Dot Blot Technique Principle, Steps & Applications

 In the realm of molecular biology, where precision and efficiency are paramount, the dot blot technique stands out as a simple yet powerful method for detecting and analyzing biomolecules. Originally developed in the 1970s by George Stark and colleagues, dot blotting has since become a cornerstone in various research fields, including genetics, immunology, and diagnostics.

Principle of Dot Blotting :

 At its core, dot blotting involves the immobilization of target molecules, such as DNA, RNA, or proteins, onto a solid support membrane. This membrane, typically made of nitrocellulose or nylon, acts as a platform for subsequent detection and analysis steps.

The procedure begins with the application of a small volume (usually a few microliters) of the sample directly onto the membrane in the form of discrete dots. The samples can be pure substances, crude extracts, or complex mixtures, depending on the experimental objectives.

Steps Involved in Dot Blotting:

1.Sample Application: The sample is carefully spotted onto the membrane, usually using a pipette or a specialized dot blot apparatus. The arrangement of the dots can be controlled to facilitate comparison and quantification.

2.Blocking: To minimize nonspecific interactions and reduce background noise, the membrane is incubated with a blocking agent, such as bovine serum albumin (BSA) or non-fat dry milk. This step saturates any unbound binding sites on the membrane surface.

3.Primary Antibody Incubation: Next, the membrane is exposed to a specific primary antibody that recognizes the target molecule of interest. This antibody binds selectively to its target, forming an antigen-antibody complex.

4.Washing: Excess primary antibody is removed through several washes with a suitable buffer. This step helps to remove any unbound antibodies and further reduces background signals.

5.Detection: The presence of the target molecule is visualized by adding a detection reagent that interacts with the primary antibody. This can involve secondary antibodies conjugated to enzymes, fluorophores, or other reporter molecules. The detection reagent generates a signal that can be detected and quantified.

6.Analysis: The dot blot results are analyzed either qualitatively or quantitatively, depending on the research goals. Quantification can be achieved through densitometry or by comparing signal intensities relative to standards of known concentration.

Advantages of Dot Blotting:

- Speed and Simplicity: Dot blotting offers a rapid and straightforward alternative to traditional methods such as Western blotting and Southern blotting. The entire procedure can be completed in a matter of hours, making it ideal for high-throughput applications.

- Versatility: Dot blotting can be adapted to detect a wide range of biomolecules, including DNA, RNA, proteins, antibodies, and antigens. This versatility makes it a valuable tool in various research areas, from gene expression analysis to disease diagnostics.

- Sensitivity: With proper optimization, dot blotting can achieve high levels of sensitivity, allowing for the detection of low-abundance molecules in complex samples.

- Cost-Effectiveness: Dot blotting requires minimal specialized equipment and reagents, making it a cost-effective option for many laboratories.

Applications of Dot Blotting:

- Gene Expression Analysis: Dot blotting can be used to study gene expression patterns by detecting mRNA transcripts or specific DNA sequences.

- Protein Detection: Dot blotting is commonly employed to screen for the presence of specific proteins in biological samples, such as cell lysates or tissue extracts.

- Antibody Screening: Dot blotting can rapidly screen for the presence of antibodies in patient sera, facilitating the diagnosis of infectious diseases or autoimmune disorders.

- Quality Control: Dot blotting is often used in quality control processes to assess the purity and identity of biomolecules, such as recombinant proteins or synthetic oligonucleotides.

Application of Biotechnology in various fields


 Biotechnology is a multidisciplinary field that combines principles of biology, chemistry, physics, engineering, and computer science to develop technologies and products that utilize biological systems, living organisms, or derivatives thereof, for various applications. These applications can range from healthcare and agriculture to industrial processes and environmental remediation. In essence, biotechnology harnesses the inherent capabilities of living organisms or their components to solve problems, create new products, or improve existing processes. Examples include genetic engineering, biopharmaceuticals, agricultural biotechnology, biofuels, and environmental bioremediation.

Application of Biotechnology in various fields 

Biotechnology, a dynamic field at the intersection of biology and technology, has revolutionized industries ranging from healthcare to agriculture. With its vast array of applications, biotechnology continues to push the boundaries of what is possible in science and innovation.

Healthcare Advancements

 One of the most impactful applications of biotechnology is in healthcare. From personalized medicine to advanced diagnostics, biotechnology plays a pivotal role in improving human health. Genetic engineering techniques, such as CRISPR-Cas9, have opened doors to precise gene editing, offering hope for treating genetic disorders and diseases. Additionally, biopharmaceuticals derived from biotechnology, such as insulin and monoclonal antibodies, have transformed the treatment of various illnesses, including cancer and autoimmune diseases.

