Micro And Biotech Fp

Blotechnology &Micro I1

Some therapedic product of rDNA technology (Gpat, Niper, DI, Pharmacis) Appllcatlon

Product Interferon

Cancer and viral infections

Appllcation

Product

Lympholines

For auto immune

functioning8 Human urokinase

Plasminogen activator used to Serum albumin

(tPA)

dissolve the clouds

Human factor IV Clotting factor for

IISsue

Attenuated

haemophilia

psudorable virus antigen

Treatment of heart attack

Somtostan

In the surgery

Yaceine against rabies

Treatment of growth

disorders

plasminogen activator

Steps and holding temperature forpofyunerase chain reactions (Gpas, Niper, DI, Ph) PCR: in vitro technique in which da resuamplification of DNA of interest in to the billion copies of DNA using a heat stablé DNApolymerase. Different heat stable DNA polymerases enzymes all these enzymes requiredmg2 as a cofactor, they are, 1. Taq DNA polymeras

bbtyined from thermus aquaticus stable upto 95°c. 2. Vent DNA

polymerase: obtained-fromgkermococcus litoralis. 3. Pfu DNA polymerase: obtained from pyrococcus furioss stable over 95°c.

Reaction mixture for PCR: 1. DNA of interest, 2. Primer which is a short fragment of DNA, 3. Heat stable DNA polymerase enzyme, 4. 4 nucleotide base pairs (A, T, G, C).

PCR use exponential synthesis of DNA strands 2". Applleatlons of PCR: diagnosis of pathogens, diagnosis of mutations, prenatal diagnosis, research, molecular archaeology. DNA fingerprinting and diagnosis of plant pathogens. RT-PCR: synthesis of DNA from RNA by reverse transcriptase, and then amplification of synthesized DNA.

Arbitrary primed PCR AP-PCR: arbitrary primers are used which are around 10-50 base pairs

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primer.

Steps Denaturatlon or melting

Comments

94-95°c for 15 seconds to Iminutes so that two strands will be

separated

Annealing

54-55°c for 62 seconds so that shot primer will be bound to the ends of separated strands of DNA.

Elongatlon or

polymertzation

72°c for 1-2 min, which is the optimum temperature to operate tag DNA polymerase.

Enzymes used In rDNA technology (Gpat, Nipe, D,

Pharmacist)

Alkallne phosphatase

Removes P04 from 5' of ds or qs DNA or RNA

DNA lIgase

Joining of sugar phosphaie backbóne of ds DNA with 5-PO4 and 3OH in ATP dependant (wo pnds of DNA must be blunt or

AYY

complementary to ach other for joining) DNA polymerase I

Synthesis of DNA oh complementary strand 5'3end direction

with pric Exonuclease IlI

Digesdied DNA 3end of linear DNA

Mung bean nuclease

Digestonly ss DNA or RNA but leaves intact double stranded.

Nuclease Si

at

rbehaves as mungbean nucleus and digests opposite to nick on complementarystrand.

Polynucleotlde kinase

Addition of 5-OH end of ds DNA or as DNA or RNA and ATP

dependant process. Restrlction endonuclease

Cuts both strands ds DNA at symmetrical recognition sites which results into sticky or blunt fragments.

Reverse transcriptase

Responsible for synthesis of DNA from RNA also known as RNA dependent DNA polymerase, synthesizes DNA S 3 direction by using ribonucleotide with primer.

Taq DNA Polymerase

3

DNA polymerase isolated from thermus aquaticus operates at 72°c,

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and it is table above 90°c it is used in PCR techniques.

Varlous type II restrictton enzymes and thelr mieroblal source (Gpat, Niper, DI, Ph) Mlcroblal source

Restriction

Microblal source

Restriction enzymes

enzymes BamHI

Bacillus amyloliquefaciens

PstI

Providencia stuartii

Bg/II

Bacillus globigii

Sall

Streptomýces Albus G

EcoRI

Escherichia coli RY 13

Sau3AI

EcoRII

E coli R2 45

Notl

Hind II

Haemophilus influenzae RD

KpnI

Hpa l

H Parainfuezeae

dbccus aureus 3AI

Nocrdia otitidis carviarus

Klebsicla pemumonae OK

Bacterlal and plants Cloplag Pectórs (Gpat, Niper, DI, Pharmacist) Vector is another DNA molecule yhich is available that may replicate in the transformed host

cell that is known as vector,. It igthe DNA in which our desired DNA is to be inserted

Propertles of cloning vectorpat 18, Niper, D) 1. Must have origin of rephicdtion site (ORI sequence) for the replication in the host cell. 2. Mast how markergènes for selection (antibiotic resistance gene Ampr. Kanr or Terr) 3. Cleavage site for restriction site (to cut using restriction enzymes) 4. Multiple coning sites, polylinkers, promoters etc. 5. They must have control system like promoters terminators ribosome binding sites etc. So that DNA

expression

is

properly.

