lecture 6-gene cloning
TRANSCRIPT
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Lecture 6:
Gene Cloning and Analysis
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Outline
1. What is gene cloning
2. Overview of the procedures
3. Extraction and purification of nucleic acids4. Plasmid vectors
5. igation and transformation
!. "ector #ased on the lam#da #acteriophage$
cosmids and supervectors
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What Is Cloning?
% &sing asexual reproduction to o#tain organisms that aregeneticall' identical to one another$ and to the (parent)
% *lones produced are onl' identical genetically+ the actual
appearance and #ehavior of the clones will #e influenced #'other factors such as their environment
% Example 1, to propagate plants #' ta-ing cuttings$ and produce a num#er
of plants that are identical to the parent. hese new plants areclones
% Example 2, Purif'ing a #acterial strain #' pic-ing a single colon' for
inoculation a series of fresh cultures is also a form of cloning
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What is Gene Cloning?
/ fragment of 0/$ containing the gene to #e cloned$ is inserted intoa circular 0/ molecule called a vector$ to produce a chimera orrecom#inant 0/ molecule
he vector acts as a vehicle that transports the gene into a host cell$which is usuall' a #acterium
Within the host cell the vector multiplies$ producing numerousidentical copies not onl' of itself #ut also the gene that it carries
When the host cell divides$ copies of the recom#inant 0/ moleculeare passed to the progen' and further vector replication ta-es place
/fter a large num#er of cell divisions$ a colon'$ or clone$ of identicalhost cells is produced. Each cell in the clone contains multiple copiesof the recom#inant 0/ molecule
*loning can provide a pure sample of an individual gene$ separatedfrom all the other genes that it normall' shares the cell with
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Overview of the Procedures
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% he 1ststep for the proceduresextract the 0/or / from the cell and to purif' it #'separating it from other cellular components
% ost commonl' used methods of purif'ing andfractionating nucleic acids are as follow,
1. rea-ing up cells and tissues
2. Purification and concentrating of the nucleicacids
3. 0etection and 6uantification of nucleic acids
Etraction and Purification of !ucleic Acids
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1. Breaking up cells and tissues
% 7eparate the cell from the growth medium 8e.g. #'centrifugation9
% *ell need to #e l'sedto release their components #'
using a com#ination of E0/$ l'so:'me and adetergent such as 707
% E0/eliminates divalent cations and thusdesta#ili:es the outer mem#rane$ allowing thel'so:'me to access the cell wall structure
% 'so:'medigest the polimer that form the rigid cellwall
% 0etergentto solu#ili:e the mem#rane lipids$releasing the cell contents
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%What do 'ou get after the l'sis;
%/ crude extracta complex miture of 0/$/$ proteins$ lipids and car#oh'drates
% ext stepto separate the desired nucleic acidsfrom other components
% emoval of proteinsextraction with a mixtureof li6uefied phenol and chloroform or digestionwith a proteol'tic en:'me such as proteinase
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2. Purification and concentrating
of the nucleic acids% Ethanol precipitation
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% *olumn purification
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3. Detection and quantification of
nucleic acids
% Estimate the concentration #' measuring thea#sor#ance in an &" spectrophotometer at
2!=nm% 2!=,2>= a#sor#ance ratio #etween 1.?5 and 2@
pure 8accepta#le9
% A&" a#sor#ance onl' gives the estimate ofconcentration$ not the integrity of the 0/
% Bel electrophoresisfor #oth detecting and6uantitating nucleic acids
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% Bel electrophoresis Ethidium #romide agarose gel
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Overview of the Procedures
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Plas"id as cloning vector:
% Plasmid 0/ can #e transferred from one #acterialcell to another during conCugation
% ransport re6uires a mo#ilit' protein 8encoded #'
the plasmidD#ornemob
gene9 and a specific site8nic9 on the plasmid
% he mobprotein nic-s the plasmid at nicand thenattaches to the nic-ed strand to conduct the
plasmid through a mating channel 8pilus9 into arecipient cell
% *loning vectors lac- #oth the mobgene and the nicsite and thus cannot #e mo#ili:ed for transport
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mob
gene
mob
proteinPlasmid
nicsite
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Plas"id #ectors
% Bene cloning re6uires speciali:ed tools andtechni6ues
% he "ehicles
ransport the gene into the host cell and isresponsi#le for its replication
ust #e capa#le of entering a host cell$ replicationto produce multiple copies of itself
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$asic features of %las"ids:
% *ircular molecules of 0/ that lead an independentexistence in the #acterial cell as extrachromosomal0/ molecules
% &suall' circular$ dou#leDstranded and supercoiled
% Occur widel' in nature$ and are found in most
#acterial species
%/lwa's carr' one or more genes$exp, anti#ioticsresistance 8ampicillin$ tetrac'cline$ -anam'cin.9 F
used as a selecta#le mar-er
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% Origin of replication, a#le to multipl' within the
cell independentl'
% 7maller plasmids use of the host cellGs own 0/replicative en:'mes$ larger ones carr' genes that
are specific for plasmid replication
% Plasmid si:e range from a#out 1.= -# to 25= -#
% *op' num#er, present in the cell as one 8largeplasmids9 or as man' as 5= or more for smallerplasmid
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% ased on small #acterial plasmids 8p&*1>Hp&*1I$plu7cripts9 F avoid pro#lems such as 0/ #rea-down
% Jigh transformation efficienc'
% *onvenient selecta#le mar-ers 8anti#iotic or lacKG gene9
% he a#ilit' to clone reasona#l' wide range of 0/
inserts 8=.1 -# F 1= -#9
% Jigh cop' num#er of 5==D1=== cop' per cell
% 0o not integrate into the host genome
% *loning sitesHmultiple cloning siteHpol'lin-er cloningsites
% &ni6ue restriction en:'me on *7 F onl' one site in thevector
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Overview of the Procedures
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Ligation and &ransfor"ation
% *loning serves two main purposes,
/llow a large num#er of recom#inant 0/molecules to #e produced from a limitedamount of starting material
/llow the purification of recom#inant 0/
molecules
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&he ligation "iture "ay contain:
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% &nligated molecules will #e degraded #' thehost en:'mes$ if go into the host cell
% Each host cell can onl' ta-en up one 0/molecule
% Each cell gives rise to a single colon'$ so each ofthe resulting clones consists of cells that allcontain the same molecule.
