lecture 6-gene cloning

7/21/2019 Lecture 6-Gene Cloning

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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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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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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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lecture 6-gene cloning

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