techniques in cell and molecular biology,bm1

8/18/2019 Techniques in Cell and Molecular Biology,Bm1

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Techniques in Cell and

Molecular Biology


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Perkembangan Teknologi untuk Diagnosa

Penentuan langsung secara optis(mikroskop cahaya atau elektron)Kultur (isolasi, koloni, sifat-sifat micro-organisme dan reaksi biokimia)Reaksi antigen-antibody (enzyme-linked,zat fluorisensi, agglutinasi)Penggunan teknologi D ! " #ybridisasi(southern blot, dot-blot, P$%&), 'nsitu#ybridisasi dan !mplifikasi D ! (P R)

*+& +!R ' R* '*+*%.*+& +!R ' R* '*+*%.


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!plikasi teknologi D !

eningitis " eisseria meningitidisDiagnose harus cepat untuk mencegahkomplikasi/

Pe0arnaan %ram dapat dilakukan,tetapi minimal 12 3amPemberian antibiotika pada stadium dini

mengganggu interpretasiTransport bahan pemeriksaanTest Kit " antigen meningococcus

tidak sensitif dan false positif


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!plikasi teknologi D !

ycobacterium tuberculosis "embedakan 3enis atypic, dengan

mikroskop hal ini tidak mungkin

Kultur " 0aktu yang lama dan bakteri harusbanyak (terutama untuk sensiti4ity test)Diagnose cepat dibutuhkan, mis/ padapenderita !'D5/Ditemui 3enis yang multi drug resistant Diagnosa dengan P R dan #ybridisasi(contoh " dot-blot)


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'nstruments

6 The light microscope

$eugen stain is specific for D ! (onion root tipcell)

agnification 4s Resolution

Total agnification 7 *b3ecti4e 8 *cular +ens


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Instruments6 Phase-Contrast Microscope

+ight micrograph under bright-field/

on4erts differences in refracti4e inde9 intodifferences in intensity (relati4e brightnessand darkness), 0hich are 4isible to the eyes/$or e9amining intracellular components of

li4ing cells at relati4ely high resolution/

Differential 'nterference ontrast (D' )omarski 'nterference deli4ers image that has

an apparent three dimensional :uality/


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Instruments6 Fluorescence Microscope

sing green flurescent protein (%$P), the nonin4asi4e approach utilizinga fluorescent protein from the 3ellyfish Aequorea victoria that 0idelyemployed recently/ This strategy pro4ides the dynamic acti4ities in 0hichthe protein participates/

.$P (yello0 fluorescent protein)$P (cyan fluorescent protein)

The arro0 indicates t0odifferent neuromuscular

3unctions on t0o differentmuscle fibers/

$luorencent neurons


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Skeletal Muscle Tissue under Light vs lectron Microscope

6 lectron Microscope - Transmission M Can provide vastly greater

resolution than the light microscope

- Scanning M

Magni!ication vary !rom a"out #$$$ time to %&$'$$$ times

Image taken "y a light microscope

By electron microscope


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Technologies ( in vitro study )

6 Cell culture%ro0 cells outside the organism/ Advantages :- ulture cells can be obtained in large :uantity/

- ost cultures contain only a single type of cell/ - any different cellular acti4ities can be studied,including endocytosis, cell mo4ement, cell di4ision,membrane trafficking, and macromolecularsynthesis/

- ells can differentiate in culture/ - ultured cells respond to treatment 0ith drugs,

hormones, gro0th factors, and other acti4esubstances/


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*ro+th Properties o! ,ormal The !!ects o! Serum eprivation on the

and Cancerous Cells *ro+th o! ,ormal and Trans!ormed Cells


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Technologies ( in vivo study )

6 Polyacrylamide *el lectrophorese

- The electrophoretic separation o! proteins' in +hichthe proteins are driven "y an applied current through agelated matri./

- *els are typically stained +ith Coomassie Blue or

silver stain to reveal the location o! the proteins/


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Polyacrylamide *el lectrophorese

- I! the proteins are radioactively la"elled'their location can "e determined "ypressing the gel against a piece o! 0-ray!ilm to produce an autoradiograph/

