sci. living sys.trascription unit i sck_21.01.15 (1)

8/16/2019 Sci. Living Sys.trascription Unit I SCK_21.01.15 (1)

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Overview of transcription, translation and

recombinant DNA technology

S. C. Kundu

Department of Biotechnology


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Eukaryotic cells

with a nucleus

• Nucleus

• Mitochondria• Chloroplast• Ribosomes• RER• SER• Golgi body• Cytoplasm

• acuoles

Prokaryotic cells

without a nucleus

• Cytoplasm

• Ribosomes• Nuclear !one• DN"• #lasmid• Cell Membrane• Mesosome• Cell $all

• Capsule %or slime layer&• 'lagellum


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DN" consists of t(o strands running anti)parallel and formingdouble hellical structure


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RN"• RN" is a polymer of ribonucleotides that contain ribose rather than

deo*yribose sugars.

• +he normal base composition is made up of guanine, adenine, cytosine,and uracil

• RN" is found in nucleus and cytoplasm

• +ypes of RN" - Messenger RN" %mRN"&

Ribosomal RN" % rRN"& +ransfer RN" %tRN"&


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'ormation of the peptide bond

O

N!

"#O

O

N!

"!O

+(o amino acid molecules4

the nature of the R group %R5

and R/& determines the amino

acid

O

N!

"#

O

O

N!

"!

O

+he molecules must be

orientated so that the

carbo*ylic acid group of one

can react (ith the amine group

of the other

O

N!

"#N

O

"!

O

O!

+he peptide bond forms (ith

the elimination of a (ater

molecule.


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SG

Y

A

V


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The levels of protein structure


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DNA

mRNA

Transcription

+he Central Dogma of Molecular Biology

Cell

Polypeptide

(protein)

Translation Ribosome

+his describes the flo( of information from DN" into RN" %most commonly

mRN"& through transcription %copying the same code from one molecule to

another&, and then e*pressing the code into a functional molecule by

translation %con3erting from a nucleic acid code into an amino acid code&.


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+R"NSCR6#+60N "ND +R"NS7"+60N-

#ro8aryotic 3s Eu8aryotic

SE#"R"+E C0M#"R+MEN+SC09#7ED


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7

Prokaryotic Gene Structure

Promoter CDS erminator

transcription

Genomic DNA

mRNA

protein

!R !R

translation

#romoter is a DN" se:uence usually present upstream of coding regions

(here RN" polymerase binds to initiates transcription.

Gene is the structural and

functional unit of heridity,

(hich carry genetic

information from onegeneration to ne*t. 6n

molecular terms Gene is a

part of chromosomes

%DN"&, (hich codes for

functional RN" or protein

Gene transcription in pro8aryotes

9+R %9ntranslated se:uences&- ;2 9+R contains ribosome binding sitesfor protein synthesis4


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RN" transcript is complementary

to template strand and identical

to coding strand

"e$uirement for transcription in

prokaryotesGene or DN" to be transcribed, RN" polymerase, rN+#sand cellular en3ironment

Di""erent genes are transcribed "rom di""erent strands


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Bacterium has

one RN"

polymerase tosynthesi=e all

three RN"-

%mRN", rRN",

tRN"&

RN" polymerase binds to promoter of a gene to initiate transcription


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1

Promoter

! Promoters se"uences can vary tremen#ously$

! RNA polymerase reco%ni&es hun#re#s of

#ifferent promoters


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Stages of +ranscription

• Chain 6nitiation

• Chain Elongation

• Chain +ermination


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Bacterial +ranscription 6nitiation

• #romoter recognition by RN" polymerase

> 'ormation of +ranscription Bubble by separating DN"

strands

> Bond formation bet(een rN+#s to start RN" synthesis

> Escape of transcription apparatus from promoter

RN" # l fi d t d 6 iti t


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o( RN" #olymerase finds promoter and 6nitiates

+ranscription

•Core en=yme can synthesi=e RN" on a DN" template but cannot initiate

transcription.

