BIOTECHNOLOGYBIOTECHNOLOGY8 HOURS 12 MARKS8 HOURS – 12 MARKS

• Karl Ereky• Karl Ereky• An Industrial or laboratory

technique which deals withthe use of living organisms org gtheir products for the welfareof mankindof mankind

• Genetic Engineering / rDNATechnologygy

• DNA Fingerprinting• Monoclonal antibodies• Monoclonal antibodies• Human Genome Project• Gene Therapy• Improvement of crop plantsImprovement of crop plants• Improvement of animals

H d d S f d• Hazards and Safeguards

GENETIC ENGINEERINGf• Manipulation of the genes at

the molecular level• TOOLS:

Vectors pUC 18Vectors – pUC 18Enzymes – RENs & Ligasesy gHost cells – Escherichia coliBi t F tBioreactors - Fermenters

pUC18

HUMULIN• Human insulin produced by

genetic engineeringg g g• pUC 18 or pBR322 – E coli –

REN – Ligase – Host cell (EREN Ligase Host cell (Ecoli) – Bioreactor withlactose – Cyanogen bromidelactose Cyanogen bromide– Sodium sulphite / Sodiumdisulphonatedisulphonate

DNA FINGERPRINTINGDNA FINGERPRINTING• For establishment of genetic

l ti hirelationship• VNTRs – Variable Number of

Tandem Repeats• RFLP Restriction Fragment• RFLP – Restriction Fragment

Length Polymrphism

MONOCLONAL ANTIBODIESI l i ll & h i ll• Immunologically & chemicallypure and similar antibodiesproduced from clonal cultureof hybridomas

• Hybridomas are hybrid cellsobtained by the fusion of twoobtained by the fusion of twodifferent cells

• PEG (Polyethylene Glycol)• PEG (Polyethylene Glycol)

GENE THERAPY• Replacement of a defective or

non – functional gene with aghealthy gene

• Somatic gene therapy & Germ• Somatic gene therapy & Germline gene therapy

• In vivo gene therapy & Ex vivogene therapygene therapy

HUMAN GENOME PROJECT (HGP)

• Identification of the sequenceof nucleotides in all the 46of nucleotides in all the 46human chromosomes and alsoidentification of the locationidentification of the locationof different genes

• Data is useful in studyingvarious genetic diseasesvarious genetic diseases

IMPROVEMENT OF CROP PLANTS• SelectionSelection• Plant introduction

H b idi ti I t ifi / • Hybridisation – Intraspecific / Interspecific / Intergeneric).

• Induction of polyploidy• Mutation breedingg• Tissue culture• Transgenesis Golden rice• Transgenesis – Golden rice

IMPROVEMENT OF ANIMALS• Traditional practices:

Inbreeding and cross breeding Inbreeding and cross breeding • Stem cell culture• Transgenesis

Multiple ovulation embryo • Multiple ovulation – embryo transfer (MOET)

HAZARDS & SAFEGUARDSHazards:• Threat to human health by GMOs

(Toxins and allergens)• Damage to native and traditional

plant species (Genetic pollution)• Accidental hybridisation of GMOs

with weeds and pests (‘Super d ’ d ‘S t ’)weeds’ and ‘Super pests’)

• Replacement of traditional crop i ti (Bi di it d l ti )varieties (Biodiversity depletion)

SAFEGUARDS: • Thorough research and

experimentationp• Regulation to ensure safety

of GMOs by regulatoryof GMOs by regulatoryauthorities before they are

l d i t th k t freleased into the market forconsumption

ONE Mark Questions

1.What is genetic engineering ?The manipulation of thegenetic material at thegmolecular level

ORORThe selective alteration ofthe genetic make up of cells /individuals.

2. Define gene cloning ?The production of multipleThe production of multiplecopies of a desired gene.

3. What are bioreactors ?Large sized containers usedLarge sized containers usedfor the bulk growth ofmicrobes & to form productsmicrobes & to form productsfrom them by biocatalysedreactions like fermentationreactions like fermentation.

