MIC 210

BASIC MOLECULAR BIOLOGY

LECTURE 4
DNA
CLONING
BY
SITI NORAZURA JAMAL (MISS AZURA)
03 006/ 06 483 2132
 Outline

Source of DNA
Vector
Restrictio□ en^^me
‫□ ه؛ أ^ وـ؛ـا‬

Bacteria bost
Transformation Selection
of recombinants
INTRODUCTION TO DNA
CLONING
What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an
original molecule or cell

• To "clone a gene" is to make many copies of it - for

example, by replicating it in a culture of bacteria.
• Cloned gene can be a normal copy of a gene (= “wild
type”).
• Cloned gene can be an altered version of a gene (=
“mutant”).
• Recombinant DNA technology makes manipulating
genes possible.
• To work directly with specific genes, scientists prepare
gene-sized pieces of DNA in identical copies, a process
called DNA cloning
 Cell containing gene
Bacterium
of interest
Gene inserted into
plasmid

Bacterial Plasmid
chromosome
ne of
Recombinant interest DNA of
DNA (plasmid) chromosome
Plasmid put into
bacterial cell

Recombinant
bacterium

^Host cell grown in culture

to form a clone of cells
containing the “cloned”
gene of interest

Gene of Protein expressed by gene of

interest
Interest Copies
Protein harvested
of gene
٠ Basic research
and various Basic
Basic
research applications research on
on gene protein

Gene for pest Gene used to alter Protein dissolves Human growth hor-
resistance inserted bacteria for cleaning blood clots in heart mone treats stunted
into plants up toxic waste attack therapy growth
Copyright ©2008 Pearson Education, Inc., publishing as Parson Benjamin Cummings.
 _

• A preview of gene cloning and some

uses of cloned genes
• Most methods for cloning pieces of DNA in the laboratory
share general features, such as the use of bacteria and their
plasmids
• Plasmids
> are small circular DNA molecules that replicate separately from the
bacterial chromcsome
• Cloned genes are useful for making copies of a particular
gene and producing a protein product
• Gene cloning involves using bacteria to make multiple copies of
a gene
• Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial cell
• Reproduction in the bacterial cell results in cloning of the
plasmid including the foreign DNA
• This results in the production of multiple copies of a single
Gene cloning, genet‫؛‬c eng‫؛‬
neering, recombinant DNA
technology tfii^liriiil ■‫'<['ا'اه‬:'‫■ ■تا[لنلم‬
iHntkdi
٢!،■ -■!=‫ا؛‬-‫اجإ آنء<اآءا!و‬
uu ‫ ن‬5‫لف هلم ءأأهءء‬،1‫ا‬
ion‫تـــ‬
‫ه‬ ‫اسوءت‬،]|‫ و;ا‬CT
they’re more or (ess the same ‫تـــومرء‬
‫آل‬
‫ءا‬

It basically means : ‫اءءآ؟م يء"ءوبما‬

‫ءـمـاـ؛ـ‬
joining together DNA from ‫الــصـيـمـمـهـهـ‬
different sources/organisms,
‫هـء‬
forming a recombinant DNA ‫ل‬
-
"
‫ا؛ه‬:‫ د'■'؛! ءا‬fT<‫ س‬.٦■‫؛؛‬،
molecule ‫صـهـأـسـمـىـ‬ :‫زـ[ ؛ـ‬
, "'‫ ةـا‬,‫ أ‬MlI ‫سـء‬
Then put this recombinant DNA ‫ ■ـا‬.
into a host cell, usually bacteria
The host cell will then replicate
many copies of this
recombinant DNA molecule
Sometimes, we might want to
ask the host cell to use the ‫آ‬1‫ء‬1‫أدء‬1|‫سءت؛ي‬,‫|ء‬1

genetic information in the ‫ت‬

‫ ■■ء‬-‫أآ‬1،‫ء‬,,‫ا‬-‫مت‬
‫ءت‬-‫ ت‬LJVH
recombinant DNA to make
‫'د‬, »■■'■
proteins
 Vector Foreign DNA

Insertion

Hybrid ve^or

Vector

Host chromosome

Many copies of
foreign DNA
Expression of
foreign DNA
(presence of
protein product)

/
Why genetic engineering

Medical &health
applications

Production of novel
and important proteins
Insulin.. See chapter
‫مـؤـمـمـيـأـ‬
‫»ـاـآـاـاـمـ‬
‫وـءـإلـ‬
A-chain
COOH