- 1lternatively' the proteins in the gel can"e trans!erred "y a secondelectrophoretic procedure to anitrocellulose mem"rane to !orm a "lot/Proteins "ecome a"sor"ed onto thesur!ace o! the mem"rane/ In Westernblot, the proteins on the mem"rane areidenti!ied "y their interactions +ith

speci!ic anti"odies/


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S S-P1* (Sodium odecyl Sul!atePolyacrylamide *el lectrophorese)

6 etergent S S (negatively charged' denaturates proteins"ut does not alter a protein2s tertiary structure) "inds inlarge num"ers to all types o! protein molecules/Consequently' each protein species' regardless o! itssi3e' has an equivalent charge density and is driventhrough the gel +ith the same !orce/

6 1s a result' proteins "ecome separated "y S S-P1* onthe "asis o! a single property- their molecular mass/


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The results o!S S-P1* usedto !ractionate the

proteins o! theerythrocyte

mem"rane' +hichare identi!ied at

the side o! the gel


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,ucleic 1cid Puri!ication

6 The first step in D ! purification isgenerally to homogenize the cells andisolate nuclei from 0hich the D ! ise9tracted/

6 5eparate D ! from R ! and protein/6 Deproteinization is accomplished by

shaking the mi9ture 0ith a 4olume ofphenol;chloroform (acti4e proteindenaturant)/

6 entrifuge to separate phases, 0hichlea4es the D ! (and R !) in solutionin the upper a:ueous phase and theprotein as a precipitate at theboundary bet0een the t0o phases/

6 Repeated cycles of shaking 0ithphenol and centrifugation until nofurther protein is remo4ed fromsolution/


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,ucleic 1cid Puri!ication

6 y addition of cold ethanol, the nucleic acid are thenprecipitated from solution/

6 The D ! is redissol4ed and treated 0ith ribonucleases

to remo4e the contaminating R !/6 R ! can be purified in a similar manner using D ! ase

in the final purification steps rather than ribonulease/


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4,1 isolation(alternati4e procedure)

6 Tissues are homogeni3ed in a solutioncontaining 5M guanidine thiocyanate/

6 4,1 e.tract is mi.ed +ith phenol and shaken+ith chloro!orm (or "romochloropropane)/

6 The suspension is then centri!used' leaving4,1 in the upper aqueous phase and "oth ,1

and protein at the inter!ace "et+een the t+ophases/


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Separation o! ,1s "y *el lectrophorese

6 5eparation of D ! molecules greater than about 1< kb isgenerally accomplished using the techni:e of pulsed-fieldelectrophorese/

6 The D ! fragments that are present in a gel can be

re4ealed by immersing the gel in a solution of ethidiumbromide and then 4ie0ing the gel under an ultra4iolet light/


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1mpli!ication o! ,1 "y PC4

6 ! sample of D ! is mi9ed 0ith an ali:uot of Ta: polymerase and allfour deo9yribonucleotides, along 0ith a large e9cess of t0o short,synthetic D ! fragments (oligonucleotides) that are complementaryto D ! se:uence at the => ends of the region of the D ! to beamplified/ The oligonucleotides ser4e as primers/

6 The mi9ture is heated to about ?= to cause the D ! molecules inthe sample to separate into their t0o component strands/

6 Then the sample is cooled to about @A to allo0 the primers to bindto the strands of the target D !, and the temperature is raised toabout B1 , to allo0 the thermophilic polymerase to add nucleotides tothe => end of the primers/


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1mpli!ication o! ,1 "y PC4

6 !s the polymerase e9tends the primer, it selecti4ely copies the targetD !, forming ne0 complementary D ! strands/

6 The temperature is raised once again, causing the ne0ly formed and theoriginal D ! strands to separate from each other/

6 The sample is then cooled to allo0 the synthetic primers in the mi9ture tobind once again to the target D !, 0hich is no0 present at t0ice theoriginal amount/

6 This cycle is repeated o4er and o4er again, each time doubling theamount the specific region of D ! that is flanked by the bound primers/

6 illions of copies can be generated in 3ust a fe0 hours using a thermalcycler that is automatically changes the temperature of the reactionmi9ture to allo0 each step in the cycle to take place/


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1mpli!ication o! ,1"y PC4

6 asic procedure ofPolymerase hain Reaction(P R) employs a heat-stable D ! polymerase,called ta: polymerase,

isolated from Thermusa:uaticus, a bacterium li4esin hot springs attemperature abo4e ?A /


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L CT46P764 SIS * L 1*146S

C1= bp C1= bp


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