first presentation of m.pharm

8/7/2019 First Presentation of M.Pharm

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Naimesh N Patel

M.Pharm Sem-PharmaceuticsKBIPER


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Content : -y D efine Biotechnology.y Major Types of Nucleic Acid in Cell.y Why do we need Isolated Pure D NA & RNA?y Methods for Isolation & Purification of D NA.y Method for Isolation & Purification of RNA.y

Method for Isolation of Specifically mRNA.


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BIOTECHNOLOGYy F usion of two words : -

y Biologicaly Technology

y It refers to as a link between the biological science,physical science, chemical science and technologicalachievement, commonly reffered as the clever science

of biology.y Various basic biological disciplines involved in the

origin of biotechnology are : -


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BIOTECHNOLOGY

Genetics

Engineering technology

Biophysics

Molecular biology

Biochemistry

Cell biology

Microbiology


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Maj or Typ es of Nucleic Acid

y

D eoxyribonucleic acid( D NA)y Ribonucleic acid(RNA)


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The Central D ogma of Genetics

ReplicationTranscription

Translation

mRNA

non-coding RNA (rRNA, tRNA, siRNA, etc.)


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Wh y do we need to isol a ted p ure DNA?

D etect, enumerate species

D etect, enumerate, clone genesD etect/sequence specific D NA regionsCreate new D NA constructs (recombinant D NA)


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Th e p urific a tion of nucleic acids bro adly involves following st ages : -

y Breaking or opening of the cells to expose nucleic acid.y Inactivation of D NA- and RNA-degrading enzymes

(D Nases, RNases)y Separation of nucleic acids from cellular components.

y Extraction/Precipitation Methodsy Adsorption Chromatography Method

y Recovery of nucleic acids in a pure form.


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1. Bre ak ing or Lysis of cells

a) Bacterial cellsb) Animal cellsc) Plant cells


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1. Bre ak ing or Lysis of cells(cont )

a) Bacterial cells : -

The bacterial cells (e.g. E.coli) can be lysed by

combination of enzymatic and chemical treatments.The enzyme lysozyme and the chemical ethylenediaminetetraacetate(E D TA) are used for this purpose.This is followed by the addition of detergent such as

sodium dodecyl sulfate(S D S).


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1. Bre ak ing or Lysis of cells(cont )

b) Animal cells : -Animal cells, paticularly cultured animal cells, can be

easily opened by direct treatment of cells with

detergent(S D S).

c) Plant cells : -Plant cells with strong cell walls require harsh

treatment to break open. The cells are frozen and thenground in a mortar and pestle. This is an effective way of breaking the cellulose walls.


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2 .S e p a ra tion of DNA from cellul a r com p a rtment

A) Extraction/Precipitation MethodI. Purification of D NA by removing cellular compartmentII. D irect purification of D NA

B) Adsorption Chromatography Method


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A) PRECIPITATION METHOD

I. Purification of D NA by Removing Cellular Components:-

y The cellular extract is centrifused at a low speed to remove thedebris(e.g. pieces of cell wall) that forms a pellet at the bottom of thetube.

y The supernatant is collected and treated with phenol to precipitateprotien at the interface between the organic and aqueous layers.

y The aqueous layer, containing the dissolved nucleic acid, is collectedand treated with the enzyme ribonuclease(RNase).

y The RNA is degraded while the D NA remain intact.y This D NA can be precipitated by adding ethanol and isolated after

centrifugation, and suspended in an appropiate buffer.


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A) PRECIPITATION METHOD(Cont )II. D irect Purification of D NA y In this approach ,the D NA itself is selectively removed from the

cellular extract and isolated.y In this method, the addition of detergent cetyltrimethyl ammonium

bromide(CTAB) results in the formation of an insoluble complexwith nucleic acid.

y This complex, in the form of a precipitate is collected aftercentrifugation and suspended in a high-salt solution to releasenucleic acids.

y By treatment with RNase, RNA is degraded.y Pure D NA can be isolated by ethanol precipitation.