1. Plasmld vectors: Plasmids are circular, self-replicating, extrachromosomal DNA present in

bacterial cel.

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Vector

Source

pACY 177 (p-for plasmid capital letters for

E. Coli

initials of scientist and numericals for the

strain)

pBR 322, pBR 324. pMB9 & pRK646

E.coli

PC194, pSA0501

Staphylococcus aureus and streptococcus aureus respectively Bacillus subtilis

pBS161

Cosmids: they are the hybrid of plasmids and bacteriophage vectors (ças site for cosmids)

pIC74,pJC720 PHC79

Plasmid form of Col EL

2

0

Derivative of pBE322

GEA

Viruses (hen virus attackk bacteria is known as bacteriophmée)

Mammalianeis

SV40

E.coli

M13, lambda phage

Artificial vectors or chromosomes

YAC yeast artificial chromosonme(hehest amount of DNA that can be incorporated in this

vector, smallest amount of DNA NSDrporated in the plasmid vector)

Bacterial artificial chromosogne. Plant vectors (Gpat, Niper, DI, Pharmacist)

Ti-plasmid

(isolated from Argobactrium tumefaciens, causes tumor or crown gall disease the plant)

Ri-plasmid

Argobacterium rhizogenes causes hairy

root

disease in the

plant Properties of Ti and Ri plasmids, They do not cause disease because removal of disease causing gene, they have site per insertion of foreign gene or gene of interest, how selectable

markers

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Diagram of cloning vectors, Diagrams of gene cloning, Diagram of construction of genomic

library, please refer this from books.

Application of rDNA technology: (Gpat, Niper, DI, Pharmacist) Production of insulin: pBR 322 vector is used, DNA sequence responsible for production insulin chain A and B are synthesized in the lab and inserted into the vector at the side of B-

galactosidage gene, vector is transferred into the host cell which is e.coli, then allowed to express bacterial cells are isolated ruptured and the two separate chains of insulin are recovered. In the similar way somatostatin, somatotropin growth hormone, and other proteins of interest are

produced. Interferon: interference are the antibodies produced against the viral infection, there are three kinds of interferon Alpha interferon beta interferon and gamma intérferonthey have various

application in Cancer and viral diseases. Monoclonal antibodies: normally after infection whatever andikody are produced polyclonal, do

not have characteristics of specificity, MCAs producedhem dcombination of myeloma cells from bones and antibodies isolated from previously iuftynised animal spleen cells.

Hybridoma technology is used for production oonoclonal antibodies.

Plant tissue culture: Totipotency: capacity of plant cells to deyelop into organism by regeneration; the concept is

important

in

plant

tissue

culture issue culture media requirements

Sr.no Requirements

Examples or comments

Inorganíc

Micronutrients: N, P, K, Ca, Mg. & S. Micronutrients: Cu, Zn, Fe, C1,

minerals

boron and molybdenum.

Organic content Source of carbon and energy: sucrose and D-glucose are commonly used.

Glycerol myo-inositol good

source

of

carbon, peptone, yeast

Cxtract, coconut water, tomato juice and malt extract.

Vitamins

Vitamin B1 is most commonly used in all PTC, other vitamins which

stimulate the growth are nicotinic acid, riboflavine, pyridoxine, ascorbic acid and cyanocobalamin. (Gpat, Niper, DI, Pharmacist) Amino acids

commonly used amino acids are I-aspartic acid 1-asparagine 1-glutamic

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acid l-glutamine and -arginine

Solidifying

gar is commonly used, gelatin is also sometime used but it has

agent

demerits of melting at temperature 25°c.

pH

Optimum pH should be between 5-6, and it should be maintained before sterilization of medium.

Different plant growth hormones use in PTC (Gpat, Niper, D, Pharmacist)

Synthetic

Natural

Category of

Functions

hormones Indole 3 beutyric

(TAA), indole 3

acid IBA,nepthalinoxy

acetonitrile (IAN),

acetic acid NOA

Indole 3 acetic acid

Auxins

Root development

phenyl acetic acid.

Cytokinins

Adepin digbohyl urea

Zeitine, N6

Shoot development

dimethylaminopurine, kinetine adenin.