Agricultural Innovations

 Biotechnology has also revolutionized agriculture, offering solutions to global challenges such as food security and environmental sustainability. Genetically modified organisms (GMOs) have been developed to enhance crop yield, improve resistance to pests and diseases, and reduce the need for chemical pesticides and fertilizers. Furthermore, biotechnology enables the development of drought-resistant and nutrient-enriched crops, addressing the challenges posed by climate change and malnutrition.

Environmental Remediation

 Biotechnology presents promising solutions for environmental remediation and conservation. Bioremediation techniques utilize microorganisms to degrade pollutants in soil, water, and air, offering eco-friendly alternatives to traditional cleanup methods. Moreover, biotechnology facilitates the development of biofuels, such as biodiesel and bioethanol, derived from renewable sources like algae and agricultural waste, reducing reliance on fossil fuels and mitigating greenhouse gas emissions.

Industrial Applications

 In the industrial sector, biotechnology drives innovation in various fields, including bio-based materials, bioinformatics, and bioprocessing. Bio-based materials, such as bioplastics and biofibers, offer sustainable alternatives to conventional petroleum-based products, reducing environmental impact and promoting circular economy principles. Additionally, bioprocessing techniques enable the production of bio-based chemicals, enzymes, and pharmaceuticals through microbial fermentation and enzymatic catalysis, fostering a greener and more efficient manufacturing industry.

Challenges and Ethical Considerations

 Despite its tremendous potential, biotechnology also raises ethical and societal concerns, particularly regarding genetic engineering, biosecurity, and bioprospecting. The manipulation of genetic material, while offering opportunities for therapeutic interventions and agricultural improvements, raises questions about safety, equity, and unintended consequences. Furthermore, the commodification of biological resources and the potential for biopiracy highlight the importance of ethical frameworks and regulatory oversight to ensure responsible innovation and equitable access to biotechnological advancements.


Biotechnology continues to shape the world we live in, offering transformative solutions to global challenges while posing complex ethical dilemmas. As we navigate the evolving landscape of biotechnological innovation, it is crucial to approach its applications with a balanced perspective, weighing the potential benefits against ethical considerations and societal implications. By harnessing the power of biotechnology responsibly, we can unlock its boundless potential to improve lives, safeguard the environment, and drive sustainable development for future generations.

Koch's postulates application in Microbiology

 In the world of microbiology, understanding the causes of infectious diseases is crucial for effective prevention and treatment strategies. One of the fundamental principles in this field is Koch's postulates, named after the German physician and microbiologist Robert Koch, who developed them in the late 19th century. Koch's postulates provide a systematic approach to demonstrate the causative relationship between a microorganism and a disease, laying the groundwork for the field of medical microbiology.

The Four Koch's Postulates:

1. The microorganism must be present in every case of the disease but absent from healthy organisms.

   This postulate emphasizes the importance of identifying a specific microorganism consistently associated with a particular disease. Koch recognized the need to isolate and characterize the microorganism responsible for causing the illness.

2. The microorganism must be isolated from the diseased organism and grown in pure culture.

 Once the microorganism is identified in a diseased individual, it must be isolated and cultivated in laboratory conditions. This step ensures that the microorganism can be studied and manipulated independently of its host.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

   To establish a causal relationship, Koch demonstrated that inoculating a healthy organism with the isolated microorganism results in the development of the same disease observed in the original host. This step confirms that the microorganism is indeed responsible for the illness.

4. The microorganism must be re-isolated from the experimentally infected organism.

   Finally, to complete the chain of evidence, Koch reisolated the same microorganism from the experimentally infected organism. This step confirms that the microorganism retrieved from the experimental host is identical to the one initially isolated from the original diseased individual.

Applications and Limitations:

Koch's postulates have been instrumental in identifying the causative agents of numerous infectious diseases, including tuberculosis, cholera, and anthrax. By rigorously applying these criteria, researchers have been able to establish causal relationships between specific pathogens and their associated diseases, paving the way for the development of vaccines, antibiotics, and other treatments.

However, it's important to note that there are limitations to Koch's postulates. For example, some microorganisms cannot be grown in pure culture or do not cause disease when introduced into a healthy host due to complex interactions with the host's immune system or other factors. Additionally, advances in molecular biology and genomics have revealed cases where multiple microorganisms or non-infectious agents contribute to disease pathogenesis, complicating the application of Koch's postulates in certain contexts.