•Core polymerase loosely binds at random sites in DN" (ithout

discriminating promoter and other se:uences.

•Binding of sigma in the polymerase %holoen=yme& reduced its ability to

loose binding sites, and the en=ymes mo3es along the DN".

•$hen it reaches the promoter se:uences, sigma factor recogni=e

specifically )


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Finding and bindingthe promoter

Closed complex

formation

RNAP bound -40 to

+20

Open complexformation

RNAP unwinds from-10 to +2

Binding of 1st NTPRequires highpurine[NTP]

Addition of next NTPs

Requires lower

purine[NTPs]

Dissociation of sigmaAfter RNA chainis 6-10 NTPslong

initiation


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%ubse$uent

hydrolysis of PPi drives the

reaction forward

"NA strand

0.

0.

DNA strand

RN" Synthesis is in the ;# to


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RNA polymeraseelongation

'()*+(,-1./*0*0 *233*4

•During Elongation

RN" polymerase

un(inds DN" ahead

of it, transcribe theregion and re(inds

the DN" at the bac8

and RN" comes out

of the comple*.

•+ranscription occursin the +ranscription•Bubble at the rate of

;@ ntsec.

•Elongation continuestill Core en=ymes

reaches the

terminator

se:uences.

•


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+ranscription +ermination

+ranscription ends after a terminator is transcribed

>

+(o types of terminators in bacteria-

& Rho)dependent terminators

& Rho)independent terminators

7

Prokaryotic Gene Structure

Promoter CDS erminator

transcription

Ge nomic DNA

mRNA

protein

!R !R

translation


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Rho-Independent TranscriptionTermination

(depends on DNA sequence -NOT a protein factor)

Stem-loop structure

$hen a nascent RN" transcript contains a series of 9 residues at the


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Rho independent transcription termination


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+he termination function of factor

+he factor, a he*amer, is a "+#ase

and a helicase.


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Rho-Dependent Transcription Termination(depends on a protein AND a DNA sequence)

G/C -rich site

RNAP slows down

Rho helicasecatches up

Elongating complex is disrupted

5& Rho binds astretch of GC rich

se:uence of

nacent RN"

upstream of the

terminator .

/& Rho acts as

he*amer, brea8s

"+# and (ith the

energy mo3es

through RN" tocatch DN")RN"

hybrid and

polymerase

comple* and

terminates

transcription.


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Lactose operon: a regulatory gene and 3stuctural genes, and 2 control elements

lac$

Regulatory gene

lacZ lacY lacA DNA

m%RNA

&%'alactosidase

Permease

ransacetylase

Protein

Structural 'enesCis%actingelements

P lac 6 P lac O lac

The Lac operon


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5. $hen lactose is absent

• A repressor protein is continuously synthesi'ed( )t sits ona se$uence of DNA *ust in the lac operon, the 0perator site

• +he repressor protein blocks the #romoter site where

the "NA polymerase settles before it starts transcribing

Re%ulator

%ene lac operon

5perator

site

y aDN"

$ O

Repressor

protein

RNA

polymeraseloc*ed


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/. $hen lactose is present

• A small amount of a sugar allolactose is formed within

the bacterial cell( +his fits onto the repressor protein atanother active site allosteric site-

• +his causes the repressor protein to change its shape a conformational change-( )t can no longer sit on the

operator site( "NA polymerase can now reach itspromoter site and initiates transcription(

Promotor site

y aDN"

$ O


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Eu8aryotic transcription is much complicated

+hree different polymerases-

RN" polymerase 6- synthesi=es rRN" in the nucleolus.

RN" polymerase 66- synthesi=es mRN" in the nucleoplasm.

RN" polymerase 666- synthesi=es tRN", ;S rRN", small RN"s in the nucleoplasm

"ll eu8aryotic RN" polymerases ha3e 5/)5 subunits %aggregates of H;@@ 8D&.

