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Friday, 25 September 2015

FSc Notes Biology Part 2 Chapter 23 Biotechnology Notes

FSc Notes Biology Part 2 Chapter 23 Biotechnology Notes

Biotechnology:

The industrial use of living organisms or their components to improve human health and food production is called biotechnology OR the utilization or exploitation of living organisms or their product for the benefit and welfare of human being is called biotechnology. The term biotechnology was first used by Karl Ereky in 1917. The history of biotechnology is as old as human civilization. The preparation of curd, ghee, wine bear, and vinegar in homes is traditional biotechnology. The modern biotechnology started during First World War. Germany was deprived of the supply of vegetable fats necessary for glycerol production. From this glycerol explosives were made by Germans. So Germany started fermentation of plant materials by yeast as an alternative source.

Recombinant DNA and Gene Cloning

The introduction of genes from one organism into the genome of another organism is called Recombinant DNA technology. Recombinant DNA is artificially produced. Recombinant DNA is artificially produced with the help of:
  1. Gene of interest which is to be cloned.
  2. Restriction enzyme or molecular scissor.
  3. Ligase enzyme or molecular glue.
  4. Vector
  5. Expression system

Isolation of DNA or obtaining gene of choice:
The first step in gen cloning is to obtain a gene of interest from a healthy organism.
DNA isolated directly from laboratory from an organism.
DNA made in the laboratory fro mRNA.

Restriction enzyme:
Gene can isolated from the DNA by using restriction enzymes. These enzymes cut the DNA into many small fragments. One of these fragments carries the gene of interest. The restriction enzyme creates sticky ends on the DNA fragments. These enzymes are specific in their recognition and cutting action. These enzymes cut specific base sequence in DNA molecule. In 1970 Hamilton D.Smith isolated the first restriction enzyme. They are called restriction enzymes because they restrict the growth of viruses. These enzymes protect bacteria from viral infection. About 400 different such enzymes have been isolated out from bacteria.

Vector:
The body which transfers the DNA molecule into another living body or host cell is called Vector. The isolated gene is then transferred into the vector. The vector may be of different kinds e.g. plasmid, pheges etc. The most common vectors are plasmids. Plasmids are small circular DNA ring present in bacteria. The plasmid ring is cut open by restriction enzyme. The DNA fragment is mixed with the open plasmid ring. The gene of choice attaches itself to the sticky end of plasmid.

Ligase Enzyme:
This enzyme joins the DNA fragment with open ends of plasmid by covalent bonds and closing the ring again. This form recombinant DNA or chimaeras DNA.

Expression system or Vector:
When bacteria are kept with calcium chloride (CaCl2) they absorb recombinant DNA. This bacteria is called expression vector. Both bacterial cell and rDNA multiply by cell division. The gene of choice will express itself by producing the desired protein in the bacterial cell. For example; a bacterium containing human insulin gen it will synthesize human insulin hormone.


Polymerase Chain Reaction

A process used to create millions of copies of a single gene or specific DNA sequence quickly in a test tube is called Polymerase Chain Reaction. This technique was developed by Kary B.Millus in California in 1983. This method is much faster than gen cloning and is performed completely invitro (in laboratory). Polymerase Chain Reaction is a chain reaction because DNA polymerase will carry out replication over and over again. The machine used in PCR is called “Thermocycler”
Requirements of Polymerase Chain Reaction: (PCR)
Following materials are required for PCR:
  1. DNA Template: A DNA having targeted sequence to be copied is called Template.
  2. Primers: it is a man made short nucleotide sequence having 20 nucleotides. With out primers DNA polymerase cannot start the DNA replication.
  3. Polymerase: The most common enzyme used in PCR is DNA polymerase. It is also called Taq polymerase. This enzyme is obtained from a bacterium called Thermus aquatic us which lives in hot springs.

Steps in Polymerase Chain Reaction: (PCR)
Following are the main steps in PCR.
  1. Denaturation of DNA: The target DNA is heated to 95oC for one minute. At this temperature the DNA denatured (separated) forming single stranded sequence. Now it is cooled for two minutes.
  2. Addition of Primers: Now primers are also added with the DNA. Primers attach it self to the target sequence by hydrogen bonding. Two primers are needed one for each strand of DNA.
  3. Primers Extension: The DNA polymerase extends the primers by adding nucleotides. A result double stranded DNA is formed. This DNA consist of one original strand and a new strand of primers. This process is repeated again and again at about 5 minute per cycle. In this away a large number of copies of the required DNA is produced.

Application or uses of Polymerase Chain Reaction: (PCR)
Diagnosis: PCR is used in the diagnosis of viral infection, cancer, genetic disorder.
Crime Investigation: PCR can copy DNA from even a single hair follicle or drop of blood left at a crime place. Thus PCR help to identify criminal.
Gene Sequencing: A process in which arrangement of nucleotides is determined is called gene sequencing. OR determination of nucleotides arrangement of DNA fragment is called gen sequencing.
Gel Electrophoresis: The separation of charged molecules on the basis of their size and charge applying an electric field in a gel is called gel electrophoresis. This technique was developed by A. Tiselium in 1937. Electrophoresis is used for separation of DNA, RNA, proteins.

Major Steps

  1. Preparation of different size DNA fragments: DNA molecule is cut into different sized fragments by using restriction enzyme.
  2. Preparation of Agarose gel: Agarose is a white powder extracted from seaweeds. Agarose is insoluble in water at room temperature. It dissolves in boiling water and on cooling it forms a gel. Ethidium bromide (Et Br) a florescent dye is also added in this gel.
  3. Loading of gel with DNA fragment: The DNA fragments are placed into the gel. An electric current is applied to the gel with +ve charge at the bottom and – ve charge at the top. DNA molecules have negative charges. Flow of electricity causes the movement of DNA fragments in the gel towards the positive pole. Smaller fragments move faster than the larger fragments. Therefore, smaller fragments accumulate at the bottom while larger fragments gather at the top. Thus different size DNA fragments are separated.
  4. Reading of nucleotide sequence from the gel: The gel is then taken into a documentation machine attached with computer. The nucleotides start gloving when the UV light fall on it due to Et Br in the gel. Nucleotides bands are seen on the computer and it is recorded.

DNA Finger Printing

A technology which help in the identification of individual at genetic level is called DNA finger printing. About 99 % DNA is similar in all human being. Only a short portion is differ in each person. This sequence is 20 – 40 bases long which is repeated several times. These bases are called Restriction Fragments Length polymorphism. RFLP was first isolated by A.Wyman and R.White in 1980. DNA fingerprinting was developed by Jeffery’s in 1984 by using RFLP.


Basic requirements:
DNA fingerprinting require the availability of biological sample such as: Skin cells, few blood drops, semen, bone marrow cells, hair with its root.

Method:
The DNA is isolated from the blood semen or other cells. This DNA is cut into fragments by restriction enzymes. DNA fragments are separated by electrophoresis. Double stranded DNA is separated into single strand by heating. A copy of fragments is transferred to a nylon membrane. A radioactive P32 single stranded DNA probe is added to the DNA bands on nylon membrane. X – Ray film is laid over the nylone membrane. The radioactive probes on the DNA develop images or prints on the film. In this way finger print of DNA is obtained called autoradiograph.
Practical Application OF DNA Fingerprinting
Parenthood Dispute: DNA fingerprinting help to solve the problem of paternity and maternity (father + mother) child dispute.
Personal identification: This technology helps in the classification of different organisms.
Immigrant dispute: it can also be used for confirming legal nationality.
Criminal Identification: It helps in the identification of criminals. The DNA of suspect is compared with the DNA isolated from skin cells, DNA from a blood or hair left at the place of crime. Similarly DNA from a single sperm is enough to identify a suspected rapist.

Transgenic Plants:

Those plants in which foreign gene have been inserted by genetic engineering are called transgenic plants. Transgenic plant will help in improving the health of malnourished people in poor countries. Transgenic plants are used by the scientists for the production of:
(i) Antibodies (ii) Protein (iii) Vitamin and (iv) Polyhydroxybutyrate used in the preparation of biodegradable plastic. Some transgenic plants are as follows:

Golden Rice: This rice contain high amount of provitamin – A. it show resistance to viral diseases.
Flavr Savr: Transgenic tomato having tough coat (epicarp). These tomato can be store for longtime.
New leaf: This is transgenic potato showing insect resistance
Roundup Ready: this transgenic plant produce enzyme which detoxify herbicide.
Boll Gard: This transgenic cotton produces an insecticidal toxin and kill many insects but harmless to bifacial insects.

Transgenic Animals:

Those animals which are produced by the introduction of foreign functional gene in zygote stage are called transgenic animals. Transgenic animals can be used as Bioreactors for large scale production of valuable recombinant chemicals such as; Protein, hormones, interferon’s etc. The main purpose to produce transgenic animal is:
  1. To produce new animal products
  2. More wool production in transgenic sheep.
  3. Increase growth rate of livestock.
  4. To Study genetic disease model.
  5. Increase feed utilization and disease resistance.