B-chain

ECO Rl ... Phe Met

A A T T C A T Insulin encoding region ‫إ‬ ^TAA TAG

GTAC §‫إ‬ fy\TT

AT
CCTAG
Bam HI
e.g. GM cr©ps
‘golden rice’ - Inserting the gene
for synthesis of carotene
(^tamin A) into rice

whole plant
Aqric^tural applications

Mobilized
region

origin E. coli
spect.
res.
Cloning genes for scientific
studies

‫ءـص‬ Cleaved Dlasmid

DNAan^
from fragment
organism DNA vector

Recombinant
DNA molecule
Bacterium 10 kb)

Transformation of a bacterium
and selection of a cell
containing the plasmid

Clones of olasmid-
containing bacter^m
Growth and
cell division
 Basic of DNA Cloning

‫بـء‬
٧٥٧ : need
Source of DNA - to be cloned )1
Ch )2©‫؛‬,ce of vectors - to carry
maintain and replicate cloned gene in host
"

Restriction enzymes - to cut DNA )3

‫ رـهـ‬DNA ligase - to join foreign and
vector DNA -> recombinant □NA

‫ (ة‬Host cell - in which the recombinant

DNA can replicate
The basics of cloning
coRI ،‫؛‬١٠ ‫ اصءء‬rMlfldkxi
£r*(>iicllon 0‫ااءم‬
٢/ ١٢
GAATTC ٠٠٠٢٢٠
__ ___ _ ‫اا'؛ررء‬
DMJ،lobnclon»d

،٠٠٠٧٠٠٠
‫اصءء‬

R»co،nbln،rt p،a،mld
1) Source of DNA

• Genomic DNA
DNA extracted from ce -‫؛؛‬s and puified

cDNA •
by reverse transcription of mRNA -

Amp(ified DNA •
- ‫صءالـ‬‫ وـ‬Po‫؛‬ymerase Chain Reaction
Synthetic DNA •
DNA made artificia -‫؛؛‬y a machine
2) Vector ‫ص‬

• to carry the ■igated foreign gene into the host

ce)>

• maintain the foreign gene in the host cell

• Replicate

• pass into new cells during cell division

• Expressed the cloned foreign gene to make

a protein
Different types of clon!ng
vectors Plasmid

‫؛‬ds @Q Insert
size -5 kb

p>asm Bacteriophage k vector ($0

kb)
bacteriophage l, M13•
‫ا‬
•Cosmids, phagemids ١
Insert size-15 kb
‫؛‬f‫؛‬c‫؛‬a[ chromosomes
Art• Cosmid vector

BAC, YAC, MAC etc.

Plasmid
pBlueacripl plasmid (2961 bp) ‫؛‬n -
An^3*cill
٢٠٠١٠١٠٨٠٠ gene

• Extrachromosomal DNA found

Plasmid origin of
in bacteria & fungi raplication

• Close circular DNA

molecules, supercoiled
‫ءـا؛‬ Multiple

• Can replicate cloning‫ء‬

‫ا‬
}(polylinker

autonomously, independent of Bs(X\ Eagi Sad Spe\ Smal Eccf\\ Cm Acc\

Ssp106l Hlnctt
'‫بـاـ‬
chromosome
• Can be transfer to other cells
511‫ ءه‬Non XbM 1‫سـهـ‬ Psti EcoRV HtndIII Sail Xho٠

‫ةـةـ‬
>ttOOCTCC4CCOOGGTQOCOOCCOCTCT4GOACT*OTOQ4TCCCCCOOOCTOCAOa<UTTOGATATC*AQCTTATCOATACOGTOGACCTCOAOQQOOOCCCCOGTACC,
.CTCOAQOTOCCOCCACCQCOGQCGAOATCTTOATCACCTAOQOOOCCCOACOTCCTTAAOCTATAGTTCCiAATAXICTATOOCAQCTQGAOCTCCCCCCCOOOCCATOCI'

by co^ugation
• Can be integrated into ‫سـء‬
Draw

the chromosome Aptri

\Kpn

• In nature, plasmids carry genes that are not essential under normal
conditions
• But confers a survival advantage under extreme conditions eg. resistance
to antibiotics, metabolism of unusual substrates

• Number of plasmid per cell - controlled by plasmid itself

High copy number > 100 /cell; low copy number < 20 /cell
• Plasmid incompatibility - the presence of one plasmid in a cell excludes other
plasmids
 pBR322 - a high copy number plasmid
‫ة‬5‫اأ‬1‫? اق‬3
Hindlll 23
EcoHI, Xap] 4359 EC032I m
BamHI 37&
Aatll 44‫ةة‬.