Gibberellins

Gibberellin A,

No synthetic derivative

gibberellic acid

Abcisic acid Abicisin &I Ethylene

Seed germination, fruit

growth, Meleic hydrochloride,

Growth inhibitor, closure

morphactins

of stomata.

l e n e

Fruit ripening hormone

Sterilization of explant is daone using sodium bypochlorite, sodium peroxide, mercuric chloride or ethanol. Glassware shops in hot air oven when 160-180°c for 2-4 hour.

Steps involved

in in

pant tissue culture:

1. Preparation of suitable nutrient medium and sterilization of medium. 2. Selection of suitable explant (part of plant). 3. Sterilization of explant, 4. Inoculation or transfer of explant, on the medium 5. Incubation at 25+ 2°c, 50-60% RH. 6. Regeneration hardening and planlets transfer.

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Types of plant tissue culture (Gpat, Niper, Di, Pharmacist) Type of PTC

r.no

Comments

Explant culture

Parenchyma is most versatile of all tissue and capable of cell division and growth, explant culture Shahar culture of plant

material example any part of plant such as young and healthy pieces of leaf, steam, and cotyledons. Callus culture

Callus is

a mass

of

loosely arranged

thin

walled parenchyma

developing from proliferating celisefnarent tissue. cells Naturally callus is developed after igteetiph to the plant, due to stimulation of hormones like auxínunedeytokinin. Callus can be developed in from explant.

culture Root culture

Generally excised rootis cultured in liquid medium,

Cell suspension

by bý trágsfering the fragments of callus into Itis prepared liquid mediup

culture

5

thelaboritory

Meristem culture or

shoot culture and

a

It is used for production of virus free plants. Apical meristem (thereiondfshoot Apex lying distal to leave perimardium)

micropropagation Protoplast culture

of protoplast (cell without cell wall) the cell wall of GCMure is removed

ell

for

mechanically or enzymatically. Lastly are used

protoplast fusion and somatic hybridization of the

FP BA protoplast are used using fusinogen eg. Most common is

polyethylene glycol PEG, and sodium nitrate. Eg. Pomatose

(potato+ tomato)

Applications of plant tissue culture: 1. Improvement of hybrids eg. Rice and wheat. 2. Production of cncapsulated seeds: covering of endospern with a gel. 3. Production of disease resistant plant: BT (bacillus thuringiensis gene responsible for production of toxin) cotton. 4. Production of virus free plant (meristem culture).

5. Production of stress resistant plant, transfer of gene in eukaryotes: various physiologically active alkaloids and glycoside).

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Transgenic animal and plants (Gpal, Niper, DI, Pharmacis)

|Dolly sheep 2

Golden rice

First

transgenic animal, hybrid of white sheep and

black head

ship.

A rice with increased vitamin A content (yellow in colour due to Pro vitamin A)

t cotton

Insect resistant cotton, crop improvement

Edible

Different antibody coding genes are transformed into the various

vaccines

vegetables. For the production of antibodies

Monoclonal antibodies and their Uses

Monoclonal

(Gpat, Niper, P

macist) Uses

Monoclonak

antibodies

antibodies

Abciximab

uses

Anti-platelet

Transtuzumab

Metastatic Breast

(Herceptin) Alemtuzumab

B-cell chronic

Gemtuzumab

Acute myelogenous leukemia

Ibritumonab

Rituximab-failed non-

lymphocytic leukemiu

Tyrosine kinase

Gefitinib

hodgkin's lymphoma

activity/Nopsnmlcell

lung cançer Infiximab

Rhchuhtyid hritiCrohn's disease

Muromonab/ Daclizumab/ Basiliximab

Allograft rejection reaction

Omalizumab

Anti-ige

Palivizumab

Respiratory syncytial virus

antibody/treatment of asthma Rituximab

Non-hodgkin's lymphoma/Folliculartype lymphoma

Types of immunity (Gpat, Niper, D1, Pharmacisl):

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Immunity is defined as resistant power of an individual against this pathogenic microorganisms, antigen is the material from pathogenic microorganisms capable of stimulation of immune response, antibodies are specific proteins that are produced tu to neutralize the

Innate

specific antigen by the body.

or natural immunity: non specific defence mechanism which is known as

inherited resistance so resistance is there which came from naturally. Eg. Diesel virus

cannot multiply in the canine cells, therefore dogs have natural immunity to the measles. Speciic immunity or Acquired immunity: This is usually developed by natural or artificial way. Which

are

further divided into active or

passive immunity.