4. What are plasmids ?Extra-chromosomal, circular,DNA molecules present in thecytoplasm of certain bacteria.y p

ORExtra chromosomal circularExtra-chromosomal, circularDNA molecules of bacteriawhich are used as vectors /which are used as vectors /cloning vehicles in rDNAtechnologytechnology.

5. Define monoclonal antibodies ?Chemically & immunologicallyChemically & immunologicallypure antibodies obtainedthrough clonal cultures ofthrough clonal cultures ofhybridomas

6. What are restriction enzymes ?6. What are restriction enzymes ?Enzymes that naturally occur inbacteria which can cut DNA atbacteria which can cut DNA atspecific regions that are knownas palindromic sequencesp q

EcoRI cutting DNA at its recognition sequenceEcoRI cutting DNA at its recognition sequence

7.What is recombinant DNA ?Hybrid DNA consisting of DNAHybrid DNA consisting of DNAfrom two different sources ororganismsorganisms

8. What is gene therapy ?technique of replacing a non– functional or defective gene– functional or defective genewith a healthy gene to cure adiseasedisease

9 Wh t i DNA fi i ti ?9.What is DNA fingerprinting ?Technique of comparing thespecific sequences ofnucleotides in DNA fromdiff t ddifferent sources andestablishing the geneticrelationship between themrelationship between them

10.What are hybridomas ?S ti ll h b id f dSomatic cell hybrids formedby the fusion of two differentcellscells

11.What is artificial insemination ?The technique of injecting theThe technique of injecting thesemen of a desired bull into theuterus of a cowuterus of a cow

12.What is ‘sperm sexing’ ?The technique used in dairyThe technique used in dairyfarm by which the spermshaving X (gynosperm) and Yhaving X (gynosperm) and Y(androsperm) chromosomes areseparatedseparated

TWO Mark Questions

1.Mention the functions of RENsand DNA ligase in recombinantand DNA ligase in recombinantDNA technology.Restriction enzymes cut theRestriction enzymes cut theDNA at palindromic sequencesand help in isolating thed i ddesired gene.DNA ligase joins the desiredgene to the plasmid or thegene to the plasmid or thegenome of the recipient toproduce rDNA.produce rDNA.

2. Draw a labelled sketch of pUC18.

3. What are bioreactors ?These are large sizedThese are large sizedcontainers used for the bulkgrowth of microbes and to formgproducts from them bybiocatalysed reactions likeyfermentation.Types:ypStirred tank bioreactorAirlift fermentation bioreactorAirlift fermentation bioreactor

4. Mention four applications of DNAfingerprintingfingerprinting.

• Solving parental disputesD t ti f i (f i• Detection of crime (forensicscience)

• Study of change in the gene(mutation) and its effects

• Identification of dead persons indisasters like flood, cyclones, y

5. What are monoclonal antibodies ?Mention two of their applications.Mention two of their applications.Chemically & immunologically pureantibodies obtained from clonalantibodies obtained from clonalcultures of hybridomasApplications:

• In the detection of specific antigensin a patient suffering from a disease.

• In ELISA technique to diagnose AIDSIn ELISA technique to diagnose AIDS

6. Mention two objectives andtwo applications of HumanppGenome Project (HGP).

Objectives:Objectives:• To sequence the 3.2 billion

base pairs in the humanbase pairs in the humangenome

• To identify all the genes• To identify all the genesand their location on thechromosomeschromosomes

(Continued)Applications:Applications:• To understand the functioning

of genes and gene regulationof genes and gene regulation• To understand, diagnose and

treat several genetic diseases

7. Mention four applications oftissue culturetissue culture.

• Micropropagation of plants ofagriculture, horticulture,floriculture importance

• To get secondary metaboliteslike alkaloids, volatile oils,like alkaloids, volatile oils,tannins, resins, etc. fromcultured tissuescultured tissues

(C ti d)(Continued)• To obtain disease - free

plants by meristem culture• To obtain interspecificTo obtain interspecific

hybrids by culturing partsof young hybrid embryosof young hybrid embryos

8. What are transgenic plants ?Mention one example with itspimportance.Genetically modified plants thatGenetically modified plants thatcontain one or more genes of adifferent organism in theirdifferent organism in theirgenome.Ex: Golden riceEx: Golden riceThis rice is used to overcomevitamin A deficiencyvitamin A deficiency

9. What is artificial insemination ?Mention two of its advantages.The technique of injecting thesemen of a desired bull into the

t futerus of a cow.Advantages:• Semen collected from a single bullcan be used to inseminate manyfemalesfemales.

