Introduction

Nucleic acids are complex biomolecules that store, transmit, and express genetic information in all living organisms. They are essential for heredity, protein synthesis, cell division, and the regulation of cellular activities. The two major types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA acts as the genetic material in most organisms, while RNA plays a crucial role in gene expression and protein synthesis.

What are nucleic acids?

Nucleic acids are polymers of nucleotides. Each nucleotide consists of three components:

• A pentose sugar

• A nitrogenous base

• A phosphate group

Nucleotides are linked together by phosphodiester bonds to form long polynucleotide chains.

Components of a nucleotide

Pentose sugar

There are two types of sugars found in nucleic acids.

The absence of one oxygen atom at the 2′ carbon of deoxyribose makes DNA more chemically stable than RNA.

Nitrogenous bases

Nitrogenous bases are of two types.

Purines

• Adenine (A)

• Guanine (G)

Pyrimidines

• Cytosine (C)

• Thymine (T) – present only in DNA

• Uracil (U) – present only in RNA

Phosphate group

The phosphate group links adjacent nucleotides through 3′–5′ phosphodiester bonds, forming the sugar-phosphate backbone.

Nucleoside and nucleotide

A nucleoside consists of a sugar and a nitrogenous base, while a nucleotide consists of a nucleoside plus one or more phosphate groups.

Examples:

• Adenosine = adenine + ribose

• Adenosine monophosphate (AMP) = adenosine + phosphate

Structure of DNA

The DNA structure was proposed by James Watson and Francis Crick (1953) based on X-ray diffraction studies by Rosalind Franklin and Maurice Wilkins.

Key features of the DNA double helix

• DNA consists of two antiparallel polynucleotide strands

• The strands are twisted into a right-handed double helix

• Sugar-phosphate backbones are located on the outside

• Nitrogenous bases face inward

Base pairing

Complementary base pairing occurs through hydrogen bonds.

Guanine-cytosine pairs are more stable due to the presence of three hydrogen bonds.

Dimensions of B-DNA

• Diameter: 2 nm

• Distance between adjacent base pairs: 0.34 nm

• One complete turn: 3.4 nm

• Number of base pairs per turn: 10

Chargaff’s rules

Erwin Chargaff discovered that:

• A = T

• G = C

• Total purines = total pyrimidines

These observations provided crucial evidence for complementary base pairing.

Types of DNA

DNA may exist in different conformations.

B-DNA is the most common form found in cells.

Structure of RNA

RNA is generally single-stranded, although it may fold into complex secondary and tertiary structures.

RNA contains:

• Ribose sugar

• Adenine, guanine, cytosine, and uracil

Because of the presence of the 2′-OH group, RNA is less stable than DNA.

Types of RNA

Messenger RNA (mRNA)

• Carries genetic information from DNA to ribosomes

• Serves as the template for protein synthesis

Transfer RNA (tRNA)

• Transfers amino acids to ribosomes

• Contains an anticodon that recognizes mRNA codons

Ribosomal RNA (rRNA)

• Forms the structural and catalytic core of ribosomes

• Catalyzes peptide bond formation

Other RNAs

• Small nuclear RNA (snRNA)

• Small nucleolar RNA (snoRNA)

• MicroRNA (miRNA)

• Small interfering RNA (siRNA)

• Long non-coding RNA (lncRNA)

DNA vs RNA

Biological functions of nucleic acids

Storage of genetic information

DNA stores hereditary information that is transmitted from parents to offspring.

DNA replication

DNA duplicates itself before cell division, ensuring genetic continuity.

Protein synthesis

RNA molecules participate in transcription and translation to produce proteins.

Regulation of gene expression

Non-coding RNAs regulate gene activity, development, and cellular differentiation.

Evolution

Mutations in nucleic acids generate genetic variation, which drives evolution.

Central dogma of molecular biology

The flow of genetic information follows the pathway:

DNA → RNA → Protein

This process involves:

• Replication

• Transcription

• Translation

DNA replication

DNA replication is semi-conservative, meaning each daughter DNA molecule contains one parental strand and one newly synthesized strand.

Major enzymes involved include:

• DNA helicase

• DNA polymerase

• Primase

• DNA ligase

• Topoisomerase

Replication proceeds in the 5′ → 3′ direction.

Transcription

Transcription is the synthesis of RNA from a DNA template.

Steps:

1. Initiation

2. Elongation

3. Termination

In eukaryotes, mRNA undergoes:

• 5′ capping

• Splicing

• 3′ polyadenylation

Translation

Translation occurs on ribosomes.

Major stages:

1. Initiation

2. Elongation

3. Termination

During translation, codons on mRNA specify the sequence of amino acids in proteins.

Importance in biotechnology

Nucleic acids have numerous applications.

Polymerase chain reaction (PCR)

Amplifies DNA fragments.

DNA fingerprinting

Used in forensic science and paternity testing.

Recombinant DNA technology

Produces genetically modified organisms and therapeutic proteins.

Gene therapy

Treats genetic diseases by introducing functional genes.

CRISPR-Cas genome editing

Allows precise modification of DNA sequences.

Nucleic acid metabolism

Purine metabolism

Purines are synthesized and degraded to uric acid.

Pyrimidine metabolism

Pyrimidines are degraded to beta-alanine and beta-aminoisobutyrate.

Disorders of nucleotide metabolism include:

• Gout

• Lesch-Nyhan syndrome

• Severe combined immunodeficiency (SCID)

Key points

• DNA contains deoxyribose; RNA contains ribose.

• Thymine is present in DNA; uracil is present in RNA.

• DNA strands are antiparallel.

• A pairs with T; G pairs with C.

• DNA replication is semi-conservative.

• mRNA carries genetic information.

• tRNA transports amino acids.

• rRNA forms ribosomes.

• The central dogma is DNA → RNA → Protein.

Conclusion

Nucleic acids are the molecular basis of life. DNA stores hereditary information, while RNA mediates the expression of that information through protein synthesis. Their structure, replication, transcription, and translation are central to genetics, molecular biology, biotechnology, and medicine.

References

1. Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 8th ed. W.H. Freeman; 2021.

2. Alberts B, et al. Molecular Biology of the Cell. 7th ed. Garland Science; 2022.

3. Watson JD, et al. Molecular Biology of the Gene. 7th ed. Pearson; 2014.

4. Lodish H, et al. Molecular Cell Biology. 9th ed. W.H. Freeman; 2021.

5. NCERT. Biology Class XI. National Council of Educational Research and Training; Latest Edition.

6. Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature. 1953;171:737–738.

1. Isolation & Extraction of Carbohydrates

Carbohydrates are classified by chain length, polarity, and solubility: low molecular weight mono- and oligosaccharides are water-soluble and polar, whereas structural polysaccharides (e.g., cellulose, chitin, starch, glycogen, pectin) require specific chemical, enzymatic, or thermal treatments for extraction.

Extraction Strategies by Carbohydrate Class

1. Free Mono- and Oligosaccharides (Glucose, Sucrose, Fructose)

• Extraction Solvent: Hot 80% aqueous ethanol (v/v) under reflux.

• Mechanism: 80% ethanol selectively dissolves low molecular weight sugars while precipitating proteins, nucleic acids, and high molecular weight polysaccharides (starch, pectins).

• Clarification / Deproteinization: Lead acetate or Carrez reagents (Carrez I: potassium ferrocyanide; Carrez II: zinc sulfate) are added to precipitate residual proteins and phenolic compounds before analysis.

2. Storage Polysaccharides (Starch and Glycogen)

• Starch Isolation:

  1. Mechanical cell disruption in cold water or mild alkali (0.05 M NaOH) to solubilize proteins.

  2. Filtration through fine mesh to separate insoluble cellulosic fiber.

  3. Sedimentation/centrifugation of starch granules.

  4. Gelatinization via heating ($>65^\circ\text{C}$) followed by enzymatic digestion ($\alpha$-amylase and amyloglucosidase) for monomer analysis.

• Glycogen Isolation:

  1. Homogenization of liver/muscle tissue in cold 5–10% trichloroacetic acid (TCA) to precipitate proteins and nucleic acids.

  2. Centrifugation to collect the supernatant containing glycogen.

  3. Precipitation of glycogen from the supernatant by adding 2 volumes of 95% ethanol in the presence of sodium chloride.

3. Structural Polysaccharides (Cellulose, Chitin, Pectin)

• Pectin Isolation: Hot aqueous extraction under acidic conditions (pH 1.5–3.0 using $\text{HCl}$ or citric acid) at $80\text{--}90^\circ\text{C}$, followed by precipitation with ethanol or isopropanol.

• Cellulose Isolation: Removal of lignin and hemicellulose via strong alkali treatment (17.5% NaOH) followed by bleaching with sodium chlorite ($\text{NaClO}_2$) and acetic acid.

• Chitin Isolation:

  1. Demineralization: Dilute $\text{HCl}$ (1–2 M) to remove calcium carbonate from crustacean shells.

  2. Deproteinization: Dilute $\text{NaOH}$ (1–2 M) at $80\text{--}100^\circ\text{C}$.

  3. Deacetylation (to form Chitosan): Concentrated $\text{NaOH}$ (40–50%) at $100\text{--}120^\circ\text{C}$.

Fractionation & Purification Techniques

• Alcohol / Solvent Precipitation: High molecular weight glycans precipitate out of aqueous solution upon addition of organic solvents (ethanol, isopropanol, or acetone) to a final concentration of 70–80%.

• Ion-Exchange Chromatography (IEC): Separates charged polysaccharides from neutral sugars.

  • DEAE-Cellulose / DEAE-Sepharose (Anion Exchange): Retains acidic polysaccharides (e.g., pectins, alginates, glycosaminoglycans, hyaluronic acid) due to negative carboxylate or sulfate groups. Eluted using a $0\text{--}1.0\text{ M NaCl}$ gradient.

• Size-Exclusion Chromatography (SEC / Gel Filtration): Separates polysaccharides based on hydrodynamic volume and molecular weight using matrices such as Sephadex (G-50 to G-200), Sepharose, or Bio-Gel.

• Lectin Affinity Chromatography: Uses immobilized lectins (e.g., Concanavalin A, Wheat Germ Agglutinin) to selectively bind specific carbohydrate motifs:

  • Concanavalin A (Con A): Binds $\alpha$-D-mannosyl and $\alpha$-D-glucosyl residues.

  • Wheat Germ Agglutinin (WGA): Binds $N$-acetylglucosamine ($\text{GlcNAc}$) and sialic acid residues.

  • Elution: Displaced using a competing monomeric sugar (e.g., $0.1\text{--}0.5\text{ M}$ methyl $\alpha$-D-glucopyranoside for Con A).

Quantitative & Analytical Methods for Carbohydrates

GC-MS Derivatization of Sugars

Carbohydrates are non-volatile and thermally unstable, preventing direct Gas Chromatography (GC) analysis. They require two major derivatization pathways:

1. Trimethylsilylation (TMS): Hydroxyl groups are reacted with trimethylsilyl reagents (e.g., BSTFA / TMCS) to yield volatile TMS-ether derivatives.

2. Alditol Acetate Conversion:

   • Acid hydrolysis of polysaccharides to monosaccharides.

   • Reduction of monosaccharides to alditols using sodium borohydride ($\text{NaBH}_4$).

   • Acetylation of alditols using acetic anhydride and pyridine to form volatile alditol acetates.

2. Isolation & Extraction of Lipids

Lipids are hydrophobic or amphipathic biomolecules. Extraction requires organic solvents that disrupt hydrophobic and electrostatic interactions between lipids and membrane proteins while maintaining lipid structural integrity.

Major Total Lipid Extraction Protocols

1. Folch Method (1957)

• Solvent Mixture: Chloroform : Methanol ($2:1\text{ v/v}$).

• Mechanism:

  1. Methanol penetrates the cellular matrix and disrupts hydrogen bonds/electrostatic interactions between membrane lipids and proteins.

  2. Chloroform dissolves neutral and polar lipids into a monophasic extraction mixture.

• Biphasic Separation: Water or dilute salt solution ($0.9\%\text{ NaCl}$ or $0.73\%\text{ NaCl}$) is added to achieve a final volumetric ratio of Chloroform : Methanol : Water ($8:4:3\text{ v/v/v}$).

• Phase Layering:

  • Upper Phase (Aqueous / Polar): Water, methanol, salts, non-lipid impurities (sugars, amino acids), and gangliosides.

  • Interphase: Denatured proteins.

  • Lower Phase (Organic / Non-polar): Chloroform layer containing total lipids (phospholipids, glycolipids, triglycerides, cholesterol).

2. Bligh and Dyer Method (1959)

• Optimized Use Case: High-water content biological samples (e.g., tissues, cell suspensions, fish muscle). Uses lower solvent-to-tissue ratios than the Folch method.

• Extraction Stage (Monophasic): Adjusts solvent proportions based on tissue water content to yield a single phase of Chloroform : Methanol : Sample Water ($1:2:0.8\text{ v/v/v}$).

• Separation Stage (Biphasic): Additional chloroform and water are added to reach a final ratio of Chloroform : Methanol : Water ($2:2:1.8\text{ v/v/v}$).

• Phase Layering: Similar to Folch; total lipids partition into the lower chloroform phase.

3. Matyash / MTBE Method (2008)

• Solvent Mixture: Methyl tert-butyl ether (MTBE) : Methanol : Water.

• Critical Advantage: MTBE has a lower density than water. Consequently, the lipid-rich organic phase forms the UPPER layer, while proteins collect in the pellet at the bottom and the aqueous phase forms the lower layer. This prevents contamination when pipetting the lipid phase through a protein interphase.

Fractionation & Chromatographic Separation of Lipids

1. Solid-Phase Extraction (SPE) / Silica Gel Column Chromatography

Lipid classes are separated based on polarity using a silica gel stationary phase by sequential elution with solvents of increasing polarity:

1. Fraction 1 (Neutral Lipids): Eluted with Chloroform or Hexane : Diethyl Ether.

   • Yields: Triacylglycerols (TAGs), free fatty acids, cholesterol, cholesterol esters, wax esters.

2. Fraction 2 (Glycolipids & Sphingolipids): Eluted with Acetone or Acetone : Methanol ($9:1\text{ v/v}$).

   • Yields: Cerebrosides, sulfatides, monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols (DGDG).

3. Fraction 3 (Polar Phospholipids): Eluted with Methanol.

   • Yields: Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Sphingomyelin.

2. Thin Layer Chromatography (TLC) of Lipids

• Stationary Phase: Silica Gel G plates (polar).

• Solvent Systems:

  • For Neutral Lipids: Petroleum Ether : Diethyl Ether : Acetic Acid ($80:20:1\text{ v/v/v}$).

    • Migration Order ($R_f$ value low to high): Monoglycerides < Diglycerides < Free Fatty Acids < Triacylglycerols < Cholesterol Esters.

  • For Polar Phospholipids: Chloroform : Methanol : Water ($65:25:4\text{ v/v/v}$).

• Visualization & Detection Reagents:

  • Iodine Vapor: Universal reversible stain for unsaturated lipids (binds carbon-carbon double bonds).

  • Ninhydrin Spray: Specific for lipids containing free primary amino groups (Phosphatidylethanolamine and Phosphatidylserine $\rightarrow$ purple/pink spots).

  • Dragendorff Reagent: Specific for choline-containing lipids (Phosphatidylcholine, Sphingomyelin $\rightarrow$ orange spots).

  • Dittmer-Lester (Molybdenum Blue) Reagent: Specific for phosphate esters (all phospholipids $\rightarrow$ blue spots).

  • Orcinol-Sulfuric Acid Reagent: Specific for glycolipids (cerebrosides, gangliosides $\rightarrow$ purple/violet spots).

Fatty Acid Methyl Esterification (FAME Analysis)

Fatty acids in complex lipids (TAGs, phospholipids) are non-volatile and must be converted into Fatty Acid Methyl Esters (FAMEs) prior to Gas Chromatography (GC-FID or GC-MS) analysis.

Complex Lipids (TAGs / Phospholipids)
       │
       ├─► Alkaline Transesterification: Methanolic NaOH or KOH (mild, preserves double bonds)
       │
       └─► Acidic Transesterification: Methanolic HCl, BF3, or H2SO4 (cleaves amide bonds in sphingolipids)
       │
       ▼
Fatty Acid Methyl Esters (FAMEs) ──► Volatilized & Separated via GC Column

• Alkaline Transesterification: Treatment with methanolic $\text{NaOH}$ or $\text{KOH}$ at $50\text{--}60^\circ\text{C}$. Cleaves ester bonds in glycerolipids without altering double bond positions or configurations (prevents isomerization).

• Acidic Transesterification: Treatment with methanolic $\text{BF}_3$ (boron trifluoride) or methanolic $\text{HCl}$. Required to hydrolyze amide bonds found in sphingolipids or esterify free fatty acids.

3. High-Yield Comparison Table: Carbohydrate vs. Lipid Extraction

 Definition

DNA fingerprinting (DNA profiling) is a molecular technique used to identify individuals based on unique patterns in their DNA. It analyzes highly variable regions called VNTRs (Variable Number Tandem Repeats) or STRs (Short Tandem Repeats). Developed by Sir Alec Jeffreys in 1984.

Principle

·        More than 99.9% of human DNA is identical, but repetitive DNA regions vary among individuals.

·        The number of repeat units differs from person to person.

·        These variations produce a unique DNA banding pattern for each individual (except identical twins).

Types of DNA Sequences Used

Modern forensic analysis mainly uses STR markers.

Steps of DNA Fingerprinting

1. Sample collection

DNA can be obtained from blood, hair root, saliva, semen, bone, teeth, and skin cells.

2. DNA isolation

DNA is extracted from the collected biological sample.

3. DNA amplification (PCR)

Specific STR/VNTR regions are amplified using the Polymerase Chain Reaction (PCR).

4. Restriction digestion (classical method)

Restriction enzymes cut DNA into fragments at specific sequences.

5. Gel electrophoresis

DNA fragments are separated according to size through an agarose gel.

6. Southern blotting

DNA fragments are transferred from the gel onto a nylon or nitrocellulose membrane.

7. Hybridization with labeled probes

Radioactive or fluorescent probes bind to specific repetitive DNA sequences.

8. Detection

Band patterns are visualized using autoradiography or fluorescence detection. The resulting pattern is the DNA fingerprint.

Workflow

Biological sample → DNA extraction → PCR amplification of STR/VNTR loci → Gel electrophoresis → Southern blotting → Probe hybridization → DNA fingerprint pattern

Applications

Forensic science: Identification of criminals, matching biological samples, and crime scene investigation.

Paternity and maternity testing: Establishing biological relationships and resolving inheritance disputes.

Identification of missing persons: Disaster victim identification and unknown body identification.

Medical genetics: Detection of inherited disorders and linkage analysis.

Wildlife conservation: Identification of endangered species and prevention of illegal wildlife trade.

Evolutionary and population studies: Genetic diversity analysis and human migration studies.

Advantages

·        Highly accurate

·        Requires a very small DNA sample

·        Useful even with degraded DNA

·        Fast analysis using PCR

·        High discriminatory power

Limitations

·        Identical twins have the same DNA fingerprint.

·        Contaminated samples may give misleading results.

·        Improper handling can affect accuracy.

·        Interpretation requires statistical analysis.

DNA Fingerprinting in India

The technique was developed in India by Dr. Lalji Singh, known as the Father of DNA Fingerprinting in India. He introduced DNA fingerprinting technology for forensic and paternity investigations in the country.

Important NEET Points

·        Inventor: Alec Jeffreys (1984)

·        Indian pioneer: Dr. Lalji Singh

·        Main markers used today: STRs

·        Technique involved: PCR + Gel electrophoresis

·        Classical method: Southern blotting

·        Used in: Forensics, paternity testing, and identification

Academic References

1.      Jeffreys, A. J., Wilson, V., & Thein, S. L. (1985). Hypervariable "minisatellite" regions in human DNA. Nature, 314, 67–73.

2.      Alberts, B., et al. (2022). Molecular Biology of the Cell (7th ed.). Garland Science.

3.      NCERT. (2025). Biology, Class XII. Chapter: Biotechnology and its Applications.

4.      Butler, J. M. (2015). Advanced Topics in Forensic DNA Typing: Interpretation. Academic Press.

5.      Jobling, M. A., & Gill, P. (2004). Encoded evidence: DNA in forensic analysis. Nature Reviews Genetics, 5, 739–751.

The five kingdom system was developed by Robert H. Whittaker in 1969 and was built on the work of previous biologists such as Carolus Linnaeus.

Living things can be classified into five major kingdoms. 

1. Kingdom prokaryote : Prokaryote is a kingdom or division in the classification organization developed for all life on Earth. This kingdom which is also elected as Monera contains all bacteria and blue-green algae (also known as Cyanobacteria). 

2. Kingdom Protista or Protoctista :   It is a kingdom of simple eukaryotic organisms usually made from a single cell or a colony of similar cells. Protists live in water, in moist terrestrial habitats, as parasites and other symbiotic in the bodies of multicellular eukaryotes. 

 3. Kingdom Fungi : A fungus is any member of the group of eukaryotic organisms that comprises microorganisms such as yeasts and molds as well as the more familiar mushrooms. These organisms are classified as a kingdom which is separate from the other eukaryotic life kingdoms of plants and animals. 

 4. Kingdom Plantae : This kingdom contains all land plants such as mosses, ferns, conifers and flowering plants etc. an amazing range of diverse forms. With more than 250,000 species they are second in size only to the arthropods. Plants have been around for a very long time. 

5. Kingdom Animalia : All animals are members of the Kingdom Animalia, also called Metazoa. This Kingdom does not contain prokaryotes (Kingdom Monera, includes bacteria, blue-green algae) or protists (Kingdom Protista, includes unicellular eukaryotic organisms).

Some branches of biology are below: 

1. Botany: The study of Plants and their features. 

 2. Zoology: The study of animals and their futures. 

 3. Microbiology: The study of Microorganisms and their features. 

 4. Taxonomy: It is the science of identification, nomenclature and classification of organisms. 

 5. Morphology: It is the study of external form, size, shape, color, structure and relative position of several living organ of living organisms. 

 6. Anatomy: It is the study of internal structure. 

 7. Histology: It is the study of tissue organization and structure. 

 8. Cytology: It is the study of form and structure of cells containing the behavior of nucleus and other organelles. 

 9. Cell Biology: It is the study of morphological, organizational, biochemical, physiological, genetic, developmental, pathological and evolutionary features of cell and its components. 

 10. Molecular Biology: It is the study of the nature of physicochemical organization, synthesis working and interaction of bio-molecules. 

11. Physiology: It is the study of different types of body functions and procedures. 

 12. Embryology: It is the study of fertilization, growth, division and distinction of the zygote into embryo. 

13. Ecology: It is the study of living organisms is relative to other organism and their environment. 

 14. Genetics: It is the study of inheritance of characters or heredity and variations. 

15. Evolution: It is the study of the origin of life as well as new types of organism from the previous ones by modifications involving genetic changes and adaptations. 

 16. Paleontology: It deals with the study of fossils or remains and impressions of past organisms. 

 17. Virology: It is the study of viruses and all their aspects.

 Introduction

Phylum Platyhelminthes (flatworms) characteristics :

Class 1- Turbellaria (L., turbella= a little string) 

• Mostly free-living but some ectocommensals and endocommensals or parasitic called.
• Terrestrial marine or freshwater.
• Body unsegmented and covered with ciliated cellular or syncytial epidermis, containing mucus-secreting cells and rod-shaped body called.
• Mouth ventral. intestine preceded by the muscular pharynx.
• Adhesive organs(suckers) abundantly present.
• Sense organ i.e. Tango, chemo, and photoreceptors common in free living forms.
• The excretory system consists of protonephridia, the flame cells.
• Mostly reproduction sexual, asexual and by regeneration.
• Life cycle simple. 

Class 2- Trematoda (Gr., trematodes= having pore) 

 Introduction

Nucleic acids are complex biomolecules that store, transmit, and express genetic information in all living organisms. They are essential for heredity, protein synthesis, cell division, and the regulation of cellular activities. The two major types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA acts as the genetic material in most organisms, while RNA plays a crucial role in gene expression and protein synthesis.

What are nucleic acids?

Nucleic acids are polymers of nucleotides. Each nucleotide consists of three components:

• A pentose sugar

• A nitrogenous base

• A phosphate group

Nucleotides are linked together by phosphodiester bonds to form long polynucleotide chains.

Components of a nucleotide

Pentose sugar

There are two types of sugars found in nucleic acids.

The absence of one oxygen atom at the 2′ carbon of deoxyribose makes DNA more chemically stable than RNA.

Nitrogenous bases

Nitrogenous bases are of two types.

Purines

• Adenine (A)

• Guanine (G)

Pyrimidines

• Cytosine (C)

• Thymine (T) – present only in DNA

• Uracil (U) – present only in RNA

Phosphate group

The phosphate group links adjacent nucleotides through 3′–5′ phosphodiester bonds, forming the sugar-phosphate backbone.

Nucleoside and nucleotide

A nucleoside consists of a sugar and a nitrogenous base, while a nucleotide consists of a nucleoside plus one or more phosphate groups.

Examples:

• Adenosine = adenine + ribose

• Adenosine monophosphate (AMP) = adenosine + phosphate

Structure of DNA

The DNA structure was proposed by James Watson and Francis Crick (1953) based on X-ray diffraction studies by Rosalind Franklin and Maurice Wilkins.

Key features of the DNA double helix

• DNA consists of two antiparallel polynucleotide strands

• The strands are twisted into a right-handed double helix

• Sugar-phosphate backbones are located on the outside

• Nitrogenous bases face inward

Base pairing

Complementary base pairing occurs through hydrogen bonds.

Guanine-cytosine pairs are more stable due to the presence of three hydrogen bonds.

Dimensions of B-DNA

• Diameter: 2 nm

• Distance between adjacent base pairs: 0.34 nm

• One complete turn: 3.4 nm

• Number of base pairs per turn: 10

Chargaff’s rules

Erwin Chargaff discovered that:

• A = T

• G = C

• Total purines = total pyrimidines

These observations provided crucial evidence for complementary base pairing.

Types of DNA

DNA may exist in different conformations.

B-DNA is the most common form found in cells.

Structure of RNA

RNA is generally single-stranded, although it may fold into complex secondary and tertiary structures.

RNA contains:

• Ribose sugar

• Adenine, guanine, cytosine, and uracil

Because of the presence of the 2′-OH group, RNA is less stable than DNA.

Types of RNA

Messenger RNA (mRNA)

• Carries genetic information from DNA to ribosomes

• Serves as the template for protein synthesis

Transfer RNA (tRNA)

• Transfers amino acids to ribosomes

• Contains an anticodon that recognizes mRNA codons

Ribosomal RNA (rRNA)

• Forms the structural and catalytic core of ribosomes

• Catalyzes peptide bond formation

Other RNAs

• Small nuclear RNA (snRNA)

• Small nucleolar RNA (snoRNA)

• MicroRNA (miRNA)

• Small interfering RNA (siRNA)

• Long non-coding RNA (lncRNA)

DNA vs RNA

Biological functions of nucleic acids

Storage of genetic information

DNA stores hereditary information that is transmitted from parents to offspring.

DNA replication

DNA duplicates itself before cell division, ensuring genetic continuity.

Protein synthesis

RNA molecules participate in transcription and translation to produce proteins.

Regulation of gene expression

Non-coding RNAs regulate gene activity, development, and cellular differentiation.

Evolution

Mutations in nucleic acids generate genetic variation, which drives evolution.

Central dogma of molecular biology

The flow of genetic information follows the pathway:

DNA → RNA → Protein

This process involves:

• Replication

• Transcription

• Translation

DNA replication

DNA replication is semi-conservative, meaning each daughter DNA molecule contains one parental strand and one newly synthesized strand.

Major enzymes involved include:

• DNA helicase

• DNA polymerase

• Primase

• DNA ligase

• Topoisomerase

Replication proceeds in the 5′ → 3′ direction.

Transcription

Transcription is the synthesis of RNA from a DNA template.

Steps:

1. Initiation

2. Elongation

3. Termination

In eukaryotes, mRNA undergoes:

• 5′ capping

• Splicing

• 3′ polyadenylation

Translation

Translation occurs on ribosomes.

Major stages:

1. Initiation

2. Elongation

3. Termination

During translation, codons on mRNA specify the sequence of amino acids in proteins.

Importance in biotechnology

Nucleic acids have numerous applications.

Polymerase chain reaction (PCR)

Amplifies DNA fragments.

DNA fingerprinting

Used in forensic science and paternity testing.

Recombinant DNA technology

Produces genetically modified organisms and therapeutic proteins.

Gene therapy

Treats genetic diseases by introducing functional genes.

CRISPR-Cas genome editing

Allows precise modification of DNA sequences.

Nucleic acid metabolism

Purine metabolism

Purines are synthesized and degraded to uric acid.

Pyrimidine metabolism

Pyrimidines are degraded to beta-alanine and beta-aminoisobutyrate.

Disorders of nucleotide metabolism include:

• Gout

• Lesch-Nyhan syndrome

• Severe combined immunodeficiency (SCID)

Key points

• DNA contains deoxyribose; RNA contains ribose.

• Thymine is present in DNA; uracil is present in RNA.

• DNA strands are antiparallel.

• A pairs with T; G pairs with C.

• DNA replication is semi-conservative.

• mRNA carries genetic information.

• tRNA transports amino acids.

• rRNA forms ribosomes.

• The central dogma is DNA → RNA → Protein.

Conclusion

Nucleic acids are the molecular basis of life. DNA stores hereditary information, while RNA mediates the expression of that information through protein synthesis. Their structure, replication, transcription, and translation are central to genetics, molecular biology, biotechnology, and medicine.

References

1. Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 8th ed. W.H. Freeman; 2021.

2. Alberts B, et al. Molecular Biology of the Cell. 7th ed. Garland Science; 2022.

3. Watson JD, et al. Molecular Biology of the Gene. 7th ed. Pearson; 2014.

4. Lodish H, et al. Molecular Cell Biology. 9th ed. W.H. Freeman; 2021.

5. NCERT. Biology Class XI. National Council of Educational Research and Training; Latest Edition.

6. Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature. 1953;171:737–738.

1. Isolation & Extraction of Carbohydrates

Carbohydrates are classified by chain length, polarity, and solubility: low molecular weight mono- and oligosaccharides are water-soluble and polar, whereas structural polysaccharides (e.g., cellulose, chitin, starch, glycogen, pectin) require specific chemical, enzymatic, or thermal treatments for extraction.

Extraction Strategies by Carbohydrate Class

1. Free Mono- and Oligosaccharides (Glucose, Sucrose, Fructose)

• Extraction Solvent: Hot 80% aqueous ethanol (v/v) under reflux.

• Mechanism: 80% ethanol selectively dissolves low molecular weight sugars while precipitating proteins, nucleic acids, and high molecular weight polysaccharides (starch, pectins).

• Clarification / Deproteinization: Lead acetate or Carrez reagents (Carrez I: potassium ferrocyanide; Carrez II: zinc sulfate) are added to precipitate residual proteins and phenolic compounds before analysis.

2. Storage Polysaccharides (Starch and Glycogen)

• Starch Isolation:

  1. Mechanical cell disruption in cold water or mild alkali (0.05 M NaOH) to solubilize proteins.

  2. Filtration through fine mesh to separate insoluble cellulosic fiber.

  3. Sedimentation/centrifugation of starch granules.

  4. Gelatinization via heating ($>65^\circ\text{C}$) followed by enzymatic digestion ($\alpha$-amylase and amyloglucosidase) for monomer analysis.

• Glycogen Isolation:

  1. Homogenization of liver/muscle tissue in cold 5–10% trichloroacetic acid (TCA) to precipitate proteins and nucleic acids.

  2. Centrifugation to collect the supernatant containing glycogen.

  3. Precipitation of glycogen from the supernatant by adding 2 volumes of 95% ethanol in the presence of sodium chloride.

3. Structural Polysaccharides (Cellulose, Chitin, Pectin)

• Pectin Isolation: Hot aqueous extraction under acidic conditions (pH 1.5–3.0 using $\text{HCl}$ or citric acid) at $80\text{--}90^\circ\text{C}$, followed by precipitation with ethanol or isopropanol.

• Cellulose Isolation: Removal of lignin and hemicellulose via strong alkali treatment (17.5% NaOH) followed by bleaching with sodium chlorite ($\text{NaClO}_2$) and acetic acid.

• Chitin Isolation:

  1. Demineralization: Dilute $\text{HCl}$ (1–2 M) to remove calcium carbonate from crustacean shells.

  2. Deproteinization: Dilute $\text{NaOH}$ (1–2 M) at $80\text{--}100^\circ\text{C}$.

  3. Deacetylation (to form Chitosan): Concentrated $\text{NaOH}$ (40–50%) at $100\text{--}120^\circ\text{C}$.

Fractionation & Purification Techniques

• Alcohol / Solvent Precipitation: High molecular weight glycans precipitate out of aqueous solution upon addition of organic solvents (ethanol, isopropanol, or acetone) to a final concentration of 70–80%.

• Ion-Exchange Chromatography (IEC): Separates charged polysaccharides from neutral sugars.

  • DEAE-Cellulose / DEAE-Sepharose (Anion Exchange): Retains acidic polysaccharides (e.g., pectins, alginates, glycosaminoglycans, hyaluronic acid) due to negative carboxylate or sulfate groups. Eluted using a $0\text{--}1.0\text{ M NaCl}$ gradient.

• Size-Exclusion Chromatography (SEC / Gel Filtration): Separates polysaccharides based on hydrodynamic volume and molecular weight using matrices such as Sephadex (G-50 to G-200), Sepharose, or Bio-Gel.

• Lectin Affinity Chromatography: Uses immobilized lectins (e.g., Concanavalin A, Wheat Germ Agglutinin) to selectively bind specific carbohydrate motifs:

  • Concanavalin A (Con A): Binds $\alpha$-D-mannosyl and $\alpha$-D-glucosyl residues.

  • Wheat Germ Agglutinin (WGA): Binds $N$-acetylglucosamine ($\text{GlcNAc}$) and sialic acid residues.

  • Elution: Displaced using a competing monomeric sugar (e.g., $0.1\text{--}0.5\text{ M}$ methyl $\alpha$-D-glucopyranoside for Con A).

Quantitative & Analytical Methods for Carbohydrates

GC-MS Derivatization of Sugars

Carbohydrates are non-volatile and thermally unstable, preventing direct Gas Chromatography (GC) analysis. They require two major derivatization pathways:

1. Trimethylsilylation (TMS): Hydroxyl groups are reacted with trimethylsilyl reagents (e.g., BSTFA / TMCS) to yield volatile TMS-ether derivatives.

2. Alditol Acetate Conversion:

   • Acid hydrolysis of polysaccharides to monosaccharides.

   • Reduction of monosaccharides to alditols using sodium borohydride ($\text{NaBH}_4$).

   • Acetylation of alditols using acetic anhydride and pyridine to form volatile alditol acetates.

2. Isolation & Extraction of Lipids

Lipids are hydrophobic or amphipathic biomolecules. Extraction requires organic solvents that disrupt hydrophobic and electrostatic interactions between lipids and membrane proteins while maintaining lipid structural integrity.

Major Total Lipid Extraction Protocols

1. Folch Method (1957)

• Solvent Mixture: Chloroform : Methanol ($2:1\text{ v/v}$).

• Mechanism:

  1. Methanol penetrates the cellular matrix and disrupts hydrogen bonds/electrostatic interactions between membrane lipids and proteins.

  2. Chloroform dissolves neutral and polar lipids into a monophasic extraction mixture.

• Biphasic Separation: Water or dilute salt solution ($0.9\%\text{ NaCl}$ or $0.73\%\text{ NaCl}$) is added to achieve a final volumetric ratio of Chloroform : Methanol : Water ($8:4:3\text{ v/v/v}$).

• Phase Layering:

  • Upper Phase (Aqueous / Polar): Water, methanol, salts, non-lipid impurities (sugars, amino acids), and gangliosides.

  • Interphase: Denatured proteins.

  • Lower Phase (Organic / Non-polar): Chloroform layer containing total lipids (phospholipids, glycolipids, triglycerides, cholesterol).

2. Bligh and Dyer Method (1959)

• Optimized Use Case: High-water content biological samples (e.g., tissues, cell suspensions, fish muscle). Uses lower solvent-to-tissue ratios than the Folch method.

• Extraction Stage (Monophasic): Adjusts solvent proportions based on tissue water content to yield a single phase of Chloroform : Methanol : Sample Water ($1:2:0.8\text{ v/v/v}$).

• Separation Stage (Biphasic): Additional chloroform and water are added to reach a final ratio of Chloroform : Methanol : Water ($2:2:1.8\text{ v/v/v}$).

• Phase Layering: Similar to Folch; total lipids partition into the lower chloroform phase.

3. Matyash / MTBE Method (2008)

• Solvent Mixture: Methyl tert-butyl ether (MTBE) : Methanol : Water.

• Critical Advantage: MTBE has a lower density than water. Consequently, the lipid-rich organic phase forms the UPPER layer, while proteins collect in the pellet at the bottom and the aqueous phase forms the lower layer. This prevents contamination when pipetting the lipid phase through a protein interphase.

Fractionation & Chromatographic Separation of Lipids

1. Solid-Phase Extraction (SPE) / Silica Gel Column Chromatography

Lipid classes are separated based on polarity using a silica gel stationary phase by sequential elution with solvents of increasing polarity:

1. Fraction 1 (Neutral Lipids): Eluted with Chloroform or Hexane : Diethyl Ether.

   • Yields: Triacylglycerols (TAGs), free fatty acids, cholesterol, cholesterol esters, wax esters.

2. Fraction 2 (Glycolipids & Sphingolipids): Eluted with Acetone or Acetone : Methanol ($9:1\text{ v/v}$).

   • Yields: Cerebrosides, sulfatides, monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols (DGDG).

3. Fraction 3 (Polar Phospholipids): Eluted with Methanol.

   • Yields: Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Sphingomyelin.

2. Thin Layer Chromatography (TLC) of Lipids

• Stationary Phase: Silica Gel G plates (polar).

• Solvent Systems:

  • For Neutral Lipids: Petroleum Ether : Diethyl Ether : Acetic Acid ($80:20:1\text{ v/v/v}$).

    • Migration Order ($R_f$ value low to high): Monoglycerides < Diglycerides < Free Fatty Acids < Triacylglycerols < Cholesterol Esters.

  • For Polar Phospholipids: Chloroform : Methanol : Water ($65:25:4\text{ v/v/v}$).

• Visualization & Detection Reagents:

  • Iodine Vapor: Universal reversible stain for unsaturated lipids (binds carbon-carbon double bonds).

  • Ninhydrin Spray: Specific for lipids containing free primary amino groups (Phosphatidylethanolamine and Phosphatidylserine $\rightarrow$ purple/pink spots).

  • Dragendorff Reagent: Specific for choline-containing lipids (Phosphatidylcholine, Sphingomyelin $\rightarrow$ orange spots).

  • Dittmer-Lester (Molybdenum Blue) Reagent: Specific for phosphate esters (all phospholipids $\rightarrow$ blue spots).

  • Orcinol-Sulfuric Acid Reagent: Specific for glycolipids (cerebrosides, gangliosides $\rightarrow$ purple/violet spots).

Fatty Acid Methyl Esterification (FAME Analysis)

Fatty acids in complex lipids (TAGs, phospholipids) are non-volatile and must be converted into Fatty Acid Methyl Esters (FAMEs) prior to Gas Chromatography (GC-FID or GC-MS) analysis.

Complex Lipids (TAGs / Phospholipids)
       │
       ├─► Alkaline Transesterification: Methanolic NaOH or KOH (mild, preserves double bonds)
       │
       └─► Acidic Transesterification: Methanolic HCl, BF3, or H2SO4 (cleaves amide bonds in sphingolipids)
       │
       ▼
Fatty Acid Methyl Esters (FAMEs) ──► Volatilized & Separated via GC Column

• Alkaline Transesterification: Treatment with methanolic $\text{NaOH}$ or $\text{KOH}$ at $50\text{--}60^\circ\text{C}$. Cleaves ester bonds in glycerolipids without altering double bond positions or configurations (prevents isomerization).

• Acidic Transesterification: Treatment with methanolic $\text{BF}_3$ (boron trifluoride) or methanolic $\text{HCl}$. Required to hydrolyze amide bonds found in sphingolipids or esterify free fatty acids.

3. High-Yield Comparison Table: Carbohydrate vs. Lipid Extraction

 Definition

DNA fingerprinting (DNA profiling) is a molecular technique used to identify individuals based on unique patterns in their DNA. It analyzes highly variable regions called VNTRs (Variable Number Tandem Repeats) or STRs (Short Tandem Repeats). Developed by Sir Alec Jeffreys in 1984.

Principle

·        More than 99.9% of human DNA is identical, but repetitive DNA regions vary among individuals.

·        The number of repeat units differs from person to person.

·        These variations produce a unique DNA banding pattern for each individual (except identical twins).

Types of DNA Sequences Used

Modern forensic analysis mainly uses STR markers.

Steps of DNA Fingerprinting

1. Sample collection

DNA can be obtained from blood, hair root, saliva, semen, bone, teeth, and skin cells.

2. DNA isolation

DNA is extracted from the collected biological sample.

3. DNA amplification (PCR)

Specific STR/VNTR regions are amplified using the Polymerase Chain Reaction (PCR).

4. Restriction digestion (classical method)

Restriction enzymes cut DNA into fragments at specific sequences.

5. Gel electrophoresis

DNA fragments are separated according to size through an agarose gel.

6. Southern blotting

DNA fragments are transferred from the gel onto a nylon or nitrocellulose membrane.

7. Hybridization with labeled probes

Radioactive or fluorescent probes bind to specific repetitive DNA sequences.

8. Detection

Band patterns are visualized using autoradiography or fluorescence detection. The resulting pattern is the DNA fingerprint.

Workflow

Biological sample → DNA extraction → PCR amplification of STR/VNTR loci → Gel electrophoresis → Southern blotting → Probe hybridization → DNA fingerprint pattern

Applications

Forensic science: Identification of criminals, matching biological samples, and crime scene investigation.

Paternity and maternity testing: Establishing biological relationships and resolving inheritance disputes.

Identification of missing persons: Disaster victim identification and unknown body identification.

Medical genetics: Detection of inherited disorders and linkage analysis.

Wildlife conservation: Identification of endangered species and prevention of illegal wildlife trade.

Evolutionary and population studies: Genetic diversity analysis and human migration studies.

Advantages

·        Highly accurate

·        Requires a very small DNA sample

·        Useful even with degraded DNA

·        Fast analysis using PCR

·        High discriminatory power

Limitations

·        Identical twins have the same DNA fingerprint.

·        Contaminated samples may give misleading results.

·        Improper handling can affect accuracy.

·        Interpretation requires statistical analysis.

DNA Fingerprinting in India

The technique was developed in India by Dr. Lalji Singh, known as the Father of DNA Fingerprinting in India. He introduced DNA fingerprinting technology for forensic and paternity investigations in the country.

Important NEET Points

·        Inventor: Alec Jeffreys (1984)

·        Indian pioneer: Dr. Lalji Singh

·        Main markers used today: STRs

·        Technique involved: PCR + Gel electrophoresis

·        Classical method: Southern blotting

·        Used in: Forensics, paternity testing, and identification

Academic References

1.      Jeffreys, A. J., Wilson, V., & Thein, S. L. (1985). Hypervariable "minisatellite" regions in human DNA. Nature, 314, 67–73.

2.      Alberts, B., et al. (2022). Molecular Biology of the Cell (7th ed.). Garland Science.

3.      NCERT. (2025). Biology, Class XII. Chapter: Biotechnology and its Applications.

4.      Butler, J. M. (2015). Advanced Topics in Forensic DNA Typing: Interpretation. Academic Press.

5.      Jobling, M. A., & Gill, P. (2004). Encoded evidence: DNA in forensic analysis. Nature Reviews Genetics, 5, 739–751.

The five kingdom system was developed by Robert H. Whittaker in 1969 and was built on the work of previous biologists such as Carolus Linnaeus.

Living things can be classified into five major kingdoms. 

1. Kingdom prokaryote : Prokaryote is a kingdom or division in the classification organization developed for all life on Earth. This kingdom which is also elected as Monera contains all bacteria and blue-green algae (also known as Cyanobacteria). 

2. Kingdom Protista or Protoctista :   It is a kingdom of simple eukaryotic organisms usually made from a single cell or a colony of similar cells. Protists live in water, in moist terrestrial habitats, as parasites and other symbiotic in the bodies of multicellular eukaryotes. 

 3. Kingdom Fungi : A fungus is any member of the group of eukaryotic organisms that comprises microorganisms such as yeasts and molds as well as the more familiar mushrooms. These organisms are classified as a kingdom which is separate from the other eukaryotic life kingdoms of plants and animals. 

 4. Kingdom Plantae : This kingdom contains all land plants such as mosses, ferns, conifers and flowering plants etc. an amazing range of diverse forms. With more than 250,000 species they are second in size only to the arthropods. Plants have been around for a very long time. 

5. Kingdom Animalia : All animals are members of the Kingdom Animalia, also called Metazoa. This Kingdom does not contain prokaryotes (Kingdom Monera, includes bacteria, blue-green algae) or protists (Kingdom Protista, includes unicellular eukaryotic organisms).

Some branches of biology are below: 

1. Botany: The study of Plants and their features. 

 2. Zoology: The study of animals and their futures. 

 3. Microbiology: The study of Microorganisms and their features. 

 4. Taxonomy: It is the science of identification, nomenclature and classification of organisms. 

 5. Morphology: It is the study of external form, size, shape, color, structure and relative position of several living organ of living organisms. 

 6. Anatomy: It is the study of internal structure. 

 7. Histology: It is the study of tissue organization and structure. 

 8. Cytology: It is the study of form and structure of cells containing the behavior of nucleus and other organelles. 

 9. Cell Biology: It is the study of morphological, organizational, biochemical, physiological, genetic, developmental, pathological and evolutionary features of cell and its components. 

 10. Molecular Biology: It is the study of the nature of physicochemical organization, synthesis working and interaction of bio-molecules. 

11. Physiology: It is the study of different types of body functions and procedures. 

 12. Embryology: It is the study of fertilization, growth, division and distinction of the zygote into embryo. 

13. Ecology: It is the study of living organisms is relative to other organism and their environment. 

 14. Genetics: It is the study of inheritance of characters or heredity and variations. 

15. Evolution: It is the study of the origin of life as well as new types of organism from the previous ones by modifications involving genetic changes and adaptations. 

 16. Paleontology: It deals with the study of fossils or remains and impressions of past organisms. 

 17. Virology: It is the study of viruses and all their aspects.

 Introduction

Phylum Platyhelminthes (flatworms) characteristics :

Class 1- Turbellaria (L., turbella= a little string) 

• Mostly free-living but some ectocommensals and endocommensals or parasitic called.
• Terrestrial marine or freshwater.
• Body unsegmented and covered with ciliated cellular or syncytial epidermis, containing mucus-secreting cells and rod-shaped body called.
• Mouth ventral. intestine preceded by the muscular pharynx.
• Adhesive organs(suckers) abundantly present.
• Sense organ i.e. Tango, chemo, and photoreceptors common in free living forms.
• The excretory system consists of protonephridia, the flame cells.
• Mostly reproduction sexual, asexual and by regeneration.
• Life cycle simple. 

Class 2- Trematoda (Gr., trematodes= having pore) 

 Introduction

Nucleic acids are complex biomolecules that store, transmit, and express genetic information in all living organisms. They are essential for heredity, protein synthesis, cell division, and the regulation of cellular activities. The two major types of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA acts as the genetic material in most organisms, while RNA plays a crucial role in gene expression and protein synthesis.

What are nucleic acids?

Nucleic acids are polymers of nucleotides. Each nucleotide consists of three components:

• A pentose sugar

• A nitrogenous base

• A phosphate group

Nucleotides are linked together by phosphodiester bonds to form long polynucleotide chains.

Components of a nucleotide

Pentose sugar

There are two types of sugars found in nucleic acids.

The absence of one oxygen atom at the 2′ carbon of deoxyribose makes DNA more chemically stable than RNA.

Nitrogenous bases

Nitrogenous bases are of two types.

Purines

• Adenine (A)

• Guanine (G)

Pyrimidines

• Cytosine (C)

• Thymine (T) – present only in DNA

• Uracil (U) – present only in RNA

Phosphate group

The phosphate group links adjacent nucleotides through 3′–5′ phosphodiester bonds, forming the sugar-phosphate backbone.

Nucleoside and nucleotide

A nucleoside consists of a sugar and a nitrogenous base, while a nucleotide consists of a nucleoside plus one or more phosphate groups.

Examples:

• Adenosine = adenine + ribose

• Adenosine monophosphate (AMP) = adenosine + phosphate

Structure of DNA

The DNA structure was proposed by James Watson and Francis Crick (1953) based on X-ray diffraction studies by Rosalind Franklin and Maurice Wilkins.

Key features of the DNA double helix

• DNA consists of two antiparallel polynucleotide strands

• The strands are twisted into a right-handed double helix

• Sugar-phosphate backbones are located on the outside

• Nitrogenous bases face inward

Base pairing

Complementary base pairing occurs through hydrogen bonds.

Guanine-cytosine pairs are more stable due to the presence of three hydrogen bonds.

Dimensions of B-DNA

• Diameter: 2 nm

• Distance between adjacent base pairs: 0.34 nm

• One complete turn: 3.4 nm

• Number of base pairs per turn: 10

Chargaff’s rules

Erwin Chargaff discovered that:

• A = T

• G = C

• Total purines = total pyrimidines

These observations provided crucial evidence for complementary base pairing.

Types of DNA

DNA may exist in different conformations.

B-DNA is the most common form found in cells.

Structure of RNA

RNA is generally single-stranded, although it may fold into complex secondary and tertiary structures.

RNA contains:

• Ribose sugar

• Adenine, guanine, cytosine, and uracil

Because of the presence of the 2′-OH group, RNA is less stable than DNA.

Types of RNA

Messenger RNA (mRNA)

• Carries genetic information from DNA to ribosomes

• Serves as the template for protein synthesis

Transfer RNA (tRNA)

• Transfers amino acids to ribosomes

• Contains an anticodon that recognizes mRNA codons

Ribosomal RNA (rRNA)

• Forms the structural and catalytic core of ribosomes

• Catalyzes peptide bond formation

Other RNAs

• Small nuclear RNA (snRNA)

• Small nucleolar RNA (snoRNA)

• MicroRNA (miRNA)

• Small interfering RNA (siRNA)

• Long non-coding RNA (lncRNA)

DNA vs RNA

Biological functions of nucleic acids

Storage of genetic information

DNA stores hereditary information that is transmitted from parents to offspring.

DNA replication

DNA duplicates itself before cell division, ensuring genetic continuity.

Protein synthesis

RNA molecules participate in transcription and translation to produce proteins.

Regulation of gene expression

Non-coding RNAs regulate gene activity, development, and cellular differentiation.

Evolution

Mutations in nucleic acids generate genetic variation, which drives evolution.

Central dogma of molecular biology

The flow of genetic information follows the pathway:

DNA → RNA → Protein

This process involves:

• Replication

• Transcription

• Translation

DNA replication

DNA replication is semi-conservative, meaning each daughter DNA molecule contains one parental strand and one newly synthesized strand.

Major enzymes involved include:

• DNA helicase

• DNA polymerase

• Primase

• DNA ligase

• Topoisomerase

Replication proceeds in the 5′ → 3′ direction.

Transcription

Transcription is the synthesis of RNA from a DNA template.

Steps:

1. Initiation

2. Elongation

3. Termination

In eukaryotes, mRNA undergoes:

• 5′ capping

• Splicing

• 3′ polyadenylation

Translation

Translation occurs on ribosomes.

Major stages:

1. Initiation

2. Elongation

3. Termination

During translation, codons on mRNA specify the sequence of amino acids in proteins.

Importance in biotechnology

Nucleic acids have numerous applications.

Polymerase chain reaction (PCR)

Amplifies DNA fragments.

DNA fingerprinting

Used in forensic science and paternity testing.

Recombinant DNA technology

Produces genetically modified organisms and therapeutic proteins.

Gene therapy

Treats genetic diseases by introducing functional genes.

CRISPR-Cas genome editing

Allows precise modification of DNA sequences.

Nucleic acid metabolism

Purine metabolism

Purines are synthesized and degraded to uric acid.

Pyrimidine metabolism

Pyrimidines are degraded to beta-alanine and beta-aminoisobutyrate.

Disorders of nucleotide metabolism include:

• Gout

• Lesch-Nyhan syndrome

• Severe combined immunodeficiency (SCID)

Key points

• DNA contains deoxyribose; RNA contains ribose.

• Thymine is present in DNA; uracil is present in RNA.

• DNA strands are antiparallel.

• A pairs with T; G pairs with C.

• DNA replication is semi-conservative.

• mRNA carries genetic information.

• tRNA transports amino acids.

• rRNA forms ribosomes.

• The central dogma is DNA → RNA → Protein.

Conclusion

Nucleic acids are the molecular basis of life. DNA stores hereditary information, while RNA mediates the expression of that information through protein synthesis. Their structure, replication, transcription, and translation are central to genetics, molecular biology, biotechnology, and medicine.

References

1. Nelson DL, Cox MM. Lehninger Principles of Biochemistry. 8th ed. W.H. Freeman; 2021.

2. Alberts B, et al. Molecular Biology of the Cell. 7th ed. Garland Science; 2022.

3. Watson JD, et al. Molecular Biology of the Gene. 7th ed. Pearson; 2014.

4. Lodish H, et al. Molecular Cell Biology. 9th ed. W.H. Freeman; 2021.

5. NCERT. Biology Class XI. National Council of Educational Research and Training; Latest Edition.

6. Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature. 1953;171:737–738.

1. Isolation & Extraction of Carbohydrates

Carbohydrates are classified by chain length, polarity, and solubility: low molecular weight mono- and oligosaccharides are water-soluble and polar, whereas structural polysaccharides (e.g., cellulose, chitin, starch, glycogen, pectin) require specific chemical, enzymatic, or thermal treatments for extraction.

Extraction Strategies by Carbohydrate Class

1. Free Mono- and Oligosaccharides (Glucose, Sucrose, Fructose)

• Extraction Solvent: Hot 80% aqueous ethanol (v/v) under reflux.

• Mechanism: 80% ethanol selectively dissolves low molecular weight sugars while precipitating proteins, nucleic acids, and high molecular weight polysaccharides (starch, pectins).

• Clarification / Deproteinization: Lead acetate or Carrez reagents (Carrez I: potassium ferrocyanide; Carrez II: zinc sulfate) are added to precipitate residual proteins and phenolic compounds before analysis.

2. Storage Polysaccharides (Starch and Glycogen)

• Starch Isolation:

  1. Mechanical cell disruption in cold water or mild alkali (0.05 M NaOH) to solubilize proteins.

  2. Filtration through fine mesh to separate insoluble cellulosic fiber.

  3. Sedimentation/centrifugation of starch granules.

  4. Gelatinization via heating ($>65^\circ\text{C}$) followed by enzymatic digestion ($\alpha$-amylase and amyloglucosidase) for monomer analysis.

• Glycogen Isolation:

  1. Homogenization of liver/muscle tissue in cold 5–10% trichloroacetic acid (TCA) to precipitate proteins and nucleic acids.

  2. Centrifugation to collect the supernatant containing glycogen.

  3. Precipitation of glycogen from the supernatant by adding 2 volumes of 95% ethanol in the presence of sodium chloride.

3. Structural Polysaccharides (Cellulose, Chitin, Pectin)

• Pectin Isolation: Hot aqueous extraction under acidic conditions (pH 1.5–3.0 using $\text{HCl}$ or citric acid) at $80\text{--}90^\circ\text{C}$, followed by precipitation with ethanol or isopropanol.

• Cellulose Isolation: Removal of lignin and hemicellulose via strong alkali treatment (17.5% NaOH) followed by bleaching with sodium chlorite ($\text{NaClO}_2$) and acetic acid.

• Chitin Isolation:

  1. Demineralization: Dilute $\text{HCl}$ (1–2 M) to remove calcium carbonate from crustacean shells.

  2. Deproteinization: Dilute $\text{NaOH}$ (1–2 M) at $80\text{--}100^\circ\text{C}$.

  3. Deacetylation (to form Chitosan): Concentrated $\text{NaOH}$ (40–50%) at $100\text{--}120^\circ\text{C}$.

Fractionation & Purification Techniques

• Alcohol / Solvent Precipitation: High molecular weight glycans precipitate out of aqueous solution upon addition of organic solvents (ethanol, isopropanol, or acetone) to a final concentration of 70–80%.

• Ion-Exchange Chromatography (IEC): Separates charged polysaccharides from neutral sugars.

  • DEAE-Cellulose / DEAE-Sepharose (Anion Exchange): Retains acidic polysaccharides (e.g., pectins, alginates, glycosaminoglycans, hyaluronic acid) due to negative carboxylate or sulfate groups. Eluted using a $0\text{--}1.0\text{ M NaCl}$ gradient.

• Size-Exclusion Chromatography (SEC / Gel Filtration): Separates polysaccharides based on hydrodynamic volume and molecular weight using matrices such as Sephadex (G-50 to G-200), Sepharose, or Bio-Gel.

• Lectin Affinity Chromatography: Uses immobilized lectins (e.g., Concanavalin A, Wheat Germ Agglutinin) to selectively bind specific carbohydrate motifs:

  • Concanavalin A (Con A): Binds $\alpha$-D-mannosyl and $\alpha$-D-glucosyl residues.

  • Wheat Germ Agglutinin (WGA): Binds $N$-acetylglucosamine ($\text{GlcNAc}$) and sialic acid residues.

  • Elution: Displaced using a competing monomeric sugar (e.g., $0.1\text{--}0.5\text{ M}$ methyl $\alpha$-D-glucopyranoside for Con A).

Quantitative & Analytical Methods for Carbohydrates

GC-MS Derivatization of Sugars

Carbohydrates are non-volatile and thermally unstable, preventing direct Gas Chromatography (GC) analysis. They require two major derivatization pathways:

1. Trimethylsilylation (TMS): Hydroxyl groups are reacted with trimethylsilyl reagents (e.g., BSTFA / TMCS) to yield volatile TMS-ether derivatives.

2. Alditol Acetate Conversion:

   • Acid hydrolysis of polysaccharides to monosaccharides.

   • Reduction of monosaccharides to alditols using sodium borohydride ($\text{NaBH}_4$).

   • Acetylation of alditols using acetic anhydride and pyridine to form volatile alditol acetates.

2. Isolation & Extraction of Lipids

Lipids are hydrophobic or amphipathic biomolecules. Extraction requires organic solvents that disrupt hydrophobic and electrostatic interactions between lipids and membrane proteins while maintaining lipid structural integrity.

Major Total Lipid Extraction Protocols

1. Folch Method (1957)

• Solvent Mixture: Chloroform : Methanol ($2:1\text{ v/v}$).

• Mechanism:

  1. Methanol penetrates the cellular matrix and disrupts hydrogen bonds/electrostatic interactions between membrane lipids and proteins.

  2. Chloroform dissolves neutral and polar lipids into a monophasic extraction mixture.

• Biphasic Separation: Water or dilute salt solution ($0.9\%\text{ NaCl}$ or $0.73\%\text{ NaCl}$) is added to achieve a final volumetric ratio of Chloroform : Methanol : Water ($8:4:3\text{ v/v/v}$).

• Phase Layering:

  • Upper Phase (Aqueous / Polar): Water, methanol, salts, non-lipid impurities (sugars, amino acids), and gangliosides.

  • Interphase: Denatured proteins.

  • Lower Phase (Organic / Non-polar): Chloroform layer containing total lipids (phospholipids, glycolipids, triglycerides, cholesterol).

2. Bligh and Dyer Method (1959)

• Optimized Use Case: High-water content biological samples (e.g., tissues, cell suspensions, fish muscle). Uses lower solvent-to-tissue ratios than the Folch method.

• Extraction Stage (Monophasic): Adjusts solvent proportions based on tissue water content to yield a single phase of Chloroform : Methanol : Sample Water ($1:2:0.8\text{ v/v/v}$).

• Separation Stage (Biphasic): Additional chloroform and water are added to reach a final ratio of Chloroform : Methanol : Water ($2:2:1.8\text{ v/v/v}$).

• Phase Layering: Similar to Folch; total lipids partition into the lower chloroform phase.

3. Matyash / MTBE Method (2008)

• Solvent Mixture: Methyl tert-butyl ether (MTBE) : Methanol : Water.

• Critical Advantage: MTBE has a lower density than water. Consequently, the lipid-rich organic phase forms the UPPER layer, while proteins collect in the pellet at the bottom and the aqueous phase forms the lower layer. This prevents contamination when pipetting the lipid phase through a protein interphase.

Fractionation & Chromatographic Separation of Lipids

1. Solid-Phase Extraction (SPE) / Silica Gel Column Chromatography

Lipid classes are separated based on polarity using a silica gel stationary phase by sequential elution with solvents of increasing polarity:

1. Fraction 1 (Neutral Lipids): Eluted with Chloroform or Hexane : Diethyl Ether.

   • Yields: Triacylglycerols (TAGs), free fatty acids, cholesterol, cholesterol esters, wax esters.

2. Fraction 2 (Glycolipids & Sphingolipids): Eluted with Acetone or Acetone : Methanol ($9:1\text{ v/v}$).

   • Yields: Cerebrosides, sulfatides, monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols (DGDG).

3. Fraction 3 (Polar Phospholipids): Eluted with Methanol.

   • Yields: Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Sphingomyelin.

2. Thin Layer Chromatography (TLC) of Lipids

• Stationary Phase: Silica Gel G plates (polar).

• Solvent Systems:

  • For Neutral Lipids: Petroleum Ether : Diethyl Ether : Acetic Acid ($80:20:1\text{ v/v/v}$).

    • Migration Order ($R_f$ value low to high): Monoglycerides < Diglycerides < Free Fatty Acids < Triacylglycerols < Cholesterol Esters.

  • For Polar Phospholipids: Chloroform : Methanol : Water ($65:25:4\text{ v/v/v}$).

• Visualization & Detection Reagents:

  • Iodine Vapor: Universal reversible stain for unsaturated lipids (binds carbon-carbon double bonds).

  • Ninhydrin Spray: Specific for lipids containing free primary amino groups (Phosphatidylethanolamine and