Course: BSc Biotech Sem-I

Subject : Fundamentals of Biotechnology

Unit – 4

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DNA profiling (also called DNA testing, DNAtyping, or genetic fingerprinting) is a techniqueemployed by forensic scientists to assist in theidentification of individuals by their respectiveDNA profiles.

DNA profiles are encrypted sets of letters thatreflect a person's DNA makeup, which can also beused as the person's identifier.

DNA profiling should not be confused with fullgenome sequencing. DNA profiling is used in, forexample, parental testing and criminalinvestigation.

The DNA profiling technique was firstreported in 1986 by Sir Alec Jeffreys at theUniversity of Leicester in England, UnitedKingdom, and is now the basis of severalnational DNA databases.

Dr. Jeffreys' genetic fingerprinting was madecommercially available in 1987, when achemical company, Imperial ChemicalIndustries (ICI), started a blood-testing centerin the U.K.

For DNA fingerprinting, repetitive nucleotidesequence which are specific for a person areimportant.

These nucleotide sequence are known as variablenumber tandem repeats (VNTR).

As every cell contains DNA, extremely smallamount of blood, semen, hair bulb or any other cellfrom the body of a person are sufficient to detectthe individual.

The system of DNA profiling used today is basedon PCR and uses short tandem repeats (STR).

DNA fingerprinting or DNA profiling, any of several similar techniques for analyzing and comparing DNA from separate sources, used especially in law enforcement to identify suspects from hair, blood, semen, or other biological materials found at the scene of a violent crime.

It depends on the fact that no two people, save identical twins, have exactly the same DNA sequence, and that although only limited segments of a person's DNA are scrutinized in the procedure, those segments will be statistically unique.

A common procedure for DNA fingerprinting is restrictionfragment length polymorphism (RFLP). In this method, DNA isextracted from a sample and cut into segments using specialrestriction enzymes. RFLP focuses on segments that containsequences of repeated DNA bases, which vary widely from personto person. The segments are separated using a laboratorytechnique called electrophoresis, which sorts the fragments bylength. The segments are radioactively tagged to produce a visualpattern known as an autoradiograph, or "DNA fingerprint," on X-ray film.

A newer method known as short tandem repeats (STR) analyzesDNA segments for the number of repeats at 13 specific DNA sites.The chance of misidentification in this procedure is one in severalbillion. Yet another process, PCR, is used to produce multiplecopies of segments from a very limited amount of DNA (as littleas 50 molecules), enabling a DNA fingerprint to be made from asingle hair. Once a sufficient sample has been produced, thepattern of the alleles from a limited number of genes is comparedwith the pattern from the reference sample.

1. Paternity and MaternityBecause a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity. The patterns are so specific that a parental VNTR pattern can be reconstructed even if only the children's VNTR patterns are known (the more children produced, the more reliable the reconstruction). Parent-child VNTR pattern analysis has been used to solve standard father-identification cases as well as more complicated cases of confirming legal nationality and, in instances of adoption, biological parenthood.

2. Criminal Identification and ForensicsDNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence. VNTR patterns are also useful in establishing the identity of a homicide victim, either from DNA found as evidence or from the body itself.

3. Personal IdentificationThe notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the foreseeable future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is both expensive and impractical. Social security numbers, picture ID, and other more mundane methods are much more likely to remain the prevalent ways to establish personal identification.

Dolly (5 July 1996 – 14 February 2003) was a femaledomestic sheep, and the first mammal to be clonedfrom an adult somatic cell, using the process ofnuclear transfer.

She was cloned by Ian Wilmut, Keith Campbelland colleagues at the Roslin Institute, part of theUniversity of Edinburgh, Scotland.

She has been called "the world's most famoussheep" by sources including BBC News andScientific American.

Clones are organisms that are exact genetic copies.Every single bit of their DNA is identical.

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Cells taken from a six-year-old Finnish Dorsetewe and cultured in a lab.

277 cells then fused with 277 unfertilized eggs(each with the nucleus removed)

29 viable reconstructed eggs survived and wereimplanted in surrogate Blackface ewes.

1 gave birth to Dolly

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Dolly with her foster mother

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Although her nuclear genome came from theFinn Dorset ewe, her mitochondria came fromcytoplasm of the Scottish Blackface ewe.Mitochondria carry their own genome and sowith respect to the genes in mitochondrialDNA, she is not a clone of the Finn Dorsetparent.