Some subunits are common to all three RN" polymerases such as +B#.

Multiple promoter types -+"+" Bo*, 6nitiator elements,

CpG island for pol 6&,

core elements,

upstream core elements %pol 6&,%" bo*, B Bo*, C Bo* for pol 666&

•Each RN" polymerase recogni=es its o(n promoter

•Many proteins %transcription factors& are in3ol3ed in promoter

recognition by RN" #olymerase

Eu8aryotic +ranscription


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6

7ukaryotic Gene Structure

+# % Promoter ,xon- $ntron- ,xon. erminator / 0#

!R splice splice !R

transcription

translation

Poly A

protein

7ukaryote Promoter 8Pol 99:


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+ranscription by #olymerase 66

+hree Steps-

6ntiation-

Binding of transcription factors and #ol 66 to promoter,DN" strand separation and beginning of RN" synthesis.

Elongation-

Continuous process of RN" synthesis by RN" pol 66.

+ermination-

Ending of transcription after transcribing a poly " signalse:uence.


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+ i i i i


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+ranscription termination


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)n eukaryotes, the primary transcript pre m"NA- must be

modified by.

& addition of a ;2 cap

& addition of a


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ppp;JNpNp

pp;J

NpNp

G+#

##i

G;J

ppp;J

NpNp

methylating at '1

methylating at C.J o" the

"irst and second

nucleotides a"ter '

"orming +2%+2

triphosphate group

remo3ing

phosphate group

mGpppNpNp

mGpppm

/JNpm

/JNp

#i

Capping at ;2 end of mRN"


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! pA ' p!+2 02+2exon 02exon

intron

p'%O4

p'pA

' p! 02!+2 O4

first transesterification

+(ice transesterification

second transesterification

!+2 p! 02

p'pA

'O4

+2

02

Splicing mechanism


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DNA

;ytoplasm

Nucleus

Eukaryotic +ranscription

,xport

G AAAAAA

RNA

ranscription

Nuclear

pores

G AAAAAA

RNAProcessing

mRNA


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! http566777%class8unl8edu6biochem6gp.6m

9biology6animation6gene6gene9a.8html

http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.htmlhttp://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.htmlhttp://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.htmlhttp://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html


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+ranslation is the process of decoding a m"NAmolecule into a polypeptide chain or protein

+ranslation- #rotein synthesis


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•Transcription and translation ineukaryotic cells are separated inspace and time. Extensive processing of primaryRNA transcripts in eukaryotic cells.

+ranslation


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+ranslation

)t is process of protein synthesis assembly of amino

acids- using m"NA as template with the help of t"NAand ribosomes r"NA with several proteins-(

+herefore, it re$uires the participation of multiple types

of "NAs.

• messenger RN" %mRN"& carries the information from DNA thatencodes proteins

• ribosomal RN" %rRN"& is a structural component of theribosome

• transfer RN" %tRN"& carries amino acids to the ribosome fortranslation

+h G ti C d


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+he Genetic Code

+he genetic code is the (ay in (hich the nucleotide se:uence innucleic acids specifies the amino acid se:uence in proteins.

" codon is a set of < nucleotides that specifies a particular aminoacid.

+herefore, mRN" carries information from DN" in a three letter

genetic code.

• " three)letter code is used because there are /@ different aminoacids that are used to ma8e proteins.

• 6f a t(o)letter code (ere used there (ould not be enough codons to

select all /@ amino acids.

• +hat is, there are ? bases in RN", so ?/ %?* ?&54

(here as for < lettered code the number is ?


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S

A ; ? > 5 N 7

> A

S 7

S

4

P O

O

4O

O

O

C4.N4.N

N4

N

N

4O4

P

O

O

4O

O

O

C4.

N4.

N

N

N

N

4

P

O

O4

4O

O

O

C4.