Despite these limitations, Koch's postulates remain a cornerstone of microbiology and have greatly contributed to our understanding of infectious diseases. They provide a systematic framework for establishing causation and have guided generations of researchers in their quest to unravel the mysteries of microbial pathogenesis. As technology continues to advance, researchers will undoubtedly refine and expand upon Koch's postulates, ensuring their continued relevance in the field of medical microbiology.

General Nature and Basic Structure of Enzyme

 A living cell is capable of performing a multitude of  biochemical reactions in order to survive grow and multiply. This involves

  • Degradation of complex nutrients into simpler forms so that they can be absorbed by the cell.
  • Uptake of these simple nutrients.
  • Chemical transformation of these simple nutrient molecules into various precursor metabolites, so that they are available for biosynthesis.
  • Generation of ATP and other bio reactive molecules, which co-operate in cellular biochemical reactions.
  • Biosynthesis of cellular molecules and structural components of the cell etc.

All these diverse types of chemical reactions can occur with a high degree of specificity and rate, under normal growth conditions for the organism. These chemical changes are brought about by the living cell through the role and participation of a special class of molecules known as enzymes . Enzymes function in the cell by acting as a biocatalyst.

Discovery of Enzymes

The term enzyme was coined by Kuhne in 1878, suggesting in yeast (en = in, zyme = yeast). It was examined that cell free extract from yeasts is capable of causing conversion of sugar into alcohol. This lead to the understanding that the molecules present inside the cells of yeasts are responsible for these chemical transformations. Kuhne called them as enzymes.

Later on, it was established that all biochemical activities of a living cell are attributed to these magic molecules called enzymes. The first enzyme to be discovered was 'amylase'. Its presence in malt extract was detected in 1833 by two French chemists Pain and Persuses.

General Nature of Enzymes

Enzymes are regarded as organic biocatalysts which are capable of functioning both extra cellular and intra cellular. Following are the major characteristics of enzymes. 

1] With exception of a few catalytic RNA molecules or ribozymes, all enzymes are protein in nature. Over 90 % enzymes are globular proteins. Many of them are conjugated proteins, having non protein component. 

Enzymes may be monomeric or multimeric proteins. As they are proteins, they share all major characteristic of proteins .

  • They are macromolecules with high MW.
  • They are non dialyzable and are unable to pass through semi permeable membrane.
  • They are amphoteric in nature. i.e. they possess both types of lonizable groups : -NH₂ and -COOH. which on ionization . yield positively charged ammonium (NH4+) ion and negatively charged carboxyl (COO-) ion.
  • As they are amphoteric, they possess specific isoelectric pH. At this pH, both -NH₂ and -COOH groups get equally ionized such that their net electrical charge becomes minimum.
  • They show electrophoretic mobility. If the enzyme solution is at pH, above isoelectric value, they acquire negative charge and hence move towards anode and vice versa.
  • They are colloidal in nature.
  • They can be salted out by salts like ammonium sulfate
  • They can be precipitated out by protein denaturing solvents like acetone and alcohol.
  • They can absorb maximum UV light at 280 um wavelength.

2]. Enzymes are mostly thermo labile and get denatured at high temperature, usually above 60°C. However, some enzymes are found thermo stable and can withstand high temperature up to 70°C - 80°C or even more.

3]. Enzymes are highly specific in action. They can act on a specific substrate molecule to bring about specific biochemical reaction .

Catalytic RNA - Ribozyme

  Apart from proteins, a few RNA have also been recognized to have catalytic activity. They are called ribozymes or catalytic RNA. They were first recognized by Thomas Cech in 1982. The ribozymes have two types of common roles :
  1. RNA processing, where the RNA is involved in RNA splicing, RNA ligation and RNA replication.  
  2. Peptide bond formation during protein synthesis. In ribosomes, they function as a part of rRNA and participate in peptide bond formation.

Basic structure of Enzymes

In general, enzyme molecule consists of two components.
  1. Apo enzyme and
  2. Prosthetic group or cofactor

Apo enzyme is protein part of enzyme.
Prosthetic group or cofactors are non protein part of enzymes. They consist of vitamin or their derivatives or metal lons. (If these non protein components are bound loosely to protein, they are called cofactors) and if they are bound firmly (covalently), are called prosthetic groups. Apo enzyme and prosthetic group form complex to form active form of enzyme, known as Holoenzyme.

Classification of Enzymes

What is GMP ?

 Good Manufacturing Practices (GMP) is the minimum standard that a pharmaceutical manufacturer must meet in their production processes. Process must:

  • Be of consistent high quality.
  • Be appropriate to their intended use.
  • Meet the requirements of the marketing authorization (MA) and product specification.

