Chapter 4.1.Concept of Molecular Cloning

7/23/2019 Chapter 4.1.Concept of Molecular Cloning

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CHAPTER 4

Cloning, Expression

and Analysis of Genesand Their Products

4.1 Concept ofmolecular cloning


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AN OVERVIEW

Molecular cloning takes advantage of te fact tat tece!ical structure of "NAis funda!entall# te sa!e in

all living organis!s$Terefore% if an# seg!ent of "NA fro! an# organis!is inserted into a "NA seg!ent containing te!olecular se&uences re&uired for "NA re'lication% andte resulting reco!(inant "NA is introduced into teorganis! fro! )ic te re'lication se&uences )ere

o(tained$Ten te foreign "NA )ill (e re'licated along )itte ost cell*s "NA in te transgenicorganis!$
http://en.wikipedia.org/wiki/DNAhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Transgenichttp://en.wikipedia.org/wiki/Transgenichttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/DNA


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THE CONCEPT

Molecular cloningis a set of e+'eri!ental !etodsin !olecular (iolog# tat are used to asse!(le

reco!(inant "NA !olecules and to direct teirre'lication)itin ost organis!s

or Molecular cloning is a (asic tecni&ue used in a!olecular (iolog# la(s$

,or eg - a tecni&ues for isolating te luciferasegene .luc/ fro! "NA using restriction digestion and

cloning it into te !ulti'le cloning region of a vector$

Te cloned lucgene is ten e+'ressed in E$ coli
http://en.wikipedia.org/wiki/Molecular_biologyhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/Recombinant_DNAhttp://en.wikipedia.org/wiki/Molecular_biology


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C0ONIN1

Te use of te )ord cloningrefers to te fact tat te!etod involves te re'lication of a single "NA

!olecule starting fro! a single living cell to generate alarge 'o'ulation of cells containing identical "NA!olecules

generall# uses "NA se&uences fro! t)o di2erentorganis!s

.i/ te s'ecies tat is te source of te "NA to (e

cloned and.ii/ te s'ecies tat )ill serve as te living ost for

re'lication of te reco!(inant "NA$
http://en.wikipedia.org/wiki/Cloninghttp://en.wikipedia.org/wiki/Host_(biology)http://en.wikipedia.org/wiki/Host_(biology)http://en.wikipedia.org/wiki/Cloning


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W RITE A3OT C0ONIN1CONVENTIONA005

In a conventional !olecular cloning e+'eri!ent% te "NA to (e cloned is o(tainedfro! an organis! of interest% ten treated )it en6#!es in te test tu(e togenerate s!aller "NA frag!ents$ 7u(se&uentl#% tese frag!ents are tenco!(ined )it vector "NA to generate reco!(inant "NA !olecules$ Tereco!(inant "NA is ten introduced into a ost organis! .t#'icall# an eas#8to8gro)% (enign% la(orator# strain of E. coli(acteria/$ Tis )ill generate a 'o'ulationof organis!s in )ic reco!(inant "NA !olecules are re'licated along )it teost "NA$ 3ecause te# contain foreign "NA frag!ents% tese are transgenicorgeneticall# !odi9ed !icroorganis!s .1MO/$:Tis 'rocess takes advantage of tefact tat a single (acterial cell can (e induced to take u' and re'licate a singlereco!(inant "NA !olecule$ Tis single cell can ten (e e+'anded e+'onentiall# togenerate a large a!ount of (acteria% eac of )ic contain co'ies of te originalreco!(inant !olecule$ Tus% (ot te resulting (acterial 'o'ulation% and te

reco!(inant "NA !olecule% are co!!onl# referred to as ;clones;$ 7trictl#s'eaking% recombinant DNA refers to "NA !olecules% )ile molecular cloningrefers to te e+'eri!ental !etods used to asse!(le te!$
http://en.wikipedia.org/wiki/Vector_(molecular_biology)http://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/Transgenichttp://en.wikipedia.org/wiki/Transgenichttp://en.wikipedia.org/wiki/Genetically_modified_organismhttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Molecular_cloninghttp://en.wikipedia.org/wiki/Genetically_modified_organismhttp://en.wikipedia.org/wiki/Transgenichttp://en.wikipedia.org/wiki/E._colihttp://en.wikipedia.org/wiki/Vector_(molecular_biology)