Important DNA elements : Sspl 4ICS,

Pael S02

1. The rop (or sometimes Xagl 2‫قء‬

ori)origin of replication, so that the Sail fiS' Boxl

plasmid can be maintained & ‫ت‬ 712

replicated in the host cell Ecn31l EcaHl

pBSI ‫و‬7‫ة‬ 3
2. Antibiotic resistance marker ‫**؛‬٦ PBR32 BveI 1063
3
genes (ApR for ampicillin resistance and 2 3
TcR for tetracycline) so that we can 4361 bp

select
‫ووت‬ Mva1269l 1353
1
‫اهو^مءالج‬ ‫اا‬،‫ا ائاا‬4‫هبم‬
1‫ك‬2‫ة‬
3. Unique restrcition sites (EcoRI, BpulOI 1580

PvuI etc) so that we can cut the Bsg]

Afllll.PacI 2473 Kpn2l mm

plasmid in one place only. ‫ اءوة‬2‫وئ‬0 / \Pvull ‫تر[)و‬4

\ ‫آـاـاـق‬
‫قـةا‬
0 BsgJI ‫ووةا‬
and insert the foreign gene we
Ndel
Bst11G7l 2244
2296
\Pio
2122
want to clone 6‫تةت اصةة‬،‫أ‬
Psyl 2217

‫ءءج‬3‫ا‬
‫ا‬
3) Restriction enzyme

< Type II Restriction endonuclease

Enzymes found • Natural • ‫؛‬n some microorganisms
role to destroy invading foreign DNA
eg bacteriophage DNA -

Recognizes very •
specific short seq٧ences
of DNA
Each enzyme has its own -
recognition sequence/ site
Sometimes two different enzymes have the same -
recognition n which case they a٢e known as
sites, ‫؛‬isoschizomers

Cuts DNA in very specific manner •

 csequences
،•■E- 1300■ ‫'ء‬

SE
I
‫ء‬0§0CUmss■،
Hindin ‫أ‬:;:‫امح?؛;;;هأ‬ S'. . . C T G C A G . . . 3 ' T
■‫ء§ وـإـ‬
3''...GJACGTC...5' ‫سـمـح؛‬

CUT
S ' . . . C f A AT T C . . . 3 ' .. 5 . . ’ C AT G . 3
‫؛‬ 3 ' . . . c T T A A . 6 . . . 5 ' ‫اآم~ا‬ 3'. Hind IN

، . G TA C ، . . . 5 '
EcoR
CUT
5'.
5 ' . . A C C W 0 S T, . , 3 '
SexA‫؛‬ 3...TQGWCC.A...S
Ssp , . A ATAT T. , , , 3 '
> 3 J J . . T TATA A . ، . S '
CUT ‫؛‬ii
p

Recognition sites - always palindromic

-Formation of hairpin loops
How REs cut DNA

Cuts symmetrically Cuts ٠ ٨ line of

symmetryw
G A AT T C TGGCCA

Separation Separation
of fragments of fragments

G A AT T C TGG C
A
sticky-end fragments Blunt-end fragments

Sticky ends can re-anneal by base-pairing

Sticky ends has complementary overhangs
- allows for proper reannealing and joining of DNA molecules
TAAT ٩٠
I I I I I I I IT?,

; t T T t t , , , , + ""111111
TTAA AATT
I ■ ■ I I
TTTTg:
TTAA ‫يــم‬ ,
annealin
1 111
I TTTT?.
g

^:nniraiiiini:
' TTAA
TAAT

TTT-r I I I I I g:

* ■ *■I
I I I I g
+
l ^ n i n j
Action٠٠
Restricted Enzyme
EcoRI

Recombinant DMA
 Inserting a DNR
Sample into a Plasmid
DNAtobe
inserted

DNA is cut with

£coRI at arrows.

Resulting DNAs have sticky

(complementary) ends.

DNA Is spliced by complementary

base pairing and sealed with DNA

‫ةـةـةـ؛ـوـاـا‬
Bacterial transformation
 ©■

،‫ م‬Add Plasmid DNA

Transferined1 bacteria] cell

1
 Bacterial TOuisiflg’nation

Antibiotic-sensitive
bacterial cell

‫ تـــ‬1‫ ق‬.treat mert

‫أإةتبم‬

‫ ؛‬to permeabilize
cell ,*,alls

‫ه‬

Seiection cri bacterial groM^th medium cort

aiming appropriate antibiotic
Inserting the recombinant DNA molecule into a Competent E.coli cell
The cells must be made competent be treating with CaCl^ or very little
DNA will be taken up.