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On 14 February 2003, Dolly was euthanisedbecause she had a progressive lung disease andsevere arthritis.

A Finn Dorset such as Dolly has a lifeexpectancy of around 11 to 12 years, but Dollylived to be 6.5 years old.

A post-mortem examination showed she had aform of lung cancer, which is a fairly commondisease of sheep and is caused by the retrovirusJSRV

Bioprocess/fermentationTechnology

Enzyme TechnologyBiofuel

Food and beverages

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Bioprocess or fermentation technology is animportant component of most ‘old’ and ‘new’biotechnology processes and will normallyinvolve complete living cells (microbe,mammalian or plant), organelles or enzymes asthe biocatalyst, and will aim to bring aboutspecific chemical and/or physical changes inbiochemical materials derived from themedium.

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The very beginnings of fermentationtechnology, or as it is now better recognised,bioprocess technology, were derived in partfrom the use of microorganisms for theproduction of foods such as cheeses, yoghurts,sauerkraut, fermented pickles and sausages,soya sauce and other oriental products, andbeverages such as beers, wines and derivedspirits.

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New products are increasingly being derived from microbial, mammalian and plant cell fermentations, namely the ability:

(1) to overproduce essential primary metabolites such as acetic and lactic acids, glycerol, acetone, butyl alcohol, organic acids, amino acids, vitamins and polysaccharides

(2) to produce secondary metabolites (metabolites that do not appear tohave an obvious role in the metabolism of the producer organism) suchas penicillin, streptomycin, cephalosporin, giberellins, etc.(3) to produce many forms of industrially useful enzymes, e.g.

exocellularenzymes such as amylases, pectinases and proteases, and intracellularenzymes such as invertase, asparaginase, restriction endonucleases,etc.(4) to produce monoclonal antibodies, vaccines and novel recombinantproducts, e.g. therapeutic proteins.

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Bioprocessing in its many forms involves amultitude of complex enzyme-catalysedreactions within specific cellular systems, andthese reactions are critically dependent on thephysical and chemical conditions that exist intheir immediate environment. Successfulbioprocessing will only occur when all theessential factors are brought together.

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ADVANTAGES

Complex molecules such as proteins and antibodies cannot be produced by chemical means.

Bioconversions give higher yields.

Biological systems operate at lower temperatures, near neutral pH, etc.

Much greater specificity of catalytic reaction.

Can achieve exclusive production of an isomeric compound.

DISADVANTAGES

Can be easily contaminated with foreign unwanted microorganisms, etc.

The desired product will usually be present in a complex product mixture requiring separation.

Need to provide, handle and dispose of large volumes of water.

Bioprocesses are usually extremely slow when compared with conventional chemical processes.

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Downstream processing refers to the isolation andpurification of a biotechnologically formedproduct to a state suitable for the intended use.

Within these products there will be considerablevariation in molecular size and chemicalcomplexity, and a wide range of separationmethods will be required for recovery andpurification.

While many of the products are relatively stable instructure others can be highly labile and requirecareful application of the methodology.

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An example of the effort expended indownstream processing is provided by theplant Eli Lilly built to produce human insulin(Humulin). Over 90% of the 200 staff wereinvolved in recovery processes.

Thus, downstream processing ofbiotechnological processes represents a majorpart of the overall costs of most processes

Improvements in downstream processing willbenefit the overall efficiency and costs of theprocesses.

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Enzymes are complex globular proteins present in living cells where they act as catalysts that facilitate chemical changes in substances.

Although enzymes are only formed in living cells, many can be extracted or separated from the cells and can continue to function in vitro.

This unique ability of enzymes to perform their specific chemical transformations in isolation has led to an ever-increasing use of enzymes in industrial and food processes, bioremediation and in medicine, and their production is collectively termed enzyme technology.

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The activity of an enzyme is due to its catalytic nature. An enzyme carries out its activity without being consumed in the reaction, while the reaction occurs at a very much higher rate when the enzyme is present.

Enzymes are highly specific and function only on designated types of compounds, the substrates and a minute amount of enzyme can react with a large amount of substrate.

The catalytic function of the enzyme is due not only to its primary molecular structure but also to the intricate folding configuration of the whole enzyme molecule.

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Enzymes are non-toxic and biodegradable (an attractive ‘green’ issue) and can be produced especially from microorganisms in large amounts.

Enzyme technology embraces production, isolation, purification and use in soluble or immobilised form.

Commercially produced enzymes will undoubtedly contribute to the solution of some of the most vital problems with which modern society is confronted, e.g. food production, energy shortage and preservation, improvement of the environment, together with numerous medical applications.

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It is estimated that the world market for enzymes is over US$2 billion and will double over the next decade.

Enzyme production and utilisation is one of the most successful areas of modern biotechnology and has increased by 12% annually over the past ten years.

There are now over 400 companies worldwide involved in enzyme production, with European companies dominating (60%) and the USA and Japan with 30%.

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Biological detergents –

Amylase enzymes in detergents for machine dishwashing to remove resistant starch residues

Baking industry –

Proteinase enzymes in Biscuit manufacture to lower the protein level of the flour

Brewing industry - Enzymes produced from barley during mashing stage of beer production.

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Dairy industry –

Rennin by Manufacture of cheese, used

to split protein

Lipases and Lactases - Break down lactose to glucose and galactose

Starch industry –

Immobilised enzymes for Production of high-fructose syrups

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Textile industry –

Various bacterial enzymes which Generally preferred for desizing since they are able to withstand working temperatures up to 100–110◦C

Leather industry –

Enzymes found in dog and pigeon dung used in process of bating for softening leather

Medical and pharmaceutical –

Pancreatic trypsin for Digestive aid formulations and treatment of inflammations

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Energy for industrial, commercial and residential purposes, electricity generation and transportation is primarily supplied by fossil fuels (coal, gas and oil) and nuclear power.

It is now widely believed that climate change is strongly linked to the increased level of greenhouse gases in the atmosphere, and that human activity especially through the combustion of fossil fuels is a major contributing factor.

Bioenergy (biofuel) is basically the production of combustible/usable energy from biological sources.

This can be liquid fuels, e.g. bioethanol, biodiesel;

gaseous fuels, e.g. methane

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Sugar crops Sugar cane, sugar beet: sugars extracted and fermented to bioethanol.

Starch crops Maize (corn), barley, wheat, oats, cassava: starch enzymatically hydrolysed to sugars and fermented to bioethanol.

Cellulose crops/wastes Straw, bagasse, woody wastes, cropped trees: the hemicelluloses can be enzymaticallyhydrolysed to sugars and fermented to bioethanol.

Oil crops Rapeseed, linseed, sunflower, castor oil, groundnut: oils extracted and transesterified to biodiesel.

Organic wastes/manures Complex microbial fermentations to methane/ methanol.

Solid energy crops Coppiced trees, sorghum, reeds, grasses, Eucalyptus: direct burning alone or with other conventional sources, e.g. coal.

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A major challenge to creating a sustainable future for the world’s populations will be to secure adequate food supplies for the majority.

By 2030 it has been estimated that the size of urban populations will be at least twice that of rural, agricultural-based populations.

What role can traditional and new biotechnology play in achieving food sustainability?

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Food biotechnology is concerned with the integration of both modern biological knowledge and techniques and current bioengineering principles in food processing and preservation.

The impact of biotechnology on the food and beverage industries can be anticipated in two directions:

(1) agronomic, i.e. increased plant and animal yields, extended growth range and environments, from which the farmers will mainly benefit

(2) non-agronomic, i.e. improving plants and microorganisms to provide benefits to the food producer, retailer or consumer

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SCP

Probiotics

Amino acids, vitamins, flavours and sweeteners

Alcoholic beverages

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AntibioticsVaccines

Monoclonal AbsBiopharmaceuticals

PharmacogeneticsDiagnostics

Gene Therapy

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Now a days in industrialised societies, infectious diseases are no longer the main threat to life but rather it is the chronic diseases (cancer, cardiovascular disease, Alzheimer’s disease, etc.)

New medical treatments based on new biotechnology are appearing almost daily in the marketplace. These include: (a) therapeutic products (hormones, regulatory proteins, antibiotics);(b) prenatal diagnosis of genetic diseases; (c) vaccines; (d) immuno-diagnostic and DNA probes for disease identification; and (e) genetic therapy.

This are the largest commercially developed area of new biotechnology with massive present and future markets.

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The discovery in 1929 by Alexander Fleming that a fungus called Penicillium notatum could produce a compound, penicillin, selectively able to inactivate a wide range of bacteria, without unduly influencing the host, set in motion scientific studies.

Antibiotics are antimicrobial compounds produced by living microorganisms, and are used therapeutically and sometimes prophylatically in the control of infectious diseases.