• Semen can be stored for a longtime by cryopreservation ( 79°C)time by cryopreservation (- 79°C).

10.What are stem cells ? Mentiontwo of their typestwo of their types.Multipotent animal cells whichh th it t di id dhave the capacity to divide andspecialize or differentiate intodiff t tidifferent tissues.Types:

Embryonic stem cellsAdult stem cellsAdult stem cells

FIVE Mark Questions

1. Explain the steps involved in theproduction of human insulin by

ti i iTwo methods:B i RNA th t h d

genetic engineering.

• By using mRNA that has codesfor proinsulinBy synthesising DNA for A and B• By synthesising DNA for A and Bchains separately usingoligonucleotides (based on theoligonucleotides (based on theknowledge of genetic code) andsubsequently using them for thepurpose

2. Explain the steps involved in DNA fingerprinting.g p g

• Collection of sample• Extraction of DNA• Amplification of DNA by PCR• Restriction digestion using REN• Separation of restriction fragments

by electrophoresisTreatment with alkali to get ssDNA• Treatment with alkali to get ssDNA

• Southern blotting• Hybridisation with DNA probe• Hybridisation with DNA probe• Autoradiography• Comparison & analysis• Comparison & analysis

3. What is gene therapy ?Explain its main types.Mention two applicationsMention two applicationsof gene therapy.

• Gene therapy is a technique ofreplacing a defective or non -p gfunctional gene by a healthygene to correct or cure agene to correct or cure adisease.Th t i t f• There are two main types ofgene therapy. They are;

Somatic gene therapy &Germ line gene therapyGerm line gene therapy

Somatic gene therapy:• Replacement of a defective gene

of somatic cells by a normalhealthy gene.

• It is non-heritable.Germ - line gene therapy:• Replacement of a defective gene

in the gametes (reproductivell ) th f tili d bcells) or the fertilized egg by a

normal healthy gene.It i h it bl• It is heritable.

Applications of gene therapy:• In the treatment of SCID

In the treatment of cystic • In the treatment of cystic fibrosis

4 Explain the technique4.Explain the techniqueinvolved in the production

f l l tib diof monoclonal antibodies.

• Mice are injected with a specificantigen to stimulate antibodyantigen to stimulate antibodyproduction.S l i d ft f• Spleen is removed after a fewdays to isolate the antibody

d i ll (B ll )producing cells (B - cells).• Cancer is induced in a different

set of mice & myeloma cells areisolated from the bone marrow.so ated o t e bo e a o

• Antibody producing cells aremixed with myeloma cells inHAT (hypoxanthine aminopterinthymidine) medium in vitro andth ll d t f i ththe cells are made to fuse in thepresence of polyethylene glycol(PEG) to get hybridomas(PEG) to get hybridomas.

• Hybridomas are screened,t d lt d d l dseparated, cultured and cloned

to get antibodies. MAbs arelater isolated and purifiedlater isolated and purified.

5. Briefly explain the technique of tissue culture.

• Cleaning and sterilisation ofglassware and instrumentsPreparation of medium• Preparation of medium

• Selection of explant & surfacesterilisationsterilisation

• Inoculation• CallogenesisCallogenesis• Morphogenesis (Rhizogenesis

& caulogenesis – Hormones togbe mentioned)

• HardeningT l t ti• Transplantation

6. Explain the steps involved in6. Explain the steps involved inthe production of golden riceby genetic engineeringby genetic engineering.