  Pharmaceutical organizations which comply with GMP must have manufacturer license. Regulatory agencies carries out in section on these pharmaceutical organizations to check if manufacturing sites comply with GMP. Sites are inspected when applied for a manufacturer license and then periodically based on risk assessments.

CGMP Legal Principles

Quality built into product

  • By "taking care" in making medicine.
  • Quality system is based on the principle of Quality by Design instead of quality by Inspection.
Without/Inadequate cGMP
  • Product(s) adulterated(defects need not be shown).
  • Firm and its management are responsible.
Current = Dynamic
  • Standards evolve over time.
Good Practices
  • Minimal standards.
  • Not "best practices". (Unless "best" is, in fact, current minimal.)


  • Is a legal requirement enforced and mandated through law, regulations and directives by each country government.
  • To protect public health of each and every individual.
  • Prevent contamination and mix-ups.
  • Prevents mislabeling and adulteration.
  • Consistent maintenance of Quality product supply throughout the product life cycle.
  • Ensure high standard quality product supplied into the market is safe and effective.
  • Satisfy stakeholders, customers and consumers.
  • To meet the requirement of the marketing authorization (MA) and product specification.
  • Enhance the organization image and reputation.

Responsibility of GMP

  • Quality and GMP compliance are independent of job title and have no boundaries.
  • Everyone involves in the process regulatory compliance, manufacturing, packing, Quality Control, distribution and supply of pharmaceuticals product has the responsibility to make sure it reaches to the patient with registered quality standards.
  • Building quality into the entire process of an operation makes sustainable compliance more achievable. However, it requires commitment by Senior management and the allocation of adequate resources (personnel, facilities, training, etc.).

Applications of Biotechnology in Various Fields

 Biotechnology is a branch of biology involving the use of living organisms and bioprocesses in engineering, technology, medicine, and other fields using bioproducts. The term biotechnology indicates the use of living organisms or their products for modifying the human health and environment.

Applications of Biotechnology in various fields

1] Medicine:

Modern biotechnology finds promising applications in medicine as in:

i) Drug Production :

  • Most of the traditional pharmaceutical drugs used for treating the symptoms of a disease are simpler molecules found through trials and errors. Small molecules are manufactured chemically, but the larger ones are created by human cells, bacterial cells, yeast cells, and animal or plant cells.
  • Modern biotechnology involves the use of genetically altered microorganisms (e.g., E.coli or yeast) for producing insulin or antibiotics via synthetic means. Modern biotechnology can also be used for producing plant-made pharmaceuticals. Biotechnology is also used in the development of molecular diagnostic devices used to define the target patient population for a given biopharmaceutical. For example, herceptin was the first drug to be used with a matching diagnostic test for treating breast cancer in women whose cancer cells expressed HER2 protein.

ii) Pharmacogenomics :

  • It is the study of how the genetic inheritance of an individual affects his/her body's response to drugs. The term pharmacogenomics was derived from the words pharmacology and genomics, thus it involves studying the relationship between pharmaceuticals and genetics. 
  • Pharmacogenomics aims to design and produce drugs adapted to each individual's genetic makeup.

iii) Gene Therapy :

  • It is used for the treatment of genetic and acquired diseases like cancer and AIDS. Gene therapy utilises normal genes for supplementing or replacing the defective genes or for strengthening immunity. This therapy targets either the somatic cells (i.e., body) or the gametes (ie., egg and sperm). 
  • In somatic gene therapy, the recipient's genome is altered; however, this alteration is not passed on to the next generation. On the other hand, in germline gene therapy, the egg and sperm cells of the parents are altered to be passed on to their offspring.

iv) Genetic Testing : 

This involves direct examination of the DNA, and is used for:
  • Carrier screening, or identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest,
  • Confirming the diagnosis of symptomatic individuals,
  • Determining sex,
  • Forensic/identity testing.
  • New-born screening.
  • Prenatal diagnostic screening.
  • Pre-symptomatic testing for determining the risk of developing adult-onset cancers, and
  • Pre-symptomatic testing for predicting adult-onset disorders.

2] Cloning :

  In this method, the nucleus from one cell is removed and is transferred to an unfertilised egg cell whose nucleus has either been deactivated or removed. Cloning can be done in the following two ways:

i) Reproductive Cloning :

  •  In this method, the egg cell after a few divisions is transferred to a uterus for its development into a foetus that is genetically identical to the donor of the original nucleus.

ii) Therapeutic Cloning :

  • In this method, the egg is placed in a petridish for its development into embryonic stem cells that are potential for treating several ailments.