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Pre%aration of co"%etent E. colicells for transfor"ation:
1. &suall' 5= m calcium chloride is used.
2. *a*l2
causes the 0/ to precipitate on
mem#rane cells
3. *a*l2responsi#le for change in the cell wall
improves 0/ #inding
4. ovement of 0/ into competent cells isstimulated #' #riefl' raising the temperature to42*
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&ransfor"ation %rotocols:
1. Preincu#ation, *ells are suspended in a solution of cations$
*a*l2$ at =o*.
2. Lncu#ation. 0/ is added$ and the cell suspension is further
incu#ated at =o
*. he cations are thought toneutrali:e negativel' charged phosphates in the0/ and the cell mem#rane
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3. Jeat shoc-
he cellH0/ suspension is #riefl' incu#ated at42o* and then returned to =o*
he rapid temperature change creates a thermalim#alance on either side of theE. coli mem#rane
7weeps plasmids into the cell.
4. ecover' #roth is added to the 0/Hcell suspension and
incu#ated at 3?o* #efore plating on selective media ransformed cells recover from the treatment$
#egin to express the anti#ioticDresistance protein
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'election for transfor"ed cells:
% / selecta#le mar-er D a gene that provides atransformed cell with a new characteristic$ notpossessed #' nonDtransformant
% he resistance gene on the plasmid must #eexpressed$ so that the en:'me that detoxifies theanti#iotic is s'nthesi:ed
% Expression of the resistance gene #eginsimmediatel'#efore the cell contains enough ofthe en:'me to #e a#le to withstand the toxiceffects of the anti#iotic
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% ransformed #acteria first placed in a smallvolume of li6uid medium$ a#sence of anti#iotic$incu#ated for a short time
% 7o that when the cells are plated out andencounter the anti#iotic$ the' will alread' haves'nthesi:ed sufficient resistance en:'mes to #ea#le to survive
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Selection based on pBR322
Lf a new fragment of0/ is ligated into oneof these sites$ theanti#ioticDresistancegenes #ecome altered
he plasmid loses thea#ilit' to confer eitherampicillin ortetrac'cline resistanceon the host
his is calledinsertionalinacti!ationof theselecta#le mar-er
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he resistance properties of colonies are tested #' transferring cellsfrom agar containing one anti#iotic onto agar containing the second
anti#iotic Lf theBamJL site has #een used then recom#inants will #e
ampicillin resistant #ut tetrac'cline sensitive
/fter transformation$ cells are plated onto ampicillin agar
/ll cells contain a p322 plasmid$ whether recom#inant or not$divide and produce a colon'
he colonies are then transferred onto tetrac'cline agar #' replicaplating
7ome colonies do not grow on the tetrac'cline agar #ecause their
cells contain recom#inant p322 molecules with a disruptedtetrac'clineDresistance gene
hese colonies contain the cloned gene. hese colonies can #erecovered #' returning to ampicillin plate
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Identification of reco"(inants
1. /nti#iotic resistance selecta#le mar-er
2. lacZG selecta#le mar-er
3. *olon' h'#ridi:ation
4. *olon'DP*
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lacZselecta(le "ar)er *(lue+white screening,:
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1. lacZ is a portion of #acterial gene$ which codes forpart of the en:'me Dgalactosidase
2. Dgalactosidase involved in the #rea-down of lactoseto glucose plus galactose
3. Lt is normall' coded #' the gene lacZ$ resides on theE. colichromosome
4. 7ome strains ofE. colihave a modified lacZgene$
which lac-s the segment referred to as lacZandcoding for the Dpeptide portion of Dgalactosidase
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5. hese mutants can s'nthesi:e the en:'me onl' whenthe' har#our a plasmid$ such as p&*1>H1IH>$ thatcarries the missing lacZsegment of the gene.
!. he proteins specified #' the gene segments on theplasmid and on the chromosome are a#le to com#ine
to produce a functional Dgalactosidase en:'me.?. he lacZG gene contains a cluster of uni6ue
restriction sites+ insertion of new 0/ into an' oneof these sites results in insertional inactivation of the
gene and hence loss of Dgalactosidase activit'.
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>. 7creening using lactose analogue called MDgal 85D#romoD4DchloroD3Dindol'DD0Dgalactop'ranoside9which is #ro-en down #' Dgalactosidase to aproduct that is coloured deep #lue
I. Lnducer of the en:'me such as isoprop'lDthiogalctoside$ LPB is used
1=.onDrecom#inant colonies$ will #e coloured #lue
11. ecom#inants colonies$ will #e in white colour
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End of Lecture 6
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