Active immunity means sensitization by antigen in the body to producèantibodies from the T lymphocytes, passive immunity means giving of

preforniedunaibodies.

1. Naturally acquired:

A. Naturally acquired active immunity: eg. Antigen enter pnurálly in the body and body produces antibodies against it. B. Naturally acquired passive immunity: Already peadiced antibodies from mother transferred

to

foetus via placenta or from

the mu

2. Artificial acquired: a. Artificialy aquard active immuáity:ämtigen is introduced to artificially inside the body against which body produces anttbodies from lymphocytes. Eg. Vaccines, toxoids, live vaccines

preformed antibodies in the blood b.isolated Artificlally acquired passipe jmmunity: and sera,

transformesipha the body patient. Eg

serum are

immunoglobulins

Mechanism of ipmnúityA. Cell mediated immunity: T (thymus) cell mediated immunity, regulated observed and cytotoxic action of T cells during specific immune response, akes almost 36 hour to accomplish the effect. B. Humoral

immunity

B cell mediated

immunity: this immunity represents immunity duly mediated by antibodies in the body fluids that is plasma and lymph; as these antibodies are or

adequately synthesized and subsequently secreted by B (none marrow) cells

C. Congenital immunity is the immunity critically present at birth which may be natural or acquired.

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D. Herd immunity: immune production duly accomplished via vaccination of a portion of population, that me eventually minimise the spread of disease by restricting the number of

potential patients for particular pathogen. Vaccines will produce artificial active immunity ond sera immunoglobulins will produce

artificial passive immunity. Interferons are proieins produced against the viral infection so they may be called as viral antibodies. Alpha beta and gamma are the three types of interferon.

Microbial assay of antibiotics

F GPAT2021

Examples of vaccines

A. Bacterial vaccine

L.Live vaccines eg. BCG vaccine for tuberculosis IL Killed vaccine eg. Cholera vaccine II. Subunit vaccine eg. Typhoid Vi antigen

B. Viral vaccines I. Live viral vaccine: Sabin polio vaccinaor Otalpolio vaccine IL. Killed viral vaccine: Salk polio ydccinewhich is injectable

M. Subunit viral vaccine: hepafitis

Waccine.

Comparison of hypersensitlvlty types

TyP

Alternatlve

often mentloned disorders

Descriptlon

Mediators

names Fast response which occurs in

minutes, rather than multiple hours

Atopy, Anaphylaxis

Allergy (immediate)

or days. Free antigens cross link the g E on mast cells and basophis

Asthma

which causes a release of vasoactive biomolecules. Testing can be done

Churg-Strauss Syndrome

via skin test for specific lgE.

II

Cytotoxic,

Autoimmune hemolytic

gM

or

IgG

antibody11

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binds

Antibody (lgM IgG) antigen on a target cell, which is

9766654377

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to

dependent

nea

Complement

actually a host cell hat 1s perceived

Rheumatic heart disease

MAC

immune system foreign, by the leading to cellular destruction via the as

MAC. Testing includes both the

Thrombocytopenia

direct and indirect Coombs test.

Erythroblastosis fetalis

Goodpasture's syndrome Graves' disease see type V explanation below

Myasthenia gravis see type V explanation below

GPAT 2021

Serum sickness, Rheumatoid arthritis, Arthus reaction Post streptococcal

glomerulonephritis Immune

II

complex disease

Antibody (IgG) binds to soluble antigen, forming a circulating

Membranous nephropathy

e

complex. This is often

Reactive arthritis, Lupus

omplement

nephritis

and kidney, NeutrophilsJoints initiating intlammatory reaction.

Systemic Ilupus erythemtosus

deposited in the vessel walls of the a

local

Extrinsic allettic

alveolitiskypersohsitivity pneupnaniti) Cantactdermatitis, including Delayed-typPRushiol-induced contact

Helper T cells (specifically Thl

hypersensitivi dermatitis (poison ivy rash).

helper t cells) are activated by an

y, cell-

mediated V.

Mantoux test, Chronic

immune

transplant rejection

memory

Multiple sclerosis, Coeliac

response,

disease

antibody-

independent

Hashimoto's thyroiditis- Some

T-cells

cell. When the antugen presenting antgen is presented again in the

future, the memory Thl cells will activate macrophages and cause an

nflammatory response. This ultimately can lead to tissue damage.

type 2. Mostly type 4.

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Autoimmune

disease,

Graves' disease

IgM or IgG

receptor mediated (see| Myasthenia gravis

Complement

below)

E

EH

Pamacist

oder &Ach

0ON

cOMING SOON

mib

S

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