Plasmid bound
to cell exterior
CaCl2 Normal bacterium
treatment

: ٠
٠
j
Competent cell
‫م‬ Plasmid transported
into the cell
Heat shock 42=0 ،‫ا‬،‫مماءا‬
for 2 minutes ‫م‬
٥١
*
١
١ Transformed cell
.
Se(ecting for transformants carrying recombinant
DNA

No vector or recombinant DNA

- will not grow on media +
ampicillin

Vector only
- will grow on media +
ampicillin

Recombinant DNA (vector + insert)

will grow on ampicillin
This is the one we want !

The goal of any cloning experiment is to obtain transformants carrying

cloned insert DNA. There are several strategies to maximise these
The goal of any cloning experiment is to obtain transformants carrying
cloned insert DNA.

There are several strategies to maximise these

1. Directional cloning
Use two different restriction enzymes to cut each end of the vector
(and also the foreign DNA you want to clone)
- Generate different sticky ends - cannot self ligate
EcoRI B^mHI H ~p
EcoRI I BamHI
1
I I—
3■ Dephosphorylation of
vector
-both the 3’OH group and
5’P04 group are required for
ligation
-if the 5’P04 groups on the
vector ends are removed
-cannot self-ligate
-Using a phosphatase
enzyme
-e.g calf
intestinal
phosphatase etc.
Blue white selection - lacZ complementation
!.replication origin

T3 I
‫كةت‬:‫ا‬
mi
'
Tt
x&ii Q
‫رـصـ‬ 3 multiple
‫ء‬
cloning
-‫> ذ‬
Psti site
LC (MCS)
.OF
557
}
2. antibiotics 1 T7
Eco
resistance gene f!(+) RV
،J-lactamase
origin [4.
‫ارــاـ‬
LacZ'
‫س‬
‫رـق‬
The vector contains a portion of the E.coli LacZ gene.
‫اـرـ‬
A multiple cloning site (MCS) sequence is inserted into
‫ س‬the LacZ’ fragment
Ac‫ا‬
 The LacZ gene codes for the /^galactosidase enzyme

Lac operon

The /-gal enzyme . :.■;١:!■■

hydrolyses lactose into
glucose and galactose
f‫؛؛‬،► galactosidase
galactose glucose
The LacZ gene can be broken into two parts, a and /
- each part encoding a fragment of the /-galactosidase enzyme

p‫ه‬

LacZP

Inserted ‫؛‬no E. coli chromosome

plasmid LacZa
vecter
٢'

mRN
A
^-fragment ‫ ء‬/- fragment
A fully active enzyme can be reconstituted from both
fragments
"U LacZp’
Inserted into
p‫؛‬asmid vector LacZa ‫ء‬. coli chromosome
IP ٠■1

mRN
A
P- fragment
،،-fragment

X-gal
The ‫؟‬-gal enzyme can
also hydrolyse a colorless o
blue ‫او‬،‫ءءمءا‬
substance called X-Gal P-
into glucose and a blue
color pigment galactosidas
e
To do blue white selection, the gene of interest is oloned into the
MCS
replication origin

Gene you
want to clone

2. antibiotics
resistance gene
jS-lactamase

Transformants are plated onto a medium oontaining

:
o Antibiotic for selection PTG to induce expression
o o X-Gal to detect the presence of ‫ ؛‬of the LacZ
’

/-galactosidase
Transformants with vector only : o LacZ is expressed ‫ج‬
a fragment is produced o Complements /-fragment to
form fully active enzyme
o Hydrolyses X-Gal ‫ج‬
Blue color colonies £ coli chromosome
R.NApotymeras
e
£
■ ^RNA
٠ ١ ‫اـ؛ـأـاـآـا‬ ‫!؛‬،‫اـه‬ mRN
A
ot-fragment

٠ X-gat
‫ح‬ ‫دجمت آ‬-‫مرسء‬.‫ا‬
p- tvrmc-5{

‫ ت‬-‫رـءـيـاـئـسـصـسـوـ‬
galactosidase glucos
e
:■ .T.‫؛؛‬#1
.٢٧+
Transformants with recombinant
o LacZ is destroyed by insertion of foreign gene ‫ ج‬no a fragment o
DNA:
Cannot form fully active enzyme
o No hydrolysis of X-Gal ‫ ج‬White color colonies ‫ ء‬coft chromosome‫ع‬
RNAoolymeras
e ٠٠
‫ا‬

^TiRNA
~^><^mRNA

inhibctOf

<x-
>
frag
‫ب‬ X-gal

QaiSfCtopp
osidase rsnos ‫ءـص‬

٢١'•‫ ر‬٠٢ ‫س‬

 blue)‫)؛‬nsertless
colony

‫ إ‬white (insert)
colony
٠ ٠ .٠ ٠
Just to remind you the basic steps.