N4.

N

NN

N

O

A 1odon

Guanine

A#enine

A#enine

Ar%inine


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GENE+6C C0DE

+herefore, there is a total of ? codons (ith mRN", 5

specify particular amino acid.

+he remaining three codons %9"", 9"G, L 9G"& are stop

codons, (hich signify the end of a polypeptide chain%protein&.

+his means there are more than one codon for each of

the /@ amino acids.

Besides selecting the amino acid methionine, the codon

"9G also ser3es as the initiator codon, (hich starts

the synthesis of a protein

Deciphering the Genetic code


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p g

Marshall Nirenberg, Khorana and their collegues deciphered the genetic code by

adding homopolymers such as 999, """, CCC, or co)polymers such as "C",

C"", ""C of synthetic nucleotide triplets to cell e*tracts containing /@ amino

acyl tRN", (hich are capable of limited translation.6n each e*pt. one amino acid is radioacti3ely labeled and rest 5 are unlabeled

and the reaction mi*ture are passed through filter. Since, ribosomes binds to filter

if the added trinucleotide caused the labeled aminoacyl tRN" to the ribosome,

then radioacti3ity (ould be detected on the filter %positi3e test& other(ise label

(ill pass through)a negati3e test)bind filter

RN" t i d hi h d f i id


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mRN" contains codons, (hich code for amino acids(

tRN" ) +ransfer RN"(


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Each tRN" molecule has / important sites of attachment.

•"nticodon binds to the codon on the mRN" molecule.

•"mino acid acceptor site attaches to a particular amino acid.

During protein synthesis, the anticodon of a tRN" molecule

base pairs (ith the appropriate mRN" codon.

t"NA Activation

tRNAStructure


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tRNA Structure

Aminoacyl tRNA synthetase

+here are !2 different aminoacyl t"NA synthetases, one for each amino acid(

Rib


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Ribosome

• Are made up of ! subunits, a large one and a smaller one,

each subunit contains ribosomal RN" %rRN"& L proteins(

• Protein synthesis starts when the t(o subunits bind tomRN"(


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+he ribosome hasmultiple t"NA bindingsites.

& # site & binds the t"NAattached to the growingpeptide chain

& " site & binds the t"NAcarrying the ne0t aminoacid

& E site & binds the t"NAthat carried the last aminoacid

T lti h 3St h ii


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Translation has 3 Steps, each requiringdifferent supporting proteins

•Initiation

–Requires Initiation Factors

•Elongation

–Requires Elongation Factors

•Termination

–Requires Termination Factor

I


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Initiation:

1. Binding of initiationfactors to small subunit.Of ribosome andattachment to mRNA

2. Binding of first tRNAto mRNA attached tosmall subunit.

3. Binding of largesubunit.


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First step in elongation

(bacteria ):

Binding of the secondaminoacyl-tRNA

S d t i


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Second step in

elongation:

Formation of the first

peptide bond


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hird step in elongation:

translocation


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A7

Large

subunitP

Small

subunit

+ranslation / )nitiation

":et

!AC

'A'888C!%A!'%%!!C%%C!!%%A'!%%''!%%A'A%%'C!%%'!A%%!'A%A 'CA888AAAAAA@mRNA


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A7

Ribosome P ! C !

Arg

Aminoacyl tRNA

PheLeu

:et

Ser

'ly

Polypeptide

CCA

+ranslation / Elongation



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A7

Ribosome P

PheLeu

:et

Ser

'ly

Polypeptide

Arg

Aminoacyl tRNA

!C!CCA




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A7

Ribosome P

CCA

Arg

!C!

PheLeu

:et

Ser

'ly

Polypeptide




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A7

Ribosome P


Aminoacyl tRNA

C ' A

Ala

CCA

Arg

!C!

PheLeu

:et

Ser

'ly

Polypeptide



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A7Ribosome

P


C C A

Arg

!C!