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Over 4000 antibiotics have been isolated but only about 50 have achieved wide usage.

The other antibiotic compounds failed to achieve commercial importance for reasons such as toxicity to humans or animals, ineffectiveness or high production costs.

Antibiotics have been extensively used in medicine since about 1945 with the arrival of penicillin.

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Antibiotics that affect a wide range of microorganisms are termed broad spectrum

for example, choramphenicol and the tetracyclines, which can control such unrelated organisms as Rickettsia, Chlamydia and Mycoplasma species.

In contrast, streptomycin and penicillin are examples of narrow spectrum antibiotics, being effective against only a few bacterial species.

Most antibiotics have been derived from the actinomycetes (filamentous bacteria) and the mould fungi.

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The production of antibiotics has undoubtedly been a highly profitable part of the pharmaceutical industries in the industrialised world.

The world market for antibiotics and anti-fungalswas worth over US$26 billion in 2001 and is the most valuable segment of the total pharmaceutical market.

In 1992, the cephalosporins were one of the largest business sectors in the global pharmaceutical market with sales at US$8.3 billion.

Due to biotechnology innovation, as world sales of antibiotics increase their production costs have decreased.

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According to the World Health Organization, each year more than 17 million people die from infectious diseases

Human ingenuity has permitted humankind to protect itself against many infectious diseases through vaccination – a process that has been successful for more than a century

Vaccines are preparations of dead microorganisms (or fractions of them), or living attenuated or weakened microorganisms, that can be given to humans or animals to stimulate their immunity to infection.

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Consequently, in more recent times, world vaccine manufacturing has become dominated by five companies:

Chiron, GlaxoSmithKline, Merck, Sanefi-Pasteur and Wyeth.

Vaccines have long processing times and are difficult and expensive to produce.

the worldwide market for vaccines in 2006 was US$10 billion.

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Vaccines have eliminated smallpox from the world and polio from the Northern hemisphere and have greatly reduced measles, rubella, tetanus, diphtheria and meningitis in many countries, saving countless millions of lives.

Vaccines still remain the most cost effective intervention available for preventing death and disease.

At the present time humankind is under severe threat of the spread of viral diseases, e.g. human immunodeficiency virus (HIV), mosquito-borne West Nile virus, severe acute respiratory syndrome (SARS) and a possible highly virulent influenza pandemic on a scale similar to the fatal outbreaks.

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A significant new development in medically related biotechnology has been the ability to produce monoclonal antibodies.

Monoclonal antibodies are now finding wide applications in diagnostic techniques requiring highly specific reagents for the detection and measurement of soluble proteins and cell surface markers in blood transfusions, haematology, histology, microbiology and clinical chemistry, as well as in other non-medical areas.

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(1) Cancer diagnosis and therapy

(2) Diagnosis of pregnancy

(3) Diagnosis of sexually transmitted diseases

(4) Prevention of immune rejection of organ implants

(5) Purification of industrial products

(6) Detection of trace molecules in food, agriculture and industry

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Gene therapy can be considered as any treatment strategy that involves the introduction of genes or genetic material into human cells to eliminate disease.

The aim of gene therapy is to replace or repress defective genes with sequences of DNA that encode a specific genetic message.

Within the cells, the DNA molecules may provide new genetic instructions to correct the host phenotype.

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Gene therapy is a complex series of events relying heavily on new biotechnological techniques.

Therapy will require a full understanding of the mechanism by which the defective or unusual gene exerts its effect on the individual, and an ability to switch off the defective gene and to substitute a healthy gene copy.

It is truly a multidisciplinary activity involving skills in molecular biology, cell biology, virology, pharmacology, clinical application and patient interaction.

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The USA is the undoubted world leader in gene therapy research and application.

Gene therapy products will present difficult challenges in development, manufacturing, testing and distribution.

Gene therapy technology is not without risk and following several high profile events there continues to be considerable focus on the ethics and safety of gene therapy trials.

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READING IMAGES

Biotechnology by John E. Smith (5th Ed.)

Biotechnology by B D Singh

Genetic Engineering by Nicholl

http://www.roslin.ed.ac.uk/public-interest/dolly-the-sheep/a-life-of-dolly/

http://www.cdfd.org.in/servicespages/dnafingerprinting.html

1 & 2: http://www.roslin.ed.ac.uk/public-interest/dolly-the-sheep/a-life-of-dolly/

3-10: Biotechnology by John E. Smith (5th Ed.)