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Overview of the Extraction/Precipitation Method


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Step 1: D isruption of cell walls by grinding

Step 2: Lysis of cells in extraction buffer

Step 1+2: mechanical disruption andhomogenization in extraction buffer

Extraction/Precipitation Method

Grind sample into a fine powder to

shear cell walls and membranes

Mix thoroughly with extractionbuffer to dissolve cell membranesand inhibit nuclease activity

A homogenizer allows cells to bemechanically disrupted within theextraction buffer

rude lysate


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Purposes of the Extraction Buffer1.D issolve cellular membranes2. Inactivation of D Nase and RNase3. Assist in the removal of contaminants

D etergents

Chaotropic saltsMetal chelatorsSaltsReducing agentsCTABPVP


+

Plasma membrane(phospholipid bilayer)

D etergent molecules

Use of D etergents to Lyse Cells: Like Dissolves LikeMixed micelle

SD S


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Crude lysate containingnucleic acids and othercell constituents

Mix thoroughly withan equal volume of organic solvent

e.g. phenol, chloroform,or phenol:chloroform

Centrifuge

The aqueous phase contains water-soluble molecules, including nucleicacids. Proteins and lipids becometrapped in the organic phase, and arethus separated away. Insoluble plantdebris become trapped in theinterphase between the two layers

Perform additional extractions for increased purity

Collect aqueous phase

Extraction/Precipitation MethodStep 3: Organic extraction

Organic

Aqueous

Interphase


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Pellet down nucleic acids. Pellet down nucleic acids. Wash pellet with 70% ethanol to removeresidual salts and other contaminants.

Pellet down nucleic acids. Wash pellet with 70% ethanol to removeresidual salts and other contaminants.D iscard ethanol and allow pellet to dry.

Af ter

Add alcohol and salt toprecipitate nucleic acidsfrom the aqueous fraction

Supernatant

Pellet

70% EtOH

D issolve pellet(H2O, TE, etc.)

Step 4: Nucleic Acid Precipitation


Bef ore Af ter

Centrifuge Wash Centrifuge


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Basic Principle

Nucleic acids within a crude lysate

are bound to a silica surface

The silica surface is washed with asolution that keeps nucleic acids bound,

but removes all other substances

The silica surface is washed with a solutionunfavorable to nucleic acid binding. The solution,

containing purified D NA and/or RNA, is recovered.

B) ADSORPTION CHROMATOGRAPHY METHOD


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Step 1: Prepare crude lysate

Silica-gel m embrane

Apply tocolumn

Step 2: Adsorb to silica surface

Adsorption Chromatography Method

Centrifuge

F low through(discard)

Nucleic acids

Surface silanol groups are weakly acidic, and will repel nucleic acidsat near neutral or high pH due totheir negative charge

Extraction Buffer composition favorsD NA and RNA adsorption to silica:

Low pHHigh i onic strengthChaotropic salt Nucleic acids bind to the membrane,

while contaminants pass through thecolumn.


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Centrifuge

Nucleic acids

Step 3: Wash away residual contaminants

Adsorption Chromatography Method

Wash buffer

Nucleic acids

F low through(discard)

Nucleic acids

Elution buffer

Elution Buffer composition isunfavorable to surface binding:H igh p H Low ionic strength

Step 4: Elute nucleic acids

Centrifuge

Nucleic acids


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Using Nucleases to Remove Unwanted D NA or RNA

Add D Nase

Add RNase

+ D Nase (protein)

+ RNase (protein)

D epending on when nuclease treatment is performed, it may be necessary torepeat purification steps for protein removal (e.g. phenol/chloroform extraction).


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App lica tions of Isol a ted DNAy After D NA is extracted, it is used as a template in

further molecular techniques such as

y PCR (polymerase chain reaction) :- Technique for generatinglarge quantities of a specified D NA.

y R F LP (restriction fragment length polymorphism)

y Southern Blotting:-Identification of desired D NA fromthousands of molecules.


