bio 30 reporting

7/24/2019 Bio 30 Reporting

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In Vitro Genetic Manipulation orRecombinant DNA Technology

Recombinant DNA Technology

the use of in-vitro techniques in the isolationmanipulation recombination an! e"pression ofDNA


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#our $asic Requirements for DNATechnology

%& Vector

'& Metho! of splicing foreign genetic

material into the vector

(& Metho! of intro!ucing the vector)foreign

DNA recombinants into the host cell

*& Metho! of Assay


3/17

%& Vector

+as the capacity to enter a host cellan! replicate ,ithin an! bemaintaine! in it

e&g

lasmi!s

.osmi!s

$A./ 0$acterial Arti1cial .hromosome2


4/17

lasmi!s

an e"tra-chromosomal DNA moleculeseparate from the chromosomal DNA,hich is capable of replicating

in!epen!ently of the chromosomalDNA

able to contain %-'3 4bp of DNA


5/17

'& Metho! of splicing foreign geneticmaterial into the vector

Ma!e use of the !iscovery of site speci1cRestriction 5n6yme 0R52

/ome R5 cut straight across the !oubleheli" pro!ucing "blunt" ends.+o,evermany restriction en6ymes cut in an o7set

fashion& The en!s of the cut have anoverhanging piece of single-stran!e!DNA& These are calle! "sticky ends"


6/17

Restriction en6ymes

0Molecular /cissors2

DNA-cutting enzymesfoun! inbacteria

recogni6es an! cuts DNA only at aparticular sequence of nucleoti!es&0restriction site2

e&g& HaeIII 0Hemophilusaegypticus)

89GG..(9


7/17

R5


8/17

R5


9/17

Metho! of intro!ucing thevector)foreign DNA recombinants intothe host cell

%& Transformation

'& 5lectroporation3. Heat-shock


10/17

Metho! of intro!ucing thevector)foreign DNA recombinantsinto the host cell

%& Transformation

.

Transfer of cell-free DNA or e"ogenousDNA from one strain to another

Nee! cells to be competent

certain stage of gro,th

In!uce! : .a.l'


11/17

Metho! of intro!ucing thevector)foreign DNA recombinants intothe host cell

'& 5lectroporation

transfer of DNA is brought aboutby the application of pulse! 1el! tothe cells


12/17

'& 5lectroporation

electroporator


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Metho! of Assay0creening2

Assay for the foreign gene pro!uct ofchoice from the population ofrecombinant clones

5"& Ampicillin-resistance that isacti!ated hen gene o# interestis connected to !ector


14/17

Metho! of Assay 0$lue an! ;hite

/creening2


15/17


16/17

ractical Applications ofRecombinant DNA Technology

harmaceutical ro!ucts

a& 5& coli

insulin

gro,th hormones

inteferons


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ractical Applications ofRecombinant DNA Technology

'&ro!uction of /peci1c $io!egra!ing=rganisms for ollution .ontrol

5"& =il-!egra!ing bacteria

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