1. DNA cloning is the process of making multiple copies of a piece of DNA by inserting it into a plasmid and using bacteria to replicate the plasmid, thereby producing many copies of the DNA fragment. 2. The DNA fragment of interest is inserted into a plasmid using enzymes that cut and join DNA fragments. The plasmid is then introduced into bacteria which replicate, making many copies of the plasmid and DNA insert. 3. DNA cloning is used to mass produce DNA or proteins, such as using E. coli bacteria to produce human insulin after inserting the human insulin gene into a plasmid. The bacteria then act as "factories" to produce large quantities of the protein.

1. DNA cloning
isthe processof makingmultiple,identical copiesof aparticularpiece of DNA.Ina typical DNA
cloningprocedure,the gene orotherDNA fragmentof interest(perhapsagene fora medically
importanthumanprotein) isfirstinsertedintoacircularpiece of DNA calleda plasmid.The insertion
isdone usingenzymesthat“cut andpaste” DNA,andit producesa molecule of recombinantDNA,or
DNA assembledoutof fragmentsfrommultiple sources.
A circularpiece of plasmidDNA hasoverhangsonitsendsthat match those of a gene fragment.The
plasmidandgene fragmentare joinedtogethertoproduce a gene-containingplasmid.Thisgene-
containingplasmidisanexample of recombinantDNA,oraDNA molecule assembledfromDNA
frommultiple
Next,the recombinantplasmidisintroducedintobacteria.Bacteriacarryingthe plasmidare selected
and grownup.As theyreproduce,theyreplicatethe plasmidandpassiton to theiroffspring,making
copiesof the DNA it contains.
What isthe pointof makingmanycopies of a DNA sequence inaplasmid?Insome cases,we need
lotsof DNA copiestoconduct experimentsorbuildnew plasmids.Inothercases,the piece of DNA
encodesauseful protein,andthe bacteriaare usedas“factories”to make the protein.Forinstance,
the humaninsulingene isexpressedinE.coli bacteriato make insulinusedbydiabetics.
Steps of DNA cloning
DNA cloningisusedformany purposes.Asanexample,let'ssee how DNA cloningcanbe usedto
synthesize aprotein(suchashumaninsulin) in bacteria.The basicstepsare:
1. Cut openthe plasmidand"paste"inthe gene.Thisprocessreliesonrestrictionenzymes(which
cut DNA) and DNA ligase (whichjoinsDNA).
2. Insertthe plasmidintobacteria.Use antibioticselectiontoidentifythe bacteriathattookup the
plasmid.
3. Grow up lotsof plasmid-carryingbacteriaanduse themas"factories"tomake the protein.
Harvestthe proteinfromthe bacteriaand purifyit.
Dolly the Sheep
Dollythe sheep,asthe firstmammal to be clonedfroman adultcell,isbyfar the world'smost
famousclone.However,cloninghasexistedinnature since the dawnof life.Fromasexual bacteriato
‘virginbirths’inaphids,clonesare all aroundusandare fundamentallynodifferenttoother
organisms.A clone hasthe same DNA sequence asitsparentand sotheyare geneticallyidentical.
Several cloneshadbeenproducedinthe labbefore Dolly,includingfrogs,mice,andcows,whichhad
all beenclonedfromthe DNA fromembryos.Dollywasremarkable inbeingthe firstmammal tobe
clonedfroman adultcell.Thiswasa major scientificachievementasitdemonstratedthatthe DNA
fromadultcells,despite havingspecialisedasone particulartype of cell,canbe usedto create an
entire organism.
To produce Dolly,scientistsusedanuddercell fromasix-year-oldFinnDorsetwhitesheep.Theyhad
to finda wayto 'reprogram' the uddercells - to keepthem alive butstopthemgrowing – which
theyachievedbyalteringthe growthmedium(the ‘soup’inwhichthe cellswere keptalive).