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7TEP7 IN MO0EC0AR C0ONIN1


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,ACT7 TO REMEM3ER

Molecular cloning is similar topolymerase chain reaction(PCR) in that

it permits the replication of DNAsequence. he fun!amental !i"erence#et$een the t$o metho!s is thatmolecular cloning in%ol%es replicationof the DNA in a li%ing microorganism&$hile PCR replicates DNA in an in vitrosolution& free of li%ing cells.
http://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/In_vitrohttp://en.wikipedia.org/wiki/In_vitrohttp://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Polymerase_chain_reaction


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7 T E P < - C H O I C E O , H O 7 T O R 1 A N I 7 M A N "

C 0 O N I N 1 V E C T O R

he great ma'ority of molecular cloningeperiments #egin $ith a la#oratory strain of the

#acteriumE. colian! a plasmi! cloning %ector.E. coli an! plasmi! %ectors are in common use#ecause they are technically sophisticate!&

%ersatile& $i!ely a%aila#le& an! o"er rapi! gro$th ofrecom#inant organisms $ith minimal equipment.

f the DNA to #e clone! is eceptionally large

(hun!re!s of thousan!s to millions of #ase pairs)&then a #acterial arti*cial chromosome oryeast arti*cial chromosome%ector is often chosen.
http://en.wikipedia.org/wiki/Plasmid_vectorhttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Yeast_artificial_chromosomehttp://en.wikipedia.org/wiki/Bacterial_artificial_chromosomehttp://en.wikipedia.org/wiki/Plasmid_vector


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7TEP < - CHOICE O, HO7TOR1ANI7M AN" C0ONIN1 VECTOR

+peciali,e! applications may call for speciali,e! host-%ectorsystems.

or eample& if the eperimental lists $ish to har%est a

particular protein from the recom#inant organism& then anepression %ector is chosen that contains appropriate signalsfor transcription an! translation in the !esire! host organism.

Alternati%ely& if replication of the DNA in !i"erent species is!esire! (for eample transfer of DNA from #acteria to plants)&then a multiple host range %ector (also terme! shuttle %ector)may #e selecte!. n practice& ho$e%er& speciali,e! molecular

cloning eperiments usually #egin $ith cloning into a#acterial plasmi!& follo$e! #y su#cloning into a speciali,e!%ector.
http://en.wikipedia.org/wiki/Expression_vectorhttp://en.wikipedia.org/wiki/Shuttle_vectorhttp://en.wikipedia.org/wiki/Subcloninghttp://en.wikipedia.org/wiki/Subcloninghttp://en.wikipedia.org/wiki/Shuttle_vectorhttp://en.wikipedia.org/wiki/Expression_vector


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7TEP < - CHOICE O, HO7TOR1ANI7M AN" C0ONIN1 VECTOR

/hate%er com#ination of host an! %ector are use!& the%ector almost al$ays contains four DNA segments that arecritically important to its function an! eperimental utility

(1) an origin of DNA replication is necessary for the %ector(an! recom#inant sequences lin0e! to it) to replicate insi!ethe host organism

() one or more unique restriction en!onucleaserecognition sites that ser%es as sites $here foreign DNA may#e intro!uce!

(2) a selecta#le genetic mar0er gene that can #e use! to

ena#le the sur%i%al of cells that ha%e ta0en up %ectorsequences& an! (4) an a!!itional gene that can #e use! forscreening $hich cells contain foreign DNA

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Chapter 4.1.Concept of Molecular Cloning