eChapters

Principle

The SDS polyacrylamide gel electrophoresis (PAGE) performs:

Separation of proteins according to their molecular weight

Ascertain whether a given protein is actually present in a sample,

Assessment of purity of the preparation

Estimation of the approximate quantity of the protein, and

Measurement of the size of the protein.

r DNA Biotechnology
    
Concept of r DNA
    
DNA Preparation from Tissue
    
Reagents
    
Preparation
    
DNA Buffer (Tris-EDTA)
    
RNase A (20 mg/ml)
    
Toe DNA Preparation for Southern Blots
    
Southern Blot
    
PCR
    
Polymerase Chain Reaction
    
The Cycling Reactions
    
Denaturation at 94°C
    
Annealing at 54°C
    
Extension at 72°C
    
DNA Sequencing
    
The Sequencing Reaction
    
Denaturation at 94°C
    
Annealing at 50°C
    
Extension at 60°C
    
Separation of the Molecules
    
Detection on An Automated Sequencer
    
Assembling of The Sequenced Parts of a Gene
    
Freezing of Buffalo Semen (Jindal, 1995)
    
Tissue Culture
    
Preparation of Media
    
Laboratory Procedure for IVF (Kharche et al., 2008)
    
Source of Oocytes
    
Preparation of Estrous Goat Serum
    
Collection of Oocytes
    
Laparoscopic Ovum Pick-up
    
Oocyte Retrieval from Ovaries Collected form Slaughter 
    
House
    
Slicing Technique
    
Follicle Puncture Technique
    
Follicle Aspiration Technique
    
Follicle Isolation Technique
    
Evaluation of Oocytes
    
In vitro Maturation of Oocytes
    
Preparation of Cells for Co-culture
    
Assessment of Nuclear Maturation After in vitro Maturation of Oocytes
    
Analysis of Nuclear Status is Ascribed to One of the Following
    
Sperm Preparation for in vitro Fertilization of Oocytes
    
In vitro Fertilization of Oocytes
    
Assessment of in vitro Fertilization of Oocytes
    
Assessment of Cleavage
    
In vitro embryo culture

1.     Determination of lmax
    
2.     Verification of Beer-Lambert Law
    
3.     Estimation of Carbohydrate by Anthrone Method
    
4.     Estimation of Reducing Sugar by Di Nitro Salicylic (DNS) Acid
    
5.     Estimation of Protein by Biuret Method
    
6.     Estimation of Protein by Lowry’s Method
    
7.     Estimation of Protein by Bradford Method
    
8.     Estimation of Cholesterol
    
9.     Estimation of DNA
    
10.     Estimation of RNA
    
11.     Determination of iso Electric Point of Amino Acid

Enzymes are biocatalysts composed of amino acids. Enzyme reactions are highly specific. It binds to the substrate to form a reversible intermediate product called enzyme substrate complex. As the concentration of the substrate increases the rate of formation of the intermediate complex also increases. This increase in velocity of the reaction is in a hyperbolic manner till it achieves the maximum velocity called Vmax .At this point all the enzymes will be in the form of ES complex. Substrate concentration will be in at its optimum level i.e. further increase in substrate concentration will not affect the rate of the reaction. The substrate concentration at which the rate of the reaction is half the maximum velocity Vmax is called the Michaelis-Menten constant, Km. This is constant for the given enzyme acting on the same substrate.

Sex Chromosomes

Sex can be considered at chromosomal, gonadal, phenotypic, and gender identity levels.

Human females are homogametic (XX) and males are heterogametic (XY).

The Y chromosome contains few identified genes, while the larger X chromosome contains several thousand genes. Pseudoautosomal regions at both tips of the Y chromosome contain genes that have counterparts on the X chromosome.

The male determining gene, SRY, has been found on the Y chromosome.

The Structure of Proteins

Proteins are composed of building blocks called amino acids. A string of amino acids is called a polypeptide chain. Once such a chain has folded into its working three-dimensional shape, it is a protein. Though there are tens of thousands of different proteins, all of them are put together from a starting set of 20 amino acids. It is the order in which the amino acids are linked in a polypeptide chain that determines which protein will be produced.

Uses of Microorganisms in Biotechnology

Somatostatin was the first human peptide hormone produced by recombinant bacteria

Many important medicines are produced by recombinant microorganisms

Bacterial species can be used as biological control agents

The release of recombinant microorganisms into the environment is sometimes controversial

