dr. Ian Wilmut (1995) of Roslin Institute, Edinberg U.K. and normal healthy lamb (DOLLY) was born in Feb, 1996. This lamb was exactly similar to her mother.

(2) The first cloned calves George and Charlie were born in January 1998.

(3) ANDI was the world’s first genetically altered primate produced by inserting a jelly fish gene into the embryo of a rhesus monkey.

(4) Scientist at Scotland cloned POLLY and MOLLY. Unlike Dolly, polly and molly were transgenic (they carried human protein gene) polly and molly were born in july 1997.

(5) Brigitte Boissliar, a 46-year old French chemist announced the creation of the world’s first cloned human baby nicknamed “Eve” (December 2002).

Few examples of applications of plant cloning in genetic engineering are given below where desired DNA has been introduced in plant genome for various purposes :

  

Applications

Examples

Herbicide resistant plants

Petunia, tobacco, tomato and corn

Insect resistant plants

Cotton, tobacco and mustard

Virus resistant plants

Tomato, potato, alfaalfa, cucumber, rice and papaya

Plants which improved storage proteins

French been and potato

Plants with improved oil and fats

Rapeseed (rich in oleic acids and sterates) and soyabean (rich in cocoa oil)

Stress tolerant plants

Tobacco

 

 Gene libraries

A gene library is a collection of gene clones that contains all the DNA present in some source. If the original source of the DNA was original DNA from a living organism, then the library seek to include clones of all that DNA, it is called a genomic gene library. Gene libraries can also be created by using RNA.  

cDNA

If a gene library is created by enzymatic copying of RNA by reverse transcriptase (RNA-dependent DNA polymerase), it would be called c-DNA library. c-DNA stands for complimentary DNA or copy DNA. c-DNA is made to use PCR to amplify an RNA. PCR does not work on RNA, so one can copy it to DNA using reverse transcriptase and then PCR amplify the c-DNA; this is called RT-PCR (reverse transcriptase PCR).

Gene bank

A gene bank is repository of clones of known DNA fragments, genes, gene maps, seeds, spores, frozen sperms or eggs or embryos. These are stored for possible use in genetic engineering and breeding experiment where species have become extinct.

DNA finger printing

Alec Jeffreys et al (1985) developed the procedure of genetic analysis and forensic medicine, called DNA finger printing. It is individual specific DNA identification which is made possible by the finding that no two people are likely to have the same number of copies of repetitive DNA sequences of the regions. It is also known as DNA profiling. The chromosomes of every human cell contain scattered through their DNA short, highly repeated 15 nucleotide segments called “mini-satellites” or variable-number Tandem Repeat (VNTR).

Applications of DNA fingerprinting

This technique is now used to :

(1) Identify criminals in forensic laboratories.

(2) Settle paternity disputes.

(3) Verify whether a hopeful immigrant is, as he or she claims, really a close relative of already an established resident.

(4) Identify racial groups to rewrite biological evolution.

Gene therapy

The use of bioengineered cells or other biotechnology techniques to treat human genetic disorders is known as gene therapy. Gene therapy is the transfer of normal genes into body cells to correct a genetic defect. It can be used to treat genetic diseases like sickle-cell anaemia and Severe Combined Immuno Deficiency (SCID). It (SCID) is caused by a defect in the gene for the enzyme adenosine deaminase (ADA). SCID patients have no functioning T lymphocytes and one treated with the injections of their white blood cells that have been engineered to carry the normal ADA alleles.

Transgenics

A gene that has been introduced into a cell or organism is called a transgene (for transferred gene) to distinguish it from endogenous genes. The animal carrying the introduced foreign gene is said to be transgenic animal and the possessor called Genetically Modified Organisms (GMOs). Most of the transgenic animals studied to date were produced by microinjection of DNA into fertilized eggs. Prior to microinjection, the eggs are surgically removed from female parent and fertilized in vitro then DNA is microinjected into the male pronucleus of the fertilized egg through a very fine-tipped glass needle. The integration of injected DNA molecules appears to occur at random sites in the genome.

The first transgenic animal produced was the ‘supermouse’ by the incorporation of the gene for human growth hormone by Richard Palmiter and Ralph Brinster in 1981.

 

Some important example of transgenic animals

Transgenic animals

Useful application

Cow, Sheep, goat

Therapeutic human proteins in their milk

Pig

Organ transplantation without risk of rejection

Fish (Common Carp, Catfish, Salmon, gold fish)

They contain human growth hormone (hGH). They attain a size twice of that shown by nontransgenic fish.

Mouse

Contains a human gene that cause breast cancer. This enables the researchers to study the very early development of cancer.

 

Some important example of transgenic plants

Transgenic plants

Useful application

Bt Cotton

Pest resistance, herbicide tolerance and high yield. It is resistant to boll worm infestation.

Flavr Savr Tomato

Increased shelf-life (delayed ripening) and better nutrient quality.

Golden rice

Vitamin A-rich

Potato

Higher protein content

Corn, Brinjal

Insect resistance

Soyabean, Maize

Herbicide resistance

 

Genomics and human genome project

The term genome has been introduced by Winkler in 1920 and the genomics is relatively new, coined by Thomas Rodericks in 1986. Genomics is the subdiscipline of genetics devoted to the mapping, sequencing and functional analysis of genomes.

Two important scientist associated with human genome are Francis Collins, director of the Human Genome Project and J. Craig Venter, founding president of Celera genomics. The complete sequencing of the first human chromosome, small chromosome 22, was published in December 1999.

  

Genome of Model organisms

Organism

No. of base pair

No. of genes

Bacteriophage

10 thousand

-

E. coli

4.7 million

4000

Saccharomyces cerevisiae

12 million

6000

Caenorhabditis elegans

97 million

18,000

Drosophila melanogaster

180 million

13,000

Human

3 billion

30,000

Lily

106 billion

-

 

Prospects and implications of human genome

(1) The genome project is being compared to the discovery of antibiotics.

(2) Efforts are in progress to determine genes that will revert cancerous cells to normal.

(3) The human genome sequencing not only holds promise for a healthier living. It also holds the prospects of vast database of knowledge about designer drugs, genetically modified diets and finally our genetic identity.

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