3] Agriculture :

Biotechnology in the agricultural field is used for the following purposes:

i) Crop Yield :

  • For increasing the crop yield, one or two genes are transferred to a highly developed crop variety for imparting a new character. The current techniques of genetic engineering are best for the effects controlled by a single gene. Some genetic characteristics related to yield (e.g., enhanced growth) can be controlled by various genes, each posing a nominal effect on the yield.

ii) Reduced Vulnerability of Crops to Environmental Stresses :

  • Such crops which can be made resistant to biotic and abiotic stresses can be developed with the help of genes; for example, drought and salty soil are the two limiting factors in crop productivity.

iii) Increased Nutritional Qualities :

  • The nutritional value of proteins contained in foods can be enhanced; for example, proteins in legumes and cereals can be transformed such that they also provide amino acids required in the balanced diet of humans.

iv) Reduced Dependence on Fertilisers :

  • Modern biotechnology can also be used to reduce the dependence of farmers on agrochemicals; for example, Bacillus thuringiensis (Bt) is a soil bacterium that produces a protein having insecticidal properties. Conventionally, these bacteria were used to produce an insecticidal spray by a fermentation process. 
  • In this form, the Bt toxin occurs as an inactive protoxin, which becomes effective when digested by an insect. There are several Bt toxins and each has a specificity for some target insects.

v) Production of Novel Substances in Crop Plants :

  • Biotechnology is also applied for novel uses apart from food; for example, oilseed is genetically modified to produce fatty acids for detergents, substitute fuels, and petrochemicals. Potatoes, tomatoes, rice tobacco, lettuce, safflowers, and other plants are genetically engineered to produce insulin and certain vaccines.

4] Biological Engineering :

 It is a branch of engineering that involves biotechnologies and biological science. It includes different disciplines such as biochemical engineering, biomedical engineering, bio-process engineering, biosystem engineering, etc.

Classification of Bacteria Based on Temperature Requirement


 The temperature at which the growth of organisms is maximum and most rapid is called the optimum growth temperature. The range of temperature between which the organisms can grow is called the temperature range for growth. Shift in temperature on either side of optimum value retards growth. Usually the maximum temperature up to which the organisms can grow, is close to the optimum growth temperature. Whereas the minimum temperature required for growth can be much lower than the optimum value. Minimum, optimum and maximum growth temperatures are called cardinal temperatures.

   Cardinal temperatures vary greatly between microorganisms. Optima normally range from 0°C to 75°C; whereas growth can take place between -20°C to 100°C. Organisms having a narrow range of growth temperature are called stenothermal while the organisms having wide range of growth temperature are called eurythermal.

Based on the temperature requirements for growth, bacteria can be divided into three groups:

  1. Psychrophiles
  2. Mesophiles
  3. Thermophiles


The organisms able to grow at low temperature i.e. below 10°C, are called psychrophiles. They can grow even at 0°C or even lower, if the medium is not frozen due to high salt concentration. The optimum temperature for growth is 15°C or lower. The maximum temperature of growth is 20°C. There are two types of psychrophiles.

  1. Obligate or true psychrophiles :  These are the organisms which grow at 0°C or lower with optimum growth temperature 15°C or below. e.g. Vibrio marinus, Vibrio psychroerythreous.
  2. Facultative psychrophiles or psychrotroph : These are the organisms which can grow at 0°C. But optimum temperature for growth is between 20°C to 30°C. e.g. Pseudoinonas flourescens.

The physiological factors for the psychrophilic nature of organisms are not much clear. But it is observed that their ribosomes and other cellular enzymes are unstable at high temperature. Hence, they cannot grow at higher temperature. Even their membrane permeability is altered at temperature above optimum value, leading to leakage of cell material, impairment of membrane permeability and hence results in loss of viability.


  These are the organisms that can grow at moderate temperatures of incubation, i.e. within range of 25°C to 40°C. Their optimum growth temperature falls within this range. All pathogenic bacteria for humans and warm blooded animals grow best at body temperature, i.e. at 37°C.


The organisms capable of growing best at temperature above 45°C are called thermophiles. They can be grouped into two categories.

1) Facultative thermophiles

These organisms can grow even in the mesophilic range of temperature.

2) Stereothermophiles or hyperthermophiles

  These bacteria cannot grow in the mesophilic range of temperature. They grow well between temperatures 45°C to 70°C. There are some organisms which can grow even at 110°C. These bacteria are also referred to as strict or obligate thermophiles. These bacteria are normally found from hot water springs, salt lakes etc.
e.g. Bacillus coaggulans, B. stereothermophilus. Thermus aquaticus can grow even at 100°C.

The basis of thermal resistance of these bacteria is the thermal stability of most of their cellular proteins.