PheLeu

:et

Ser

'ly

Polypeptide

C'A

Ala



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Termination:

1. Binding of

Release Factor toStop Codon UGA,UAA, UAG.

2. Disassembly


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Overview of Prokaryotic Translation


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Summary


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Summary


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http-((()class.unl.edubiochemgp/

mObiologyanimationgenegeneOa


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Recombinant DN"

• #roduction of a uni:ue DN" molecule by

Poining together t(o or more DN" fragmentsnot normally associated (ith each other

• DN" fragments are usually deri3ed fromdifferent biological sources

•" series of procedures used to recombineDN" segments and are called Recombinant

DN" +echnology


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History of recombinant DNAtechnology

Recombinant DNA technology is one

of the recent advances inbiotechnology, which was developedby two scientists named Boyer and

Cohen in 197!

DNA Bi


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DNA recomBinant

technolo%y

asic principle o" recombinant DNA


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p p

technology

0ne DN" molecule %called insert& is isolated from one

sources and then this DN" is inserted into another DN"

molecule called 13ector2

Mostly bacterial plasmid is used as 3ector

+he recombinant 3ector is then introduced into a host

cell (here it replicates, transcribes and translates to

produce protein.

3asic steps in "ecobinant DNA


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3asic steps in "ecobinant DNA

+echnology

#( )solate the gene

!( )nsert it in a host using a vector plasmid-

4( Produce as many copies of the host as

possible

5( %eparate and purify the product of the gene

S+E# 5 D6GES+60N 0' DN"


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S+E# 5. D6GES+60N 0' DN"

S"M#7E +0 6S07"+E GENE

S+E# / D6GES+60N 0'


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S+E# /. D6GES+60N 0'

#7"SM6D DN"

%tep 4. )nserting gene into digested


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6

Recombinant

#lasmid

E. co R5

digestedpector

!"co R6 digested 6nsert% gene&

%tep 4. )nserting gene into digested

vector to create recombinant plasmid

7igase

%tep 5. inserting recombinant plasmid into


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"mpicillin resistance gene %"mpr &

and target gene on bacterial plasmid

Bacterial clones

+ransformation mi*ture is plated

on to agar plate containing

"mpicillin

0nly E. coli containing plasmid

sur3i3e on "mpicillin plates

6ndi3idual colony is selected and

cultured to amplify recombinant DN"

#lasmid enters some

bacteria

host E. coli bacteria and selection

Overall cloning strategy


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7Ctracte# DNA from

any or%anism


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Applications of


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Applications of

Recombinant DNA

"echnology#arge$scale prod%ction of h%man

proteins by genetically

engineered bacteria!

&%ch as ' ins%lin, (rowthhormone, )nterferons and

Blood clotting factors *+))) )-.


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/rod%ction of 0%man

)ns%lin*???

.1) Obtaining the human insulin gene0%man ins%lin gene can be obtained bymaing a complementary DNA *cDNA. copyof the messenger RNA *mRNA. for h%manins%lin!

) i i h h i li


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2)Joining the human insulin geneinto a plasmid( ) vector

"he bacterial plasmids and the cDNA aremi2ed together! "he h%man ins%lin gene

*cDNA. is inserted into the plasmid thro%gh

complementary base pairing at sticy ends!

3)I t d i th bi t


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3)Introducing the recombinant

DNA plasmids into bacteria

"he bacteria E.coli is %sed as the host cell! )f E.coli and the recombinant plasmids are mi2ed

together in a test$t%be!

4)electing the bacteria !hich


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4)electing the bacteria !hich

have ta"en up the correct

piece o# DNA"he bacteria are spread onto n%trient agar! "he

agar also contains s%bstances s%ch as an

antibiotic which allows growth of only the

transformed bacteria!

#urification of recombinant protein by affinity

h h


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chromatography

sci. living sys.trascription unit i sck_21.01.15 (1)

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