2. Thentheyinjectedthe cell intoanunfertilisedeggcell whichhadhaditsnucleusremoved,and
made the cellsfuse byusingelectrical pulses.The unfertilisedeggcell came fromaScottish
Blackface ewe.Whenthe researchteamhadmanagedto fuse the nucleusfromthe adultwhite
sheepcell withthe eggcell fromthe black-facedsheep,theyneededtomake sure thatthe resulting
cell woulddevelopintoanembryo.
Polymerase chain reaction (PCR)
is a commonlaboratorytechnique usedtomake manycopies(millionsorbillions!) of aparticular
regionof DNA.ThisDNA regioncanbe anythingthe experimenterisinterestedin.Forexample,it
mightbe a gene whose functionaresearcherwantstounderstand,ora geneticmarkerusedby
forensicscientiststomatchcrime scene DNA withsuspects.
Typically,the goal of PCRis to make enoughof the targetDNA regionthatit can be analyzedorused
insome otherway.For instance,DNA amplifiedbyPCRmaybe sentfor sequencing,visualizedbygel
electrophoresis,orclonedintoaplasmidforfurtherexperiments.
PCR isusedinmany areasof biologyandmedicine,includingmolecularbiologyresearch,medical
diagnostics,andevensome branchesof ecology.
Biotechnology products
•AntibioticsMedicines whichare useful againstbacterialactionsandare obtainedfromliving
organismsVaccinesWeakenedordeadmicrobesforinoculationinthe bodytoproduce immunity
againstthe infectionthatrespectivehealthymicrobe cancause
•GeneticallyModifiedOrganisms(GMO’s) Organismswithmodifiedgeneticmaterial ororganisms
withforeignDNA.Types: GeneticallyModifiedAnimals, GeneticallyModifiedBacteria,
GeneticallyModifiedPlants,GeneticallyModifiedFlowers,GeneticallyModifiedFruit
•ClonedAnimals:Animalswhichhave same characteristicsasthatof theirparentsDollysheep
•HormonesVitaminsArtificialOrgans
• ProteinsIndustrial biotechnologyhasproducedenzymesforuse inourdailylives.Alcohol
productionisone of the mostbasicapplicationsof industrialbiotechnology.Enzymesandmicrobes
are twocommontoolsusedinindustrial biotechnology. Bioplastics,made frombiopolymersare
alreadyutilizedinplasticfoodpackaging,mobilephone cases,sunglasses.Polyesterisa synthetic
polymerfiberproducedfromfossil fuel andisusedtomake clothing,blankets,carpets,andother
fabrics. Industrial Biotechnologycanpresentasignificantopportunitytodevelopmedicinesthat
have beendifficulttoproduce viaothermeansdue topurityissues.
Tissue Culture
A methodof biological researchinwhichfragmentsof tissue fromananimal orplantare transferred
to an artificial environmentin whichtheycancontinue tosurvive andfunction.The culturedtissue
may consistof a single cell,apopulationof cellsorpartof an organ.
Transformationisthe stepinthe geneticengineeringprocesswhereanew gene (transgene) is
insertedintoasingle plantcell.
There are several thingsthatmusthappencorrectlyfora cell tobe successfullytransformed:
The newgene mustbe deliveredintothe nucleusof acell andinsertintoa chromosome.

3. The cellsthat receive the newgene muststayalive.
The cellsand plantsthatcontainthe new gene mustbe easilyidentifiable(selectablemarkers).
The transformedcell mustdivide andgive rise toanentire plant.
The locationwhere the transgene insertsintothe chromosome mustnotinterfere withthe
expressionof the gene.
The newgene mustnot insertintoanexistinggene inthe chromosome thatinfluencessurvival of
the plantcell or productivityof the entire plant.
Tissue culture iswhenclustersof undifferentiatedcells,calledcallus,are grown inculture.
What is gene therapy?
Gene therapyiswhenDNA isintroducedintoapatientto treata geneticdisease.The new DNA
usuallycontainsafunctioninggene tocorrectthe effectsof adisease-causingmutation.Gene
therapyusessectionsof DNA (usuallygenes)totreator preventdisease.The DNA iscarefully
selectedtocorrectthe effectof a mutatedgene thatis causingdisease.