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y The problem(s) with RNA:

y RNA is chemically unstable -- spontaneous cleavage of phosphodiester backbone via intramolecular transesterification

y RNA is susceptible to nearly ubiquitous RNA-degrading enzymes(RNases)

y

RNases are released upon cell lysisy RNases are present on the skiny RNases are very difficult to inactivatey -- disulfide bridges conferring stability y -- no requirement for divalent cations for activity

3 . S

e p a ra tion of RNA from cellul a r com p a rtment


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Common sources of RNase and how to avoid them

Contaminated solutions/buffers

USE GOOD STERILE TECHNIQUETREAT SOLUTIONS WITH D EPC (when possible)MAKE SMALL BATCHES OF SOLUTIONS

Contaminated equipment

USE RNA-ONLY PIPETS, GLASSWARE, GEL RIGSBAKE GLASSWARE, 300C, 4 hoursUSE RNase-free PIPET TIPSTREAT EQUIPMENT WITH D EPC

3 . S e p a ra tion of RNA from cellul a r com p a rtment(Cont )


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Top 10 sources of RNAse contamination(Ambion Scientific website)

1) Ungloved hands2) Tips and tubes3) Water and buffers4) Lab surfaces5) Endogenous cellular RNAses6) RNA samples

7) Plasmid preps8) RNA storage (slow action of small amounts of RNAse9) Chemical nucleases (Mg++, Ca++ at 80C for 5 +)10) Enzyme preparations



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Inhibitors of Rnase

D EPC: diethylpyrocarbonate

alkylating agent, modifying proteins and nucleic acids

fill glassware with 0.1% D EPC, let stand overnight at room temp

solutions may be treated with D EPC -- add D EPC to 0.1%, then autoclave(D EPC breaks down to CO2 and ethanol)



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Inhibitors of Rnase

Vanadyl ribonucleoside complexescompetitive inhibitors of RNAses,but need to be removed from the final preparation of RNA

Protein inhibitors of RNAse:-horseshoe-shaped, leucine rich protein,found in cytoplasm of most mammalian tissuesmust be replenished following phenol extraction steps



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y Break the cells/solubilize components/inactivate RNAsesby the addition of guanidinium thiocyanate (very powerfuldenaturant)

y Extract RNA using phenol/chloroform (at low pH)

y Precipitate the RNA using ethanol/LiCl

y Store RNA:1. in D EPC-treated H 20 (-80C)2. in formamide (deionized) at -20C



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y The purification of mRNA can be achived by affinity

chromatography using oligo(dT)-cellulose.y This is based on the principle that oligo(dT)-cellulose can

specifically bind to the poly(A) tails of eukaryotic mRNA.y Thus , by this approach, it is possible to isolate mRNA from

D NA, rRNA and tRNA.

4 . S elective se p a ra tion of mRNA from cellul a r

com p a rtment


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y As nucleic acid solution is passed through an affinity chromatography column, the oligo(dT) binds to poly(A) tails of

mRNA.y By washing the column with high-salt buffer , D NA, rRNA and

tRNA can be eluted, While the mRNA is tightly bound .y This mRNA can be eluted by washing with low salt buffer.y The mRNA is precipitated with ethanol and collected by

centrifugation.

4 . S elective se p a ra tion of mRNA from cellul a r

com p a rtment(Cont . )


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References :-1 . Dr. Satyanaran U. and Dr. Chakrapani U. Biochemistry

, 2 nd edition , Chapter 24 to 27, Page no.523 to 578.

2. Dr. Vyas S. P. and Dr. Dixit V. K. PharmaceuticalBiotechnology CBS Publisher & Distributors, NewDelhi, 1 st edition, Chapter 10 , Page no. 34 1 to 4 00 .

3. Ambion Scientific Website.


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