DNA fragment Cleaved plasmid

٠٢٠٨١ a ٢٦۶ organism DNA
vec

Recombinant
DNA molecule
Bacterium (5-10 kb)

Host chromosome (-
Transformation of a bacterium
4600 kb) ;
and selection of a cell containing
the plasmid

Clones of olasmid-
containing
bacter^m
Growth and
cell division

‫ص‬
Sometimes, a simple cloning vector is not good
enough
pBlueacrtpt plasmid (2961 bp) AmpicBlin arc،-
‫ءءأ‬1‫اه‬gene

‫يم‬

Ptasmid
origin ٠١
pha‫ ءو‬origin
raplication
of
replication

‫ظ^ءممأم‬،‫ ت‬fragment
‫الس‬،‫ماءاا‬
‫م‬
(potylinker)
Eccoiosi"
Ssp106l Htnci I "
‫ د‬$‫ اءه‬٨١٠٢١ BBOU ‫وكء‬ Spe I Smal ‫ اصءء‬Cm
XbM BamH\ Psti ecoRV Wndlll SaH Xht*
‫اءءم‬
٠٢٠١١
GAOCTCCACCGC<^GTQ»Q» 30GCCOCTCTAGAACTAOTOQATCCCCC«jOCTGOAOQAATTCX»ATATCAAGCTTATCQATACC>CiTCG ،،CCTCGA(iGOOO«iCC.COGT،iCC،
Apm
.CTCQAOOTQ،iCOCCACCQCOGGCGAaATCTTQATCACCTAOQ،KiOCCCaACQTCCTTAAOCTATA،GTTC^AATAjQCTATaOCAQCTQQAQCTCCCCCCCOOQCCATQO

KprA

We might want to ask the bacteria cell to make proteins using

information on the cloned gene We need to use an
expression vector
Expression vector
- clone foreign gene AND make
foreign pro،ein
- reguires ex،ra DNA elements
Promoter - to Initiate transcription
-synthesis ofmRNA
Terminator-to stop transcription
Fusion tags - for making fusion
proteins e.g. Histidinex6, c-myc,
HA, GFP
In frame MCS

٥٤٠١٠٢ things - e.g. Poly-A sites

Recomb‫؛‬nant Insulin - not as easy as it looks
The insulin molecule as coded by DNA

.‫'أءق‬
.‫ص‬
‫ ءو‬gfn' ‫؛ ئء‬
٠١٣'
‫ءء‬

‫\ءم‬
‫ ■ت‬٢□

‫ ء ت‬A-chain

COOH
 10.26 BIOSYNTHESIS ٠٢ INSULIN

PROTEM SYNTHESIS

‫إ‬
C ‫اـ^ ل‬ I ‫يب‬.‫ أ!ء‬.."‫عم‬
■ ‫دم م‬
Prep rc>
insulin
‫• مذءمء»ء‬fio ‫ممؤة‬1‫مءءمءمبمو‬،‫ممم‬0‫ءهءء‬
icqiicd for
pa*G»n«

Proins
1‫تسء‬ ulin
Active insulin molecule

C-pept‫؛‬de is removed
‫ع‬
Disulfide bonds formed between Peptide A & B

Not done by bacterial cell !

COOH

I B-chain

‫سح‬.>-
Production of recombinant insulin - ‘Humulin’ in E.coli

DNA for peptide A and Peptide B - synthesized chemically

Peptide A - 21 amino acids - 63 nucleotides + ATG + stop codon
Peptide B - 30 amino acids - ^nucleotides +ATG +stop codon

Clone into a different plasmid vector s- into the gene for B- ‫؛‬dase
galactos

Both DNA’s were cloned in frame with the /-gal gene

Expressed as fusion proteins - Peptide (A or B) + part of /-gal

This is necessary - otherwise the small peptides will be quickly degraded

Fusion with /-gal stabilises the peptides
Expression driven by the LacZ promoter Fusion proteins are purified

from the cells

The B-gal part is then cleaved off by reacting with cyanogen bromide
which cleaves methionine

>

The peptide and then purified and chemically reacted to form disulfide bonds

■\ ‫؛؛‬flAJN )‫ات‬
‫ءسـد‬ at 1‫ |ةآآ‬F I

‫ ء‬CMAIN [‫مد‬
AMINO‫مـ‬
‫ب‬ 1‫آل‬
‫اقآ‬

What is the problem of this approach ?