Microorganisms can reduce environmental pollutants

1.     Preparation of Liquid and Solid Media
    
2.     Isolation of Pure Culture
    
3.     Analysis Bacterial Growth Curve
    
4.     Study of Effect of Carbon Source on Bacterial Growth
    
5.     Gram Staining
    
6.     Aseptic Cell Transfers
    
7.     Prokaryote Cell Number by Dilution Plating
    
8.     Cell Mass by Measurement of Turbidity
    
9.     Isolation of Bactriophage from Sewage Sample
    
10.     Standard Plate Count
    
11.     Imvic Test
    
12.     Antibiotic Sensitivity Test on Microbes
    
13.     Study of Transformation In Bacteria
    
14.     Study of Bacterial Conjugation
    
15.     Effect of pH and Temperature on Bacterial Growth
    
16.     Immobilization of Yeast Cells and Alcohol Production
    
17.     Citric Acid Production using Aspergillus Niger
    
18.     Alcohol Production from Yeast and Estimation of Alcohol

1.     Plant Tissue Culture Laboratory
    
2.     Cleaning and Sterilization of Glass Wares
    
3.     Media Sterilization
    
4.     Media Composition and Media Preparation
    
5.     Callus Culture in Carrot
    
6.     Culture of Shoot Tip (Vanilla)
    
7.     Anther Culture of Datura
    
8.     Subculture of Carrot Callus
    
9.     Hardening of Plantlets
    
10.     Isolation of Protoplast from Plant Leaves

1.     Isolation of Plant DNA
    
2.     RAPD Analysis of Genomic DNA
    
3.    Agro Bacterium based Transformation in Plants
    
4.     Assay for Gus Operon
    
5.     Confirmation of Transformation using Polymerized Chain Reaction (PCR)
    
6.     Primer Designing
    
7.     Isolation, Purification and Identification of Plant Secondary Metabolites
    
8.     Evaluation of Antioxidant Activity of Plant Extract
    
9.     Estimation of Total Phenolic Content in A Plant Extract
    
10.     Estimation of Total Flavanoid Content

1.     Tissue Collection and Culture
    
2.     Trypsinization of Cells
    
3.     Cell Separation by Density Gradient Method
    
4.     Low Speed Separation of Cells
    
5.     Isolation of Lymphocyte
    
6.     Cryo Preservation
    
7.     Isolation of Spleen Cells
    
8.     Isolation of Peritoneal Macrophage
    
9.     Isolation of Blood Dna
    
10.     Differential Count of Leucocytes
    
11.     Slide Gel Electrophoresis
    
12.     Haemagglutination Assay
    
13.     Elisa
    
14.     Western Blotting
    
15.     Widal Test
    
16.     Identification of Y-chromosomes in Cattle using Pcr

Genetic Analysis of Diseases

A genetic basis for a human diseases may be suggested from a variety of observations

Inheritance patterns of human diseases may be determined via pedigree analysis

Many genetic disorders are heterogeneous

Genetic testing can identify many inherited human diseases

Prions are infectious particles that alter protein function posttranslationally

Genes in Populations

• A population is a group of interbreeding individuals who share a gene pool
• Some genes are monomorphic, and others are polymorphic
• Population genetics is concerned with allele and genotype frequencies

The Hardy-Weinberg Equilibrium

The Hardy-Weinberg equation can be used to calculate genotype frequencies based on allele frequencies

Nonrandom mating may occur in natural and human populations

Origin of Species

A biological species is a group of reproductively isolated individuals

Speciation usually occurs via a branching process called cladogenesis

Divergent evolution can be allopatric, parapatric, or sympatric

Müntzing was able to re-create an allotetraploid species

Evolution can proceed gradually or be punctuated by periods of rapid change

DNA (deoxyribonucleic acid), the giant molecule that carries genetic information in living things, is made up of just a few chemical building blocks that bond together in very particular ways. A typical molecule of DNA consists of two strands that are linked together. A segment can be visualized as a ladder-like structure:

The DNA as made of two strands, a left and a right, which are linked in the middle. In reality, since the ladder is twisted, there really is no “right” or “left,” but we use that terminology here since it applies to the model graphs. The square shapes and diamonds you see in the middles of the “rungs” are the links, which are meant to represent the different chemical bonds between the two strands of DNA.

Acetic acid            
    

Molecular weight    60.05        
    Melting point (^ºC)    17        
    Boiling point (^ºC)    118        
    Flash point (^ºC)    40        
    Auto-ignition point (^ºC)    426        
    Specific gravity at 20^ºC    1.05        
    Molarity of the concentrated acid (M)    17.4    
    1 M solution    57.5 ml concentrated acid per litre

Cells Contain 4 Major Types of Giant Molecules (Macromolecules, Polymers)

Carbohydrates

Lipids

Proteins

Nucleic Acids

BIOLOGICAL POLYMERS ARE MADE FROM ABOUT 60 SMALL BUILDING BLOCKS

Cells contain thousands of types of giant molecules (macromolecules, polymers)

Polymers are macromolecules made from a single building block molecule Most of the macromolecules are made from about 60 types of small building block molecules

Most macromolecules are linear: building blocks linked head to tail- a few have branches (some polysaccharides)

This is what you would get if you heated cells in hydrochloric acid overnight at 110 degrees C:

ENZYMES

Enzymes are the heart of Biochemistry

Protein based catalysts.

Enormously effective catalysts: typically enhance rates by 10⁶ to 10¹² fold.

Operate under mild conditions: 0 - 100 °C (or perhaps even 300+ °C for some bacteria in deep ocean), 20 - 40 °C for most organisms; and low pressures (atmospheric).

Very specific: Generally catalyze reaction for a very restricted group of molecules, sometimes for a single naturally occurring molecule of a single chirality.

There are other groups that contribute to the reactivity of enzymes beside amino acid residues.

These groups are called cofactors - chemicals required by apoenzymes (inactive) to become holoenzymes (active).

There are two types of cofactors:

1. Essential ions - metal ions -inorganic
2. Coenzymes - organic molecules that act as group-transfer reagents (accept or donate groups)- can also be H⁺ and/or e^-

Both provide reactive groups not found on amino acids side chains.

Coenzymes can be either cosubstrates (loosely bound to enzyme; is altered, then regenerated) or prosthetic groups (tightly bound to enzyme).

Coenzymes can be classified by their source: