Analytical/Preparative Centrifugation

 Analytical/Preparative Centrifugation:-

The 2 most common types of centrifugation are analytical and preparative; the distinction is between the 2 is based on the purpose of centrifugation. Analytical centrifugation involves measuring the physical properties of the sedimenting particles, such as sedimentation coefficient or molecular weight. Optimal methods are used in analytical ultracentrifugation. Molecules are observed by optical system during centrifugation, to allow observation of macromolecules in solution as they move in the gravitational field. The samples are centrifuged in cells with windows that lie parallel to the plane of rotation of the rotor head. As the rotor turns, the images of the cell (proteins) are projected by an optical system onto film or a computer. The concentration of the solution at various points in the cell is determined by absorption of a light of the appropriate wavelength. This can be accomplished either by measuring the degree of blackening of a photographic film or by the deflection of the recorder of the scanning system and fed into a computer. The other type of centrifugation is called preparative and the objective is to isolate specific particles that can be reused. There are many type of preparative centrifugation such as rate zonal, differential, and isopycnic centrifugation.

The major categories of innate lymphoid cells (ILCs) and their properties

 

Innate immune recognition by Toll-like receptors.

Properties of Vectors in RDT

Vectors

The term vector refers to the DNA molecules that act as transporting vehicle which carries target DNA into a host cell for the purpose of cloning and expression. Cloning vectors are used to clone target DNA whereas expression vectors are engineered so that desirable target DNA can be transcribed in RNA and translated into protein, A viral DNA or plasmid is generally used as a vector. The important features of a cloning vector are as follows:

1. Ability to replicate in host cells. All cloning vectors have origin of replication for autonomous replication within the host cell. The origin of replication is a specific sequence in DNA from where replication starts. When target DNA is linked to vector containing origin of replication then along with vector replication, desirable target DNA also starts replicating within the host cell.

2. Unique restriction sites for insertional cloning, All cloning vectors have features that allow a target DNA to be conveniently inserted into the vector. This may be a multiple cloning site (also called polylinker site), which contains many unique restriction sites. The restriction site(s) in the polylinker site are first cleaved by specific restriction enzyme(s), and a target gene is then ligated into the vector using DNA ligase.

3. Genetic marker to select for host cells containing the vector Genetic marker is a gene that allow the selection of transformed cell from non trans formed cells and recombinant containing transformed cells from non-recombinant containing transformed cells. Marker genes belong to two broad categories: selectable markers and screen able markers. A selectable marker gene encodes a product that allows the growth of one type of cells under specific conditions that kill or restrict the growth of other types of cells. A screenable marker gene, also called reporter gene, gives a product that can be detected using a simple and often quantitative assay. 

4. Low molecular weight (minimum amount of non-essential DNA). The advantages of a low molecular weight are several. First, the plasmid is more resistant to damage by shearing and is readily isolated from host cells. Secondly, low molecular weight plasmids are usually present as multiple copies. Finally, with a low molecular weight there is less chance that the vector will have multiple sites for any restriction endonuclease.

MCQ Quiz: Vectors

1. A vector is best defined as:

Protein carrier DNA molecule that carries foreign DNA into host RNA molecule Enzyme

2. Cloning vectors are mainly used for:

Protein degradation DNA replication and maintenance Cell death RNA splicing

3. Expression vectors are designed to:

Destroy target DNA Express target DNA as RNA and protein Prevent transcription Inhibit translation

4. Which of the following is commonly used as a vector?

Ribosome Plasmid Mitochondria Lysosome

5. Origin of replication (Ori) is required for:

Protein synthesis Autonomous replication of vector DNA degradation Cell signaling

6. Multiple cloning site (polylinker) contains:

Promoter sequences Multiple unique restriction sites Antibiotic genes only RNA genes

7. Selectable marker genes help in:

Killing transformed cells Selecting transformed cells Preventing ligation DNA sequencing

8. Screenable marker genes are also known as:

Housekeeping genes Reporter genes Structural genes Repressor genes

9. Low molecular weight of a vector is advantageous because:

Difficult to isolate Present in multiple copies Easily degraded Contains many restriction sites

10. Which is NOT an advantage of low molecular weight vectors?

Easy isolation High copy number Multiple restriction sites for same enzyme Resistance to shearing

CHEMICAL COMPOSITION OF LIVING ORGANISMS

Synopsis:-

• Introduction
• Composition of elements in living system
• Biogenic elements
• Water and Mineral substances
• Organic Matters
• Conclusion
• Reference 

        1.      Introduction to the topic:-                                                                                                                               The life on Earth can be understood as a form of existence of matter. All the living matter, i.e. living organisms, is composed of the same particles (atoms, ions, molecules) as the non-living organisms and chemical laws and laws of physics are applicable to both of them. There is a close connection between living and non-living nature, however they differ in chemical composition, structure, complexity and organization. While the chemical composition of the non-living nature is varied, the existence of living organisms is based on the presence of a few chemical elements, especially carbon, oxygen, nitrogen and hydrogen.

       All the chemical compounds in living organisms are composed of chemical elements. In these days almost 120 chemical elements are known. Out of this number 92 elements are naturally present in the nature (the rest were made in laboratories). Out of the 92 elements only 30 elements create the living matter and they are called biogenic elements.

There are 92 elements in the Earth´s crust. Oxygen and silicon represent the highest percentage - 75% of all elements. Both these elements of the Earth´s crust, as well as the other elements, are bonded especially in minerals (e.g. oxides, silicates) and rocks. The remaining 90 elements represent about 25 % of all elements.

Fig.: Representation of chemical elements in the Earth´s crust

2.     Composition of elements in living systems:-

                                     In all living systems we can always find 4 basic elements: carbon, oxygen, nitrogen and hydrogen. Carbon is the basic building unit contained in living matter. The percentage of carbon in the mass of living matter is 19.4 %. Oxygen and hydrogen are present in almost all organic compounds which create living organisms. The percentage of oxygen in the mass of living systems is 62.8 %, the percentage of hydrogen is 9.3 %. The source of hydrogen for organisms is water, the source of oxygen is water and the atmosphere. Nitrogen is bonded mainly in amino acids, proteins and nucleic acids. Its percentage is 5.1 %.

Chemical element	Average representation in living matter (%)	Average representation in non-living matter (%)
Carbon	19.37 %	0.18 %
Oxygen	62.80 %	49.40 %
Hydrogen	9.31 %	0.95 %
Nitrogen	5.14 %	0.63 %

2.1  Biogenic elements

 

       All elements contained in living matter are called biogenic elements. They are present in compounds, in the form of ions and in some special cases they are unbound (e.g. oxygen). According to their representation in organisms, the biogenic elements are divided into 3 groups: macrobiogenic, microbiogenic and trace elements. Trace and microbiogenic elements are sometimes also called oligobiogenic elements.

 

       I. Macrobiogenic elements – C, O, H, N, S, P, Na, K, Ca, Mg, Cl, Fe.  Four of these elements – O, C, H, N – represent up to 95 % of living matter. The rest of the elements mentioned above represent up to 4.9 %. Macrobiogenic elements have a building function.

      Carbon is the basis for all living matter. The typical feature of carbon atoms is the ability to bond to each other or to atoms of other elements. That is why there are many organic compounds of carbon. Carbon is also present in carbon dioxide and carbonates.

      Oxygen and hydrogen in organisms they are present both in the form of organic and inorganic compounds and they are a part of the basic micromolecule – water. Oxygen is produced by autotroph organisms (especially by plants and cyanobacteria) during the process of photosynthesis.

Nitrogen is a component of proteins and nucleic acids. It is also a part of nitrates and ammonium carbonate, which are necessary for the mineral nutrition of plants and also the synthesis of plant proteins.

      II. Microbiogenic elements – Cu, I, Mo, Mn, Zn, Co. The average content of these elements in living organisms is less than 0.1%. Microbiogenic elements have catalytic function, i.e. they are part of enzymes.

      III. Trace elements – e.g. Al, As, B, Br, F, Li, Ni, Se, Si, Ti, V. Their content in organisms is less than 0.001 %. As well as microbiogenic elements, trace elements are parts of enzymes and their function is catalytic.

 

3.  Chemical composition of living systems

 Living organisms are composed of several types of substances called biomolecules. According to their molecular weight, substances in living organisms are divided into two groups:

 1. Low molecular substances (M_r < 10 000)

•         water

•         inorganic (mineral) substances

•         intermediates of metabolic pathways (carboxylic acids etc.)

• final products of metabolic pathways (amino acids, monosaccharides, lipids, nucleotides)

 2. High molecular substances (M_r > 10 000)

•         proteins

•         polysaccharides

•         nucleic acids

      High molecular substances, which are present in living organisms, are also named as biological macromolecules or biopolymers. The building units of proteins are amino acids, the building units of polysaccharides are monosaccharides, and the building units of nucleic acids are nucleotides.

      According to their origin, the substances included in the living organisms are divided into inorganic substances (water, carbon dioxide, mineral substances) and organic substances (the most important are nucleic acids, proteins, saccharides, lipids).

  

Fig.: Average representation of the main groups of substances in organisms

3.1.  Water and mineral substances

The most frequent and the simplest biomolecule in living systems is water. Water  is basic and the most spread inorganic compound contained in living organisms. The average content of water in organisms is 60-70 %. The amount of water depends on the surroundings  in which the organism lives, on a kind of organism, on its age. The amount of water also depends on specific parts of body, e.g. the highest percentage of water in human body is in body fluids and the lowest in fatty, dental or bone tissue. Water in organisms helps to create their inner environment and keep their stability. Water is a dissolving agent, transporting medium and a thermoregulator. Bochemical reactions in living systems happen in water environment.

      

Inorganic salts can be either water-soluble, i.e. dissociated into ions, or insoluble. Insoluble salts are present in hard connective tissue such as teeth, bones or shells. Examples: Ca₃(PO₄)₂ (bones, teeth), CaCO₃ (bones, shells of invertebrates), CaF₂ (teeth). Soluble salts in the form of ions are mainly in body fluids. The main extracellular ions are cation Na⁺ and anion Cl^-.  The main intracellular ions are cation K⁺ and cation  Mg²⁺ .

     Very important compound is carbon dioxide, which is necessary for photosynthesis. It is produced in metabolic (catabolic) processes, e.g. when breathing.

 

3.2.  Organic matters                  

 

The most important organic matters necessary for organism structure and function are:

ü  proteins

ü  nucleic acids

ü  saccharides

ü  lipids

       Organic matters represent more than 30% of organism mass. The rest is represented by water and mineral matters.

Key Characteristics of Biosensors — Explained Simply

Biosensors are analytical devices that combine a biological component with a physicochemical detector to identify and measure specific substances. For a biosensor to be effective in medical diagnosis, environmental monitoring, or food safety, it must possess certain important characteristics.

1. Sensitivity

Sensitivity refers to how strongly a biosensor responds to a small change in the concentration of the target analyte.

A highly sensitive biosensor can detect even very low amounts of a substance, making it extremely useful in early disease detection and trace analysis.

2. Selectivity

Selectivity is the ability of a biosensor to recognize and respond only to the target analyte, even in the presence of other similar or interfering substances.

An ideal biosensor shows minimal or no response to unwanted chemicals, ensuring accurate and reliable results.

3. Range

The range refers to the span of analyte concentrations over which the biosensor provides a reliable and proportional response.

A good biosensor should work efficiently across a broad concentration range without losing accuracy.

4. Response Time

Response time is the duration a biosensor takes to produce a measurable output after exposure to the analyte.

A faster response time is always preferred, especially in real-time monitoring and medical diagnostics.

5. Reproducibility

Reproducibility means the biosensor should give consistent results when the same measurement is repeated under identical conditions.

This ensures reliability and trustworthiness of the sensor’s performance.

6. Detection Limit

The detection limit is the minimum concentration of an analyte that the biosensor can detect with a measurable signal.

Lower detection limits indicate higher performance and sensitivity of the biosensor.

7. Lifetime

Lifetime refers to how long a biosensor can function effectively without significant loss of performance.

A longer lifespan makes the biosensor more practical, economical, and suitable for commercial applications.

8. Stability

Stability describes how well a biosensor maintains its baseline signal and sensitivity over time.

A stable biosensor shows minimal drift or variation in its readings during storage or repeated use.

Conclusion

The performance of a biosensor depends largely on these key characteristics. An ideal biosensor should be sensitive, selective, fast, stable, reproducible, and long-lasting. These properties make biosensors invaluable tools in biotechnology, healthcare, environmental science, and industrial monitoring.

 

The DNA Blueprint: Understanding Molecular Markers in Plant Breeding

In the world of modern agriculture, identifying the best traits in plants isn't just about looking at them—it’s about reading their genetic code. This is where Molecular Markers come into play, serving as the ultimate "GPS" for a plant's genome.

What is a Molecular Marker?

A molecular marker is a specific DNA sequence within a genome that can be easily located and identified. Think of it as a unique "landmark" on a long highway of DNA.

Because of genetic changes like mutations, insertions, or deletions, the DNA sequence at a specific spot can vary between individual plants. These variations are known as polymorphisms. By mapping these polymorphisms, scientists can identify which plants carry specific traits, such as drought tolerance or high yield.

Why are Molecular Markers a Game-Changer?

While plant breeders would love to look directly at the gene responsible for a trait, it’s not always possible. Instead, they use markers that are "linked" to those genes. These markers offer several massive advantages:

• Genetic Truth: They provide a 100% accurate representation of the genetic makeup at the DNA level.
• Weather-Proof: Unlike physical traits (like height or color), markers are consistent and never affected by environmental factors or soil quality.
• Early Detection: You don't have to wait for a plant to grow or fruit. Markers can be detected at the seedling stage.
• Infinite Variety: We can generate a nearly unlimited number of markers to suit different breeding needs.

The Core Principle: How Detection Works

Imagine you have two plants of the same species: one is disease-resistant and the other is disease-sensitive. How do you tell them apart before a disease strikes?

1.     Extraction: DNA is extracted from both plants.

2.     Digestion: The DNA is cut into pieces using restriction enzymes.

3.     Separation: These pieces are separated by size using a process called gel electrophoresis.

4.     Identification: If the marker is effective, you might see a clear difference—for example, the disease-resistant plant might show a shorter DNA fragment, while the sensitive one shows a longer one.

Types of Molecular Markers

Molecular markers are generally categorized into two main "technological families" based on how they are detected:

1. Hybridization-Based (Non-PCR)

These are older, classic methods where DNA is detected using a labeled "probe" that sticks (hybridizes) to a specific sequence.

• Example: RFLP (Restriction Fragment Length Polymorphism).

2. PCR-Based Approaches

These modern methods use Polymerase Chain Reaction (PCR) to amplify specific segments of DNA millions of times, making them much faster and easier to analyze.

• Examples: RAPD, AFLP, and SSR (Simple Sequence Repeats).

Summary Table: Traditional vs. Molecular Selection

Feature	Traditional Selection	Molecular Marker Selection
Accuracy	Subjective / Environmental	Highly Accurate / Genetic
Time	Years (Wait for maturity)	Days (Seedling stage)
Reliability	Variable	Highly Reliable

Human Genome Project: Understanding Chromosome Maps in an Easy Way

The Human Genome Project (HGP) was one of the most ambitious scientific endeavors in modern biology. One of its primary objectives was to construct detailed maps of each human chromosome. These maps helped scientists understand the structure, organization, and function of genes in the human genome.

To achieve this goal, four major types of chromosome maps were developed. Let us explore them in a simple and engaging way.

1. Cytogenetic Map

A cytogenetic map is a visual map of a chromosome based on its staining pattern.

When chromosomes are treated with specific chemical dyes, they form light and dark bands. These bands represent different regions of the chromosome and help scientists locate genes and structural landmarks.

This map provides a broad overview of chromosome structure and is especially useful in identifying large chromosomal abnormalities such as deletions, duplications, or translocations.

2. Gene Linkage Map

A gene linkage map shows the relative positions of genes on a chromosome based on how frequently they are inherited together.

Genes that are close to each other tend to be passed on together, while those far apart are more likely to separate during recombination.

To construct this map, scientists used special DNA markers such as:

• RFLP – Restriction Fragment Length Polymorphism
• VNTRs – Variable Number Tandem Repeats (also called minisatellites)
• STRs – Short Tandem Repeats (also called microsatellites)

These markers act like signposts along the chromosome and help track gene locations.

3. Restriction Fragment Map

A restriction fragment map is created by cutting DNA into smaller pieces using restriction enzymes.

These fragments are then analyzed and sequenced to understand how different DNA segments are arranged along the chromosome. This map serves as an important intermediate step toward building a high-resolution physical map.

4. Physical Map (Most Detailed Map)

The physical map is the most advanced and precise chromosome map produced by the Human Genome Project.

It provides:

• Exact location of genes
• Actual DNA base sequence (A, T, G, C)
• Number of nucleotide bases between genes

This map forms the foundation of modern genomics, genetic medicine, and biotechnology. The techniques used to generate this map are based on various DNA sequencing methods, which are discussed in detail in advanced genomics studies.

Conclusion

The mapping of human chromosomes through cytogenetic, linkage, restriction, and physical maps was a landmark achievement of the Human Genome Project. These maps have revolutionized our understanding of genetics, heredity, disease, and evolution.