Human Genome Project

An international research effort to amp and sequence the genes of entire human genome is called human genome project. Human genome project was started in 1990 in U.S.A. This project completed in thirteen years.

Objectives of HGP:

Identification of all genes (30000 – 35000) in the human DNA. Storing of this information in a data base. Locating the 50,000 – 100,000 genes within human DNA.

Gene Therapy

The introduction of normal gene is place of defective gene in the patient body is called gene therapy. OR A technique in which an abnormal or defective gene is replaced by a healthy and dominant in the patient body is called gene therapy. The first gene therapy experiment was done in 1990 in a four year old girl suffering form sever immunodeficiency disease called adenosine deaminase deficiency. The main goal of gene therapy is to cure all genetic disease. It can also be used to study cell functions.
Types:
  1. Somatic Gene therapy: The introduction of functional gene into the somatic cells of the body.
  2. Reproductive Gene Therapy: The introduction of functional genes into germ cells or zygote to correct the genetic defects in the offspring.

Strategies for Gene Transferring
Gene can be transfer into the cells by the following method:
Microinjection: Used to transfer gent into, oocytes, eggs, embryos.
Gene Gun: Used to transfer gene into tissues such as; liver, skin, muscles.
Detergent Mixture: Dextrin or liposome’s can also be used for gene transfer.
Viruses Vectors: Different harmless viruses are also used as gene transfer vectors. e.g. inactivated retroviruses.

Methods of Gene Therapy

Two methods are used in gene therapy.
Ex Vivo Gene Therapy: The transferring of gene into the patient cell outside the body is called ex vivo gene therapy. The main steps in ex vivo are;
Normal gene is isolated and is cloned. Gene is inserted in retrovirus vector. Bone marrow cells are taken from patient with genetic defect. Marrow cells are infected with retrovirus. These infected cells are returned to the patient by injection in into a vein.
In Vivo Gene Therapy: The transfer of gene into the cells inside the patient body is called in vivo gene therapy. Main steps in this gene therapy:
Normal gene is mixed with liposome. This mixture is spread over the surface of defective organ. Liposome widens pore size of the cell membrane. The gene in liposome enters the body cells.

Amniocentesis:

A diagnostic method in which fluid is taken by a needle from fetal sac for tests is called amniocentesis.
The test of this fluid shows us the following defects.
  1. Down’s Syndrome: Chromosomal disorder.
  2. Spina bifida: Vertebrate fail to attach with each other.
  3. Ancencephaly: missing or incomplete brain.
  4. Sex of the baby: male or female baby.
  5. To determine Rh incompatibility or infection.

Application of Biotechnology in Agriculture and Medicine

Biotechnology has great use and application in the field of agriculture and medicines.

Biotechnology in Agriculture: 

Pests and herbicide resistant Some plants like corn, cotton, potato, and soyabean have been produced that both insect and herbicide resistant. Because their cells now can produce toxin.
Salt tolerant: salt tolerant plants have been developed which grow on salty soil. Improving Food Quality: Biotechnology had improved food quality in many crops. Soybean produced oleic acid, vernolic acid, recinolic acid. These acids are used in paints and plastic to make them.
Producing Biodegradable Plastic: A weed called mouse eared cress has been developed that produce biodegradable plastic.

Biotechnology in Medicine:

Hormones Preparation: Biotechnology is also used in the production of certain hormones such as growth hormones insulin.
Vaccine Preparation: Biotechnology is also used in the production of certain hormones such as growth hormones, insulin.
Antibody Production: one type of antibody made by corn can deliver radio isotopes to tumor cells. Transgenic soybeans made antibodies which can be used for treatment of genital herpes.

Gene Bank

Gene bank is a facility to conserve individual tissue or reproductive cells of plants or animals. The function of gene is not easy to study because there are thousands of genes. But one can study only one gene at a time in one experiment. A new technology has been developed called “DNA Microarray”. In DNA microarray a single chip can have whole genome of an individual. Thus researchers are able to now the interactions among thousands of genes at the same time. The DNA microarray can be seen on the screen as DNA as DNA fingerprinting.


Written by: Asad Hussain

3 comments:

  1. Biotechnology covers a lot of tech employed in the field of biology and life science, assisting greatly in drug discovery and other aspects to boost the life experience. This post is good summary on that.

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  2. Current progress in biotechnology depends less today on marginal advances in knowledge we've accumulated about a particular species. So as is known, biotechnology has involved in a wide range of areas, such as clinical medicine, biochemistry, etc. Anyway, this post has displayed a new insight for us to better understand biotech.

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  3. Thank you so much for sharing these detailed info!

    ReplyDelete