I l ti f t f i t t• Isolation of two genes of interestfrom daffodil and one gene frombacterium Erwinia uredovorabacterium, Erwinia uredovorausing specific RENs

• Ti plasmid of Agrobacterium• Ti plasmid of Agrobacteriumtumefaciens is cut open usingspecific REN and these genes arespecific REN and these genes areinserted into it using DNA ligaseto get recombinant DNAg

• The rDNAs are introduced backinto bacteria by electroporationyand cultured

• Culture is screened, transformedbacteria are isolated and arebacteria are isolated and areallowed to transfect rice embryosto pass on the desired genes.

• The three foreign genes ofinterest get incorporated into the

fgenome of rice and the embryosdevelop into transgenic plant(golden rice) having the capacity(golden rice) having the capacityto synthesise β – carotene whichis a precursor of vitamin A.is a precursor of vitamin A.

7.Explain the technique ofmultiple ovulation andembryo transfer employedembryo transfer employedin cattle breeding.

• Superovulation:FSH and LHFSH and LH

• Artificial insemination:• Embryo recovery:

Embryos that develop areEmbryos that develop areflushed out on 6th or the7th day (can be preserved7 day (can be preservedby cryopreservation forfuture use)future use)

• Estrous synchronization:Prostaglandin injectiong jto surrogate cows.

• Embryo transfer:Embryo transfer:Into the uterine hornsof surrogate cowsof surrogate cows

Give reasons:

1.Plasmids are considered asvery important tools in rDNAvery important tools in rDNAtechnology. OR Plasmids are

ll d t i DNAcalled vectors in rDNAtechnology.They are used as a carriers ofdesired DNA to produce rDNApwhich can be subsequentlyused for gene cloning.used o ge e c o g

2.Restriction endonucleases2.Restriction endonucleasesare called ‘molecular knives’or ‘molecular scissors’or ‘molecular scissors’.They cut the DNA at specificsites called palindromicsequences and help inq pisolating the desired gene.

3. DNA fingerprinting cannot be3. DNA fingerprinting cannot beemployed for distinguishingidentical twins.identical twins.Identical twins are producedf i l t dfrom a single zygote andtherefore, the genetic materiali b th ill b thin both will be the same.

4. Plantlets raised through tissue4. Plantlets raised through tissueculture can be considered asclonesclones.All the Plantlets obtained are

d t f it ti di i iproducts of mitotic divisions(no recombinations occur) ofthe cells in explants.

5 Golden rice is a transgenic5.Golden rice is a transgenicplant.Golden rice has three foreigngenes (two from daffodil andone from Erwinia) in itsgenome which direct thegsynthesis of β-carotene.

6. Prostaglandin is injected intoth i i t d ithe recipient cows duringMOET procedure.It is to bring all the recipientcows to estrous or reproductivepstage simultaneously so thatembryos could be transferred toembryos could be transferred tothem.

Define palindromic sequences ?‘Invert repeats’ with bifold‘Invert repeats’ with bifoldsymmetry that have the samenucleotide sequences whennucleotide sequences whenread in 5’ 3’ on both thestrands of DNAstrands of DNA

Define transgenesis.T h i f t f iTechnique of transfering adesired gene from one organismto another is called transgenesis.

What is callus ?It is an unorganized mass ofundifferentiated cells producedby the explant in tissue culture.

Mention four applications ofbiotechnology.biotechnology.• Manufacture of alcoholic drinks• Manufacture of antibiotics• Manufacture of antibiotics,vaccines, vitamins, etc.

• In multiplying high yielding or• In multiplying high yielding ordisease resistant or draughttolerant plants that aretolerant plants that areobtained by GE

• Treatment of genetic disordersTreatment of genetic disorders

What are transgenic animals ?Mention two examplesMention two examples.These are genetically modifiedanimals which contain one ormore genes of a differentmore genes of a differentorganism in their genome.Ex: “Rosie” (C l t lb i )Ex: “Rosie” (Cow – α lactalbumin)

“Tracy” (α, 1 – antitrypsin)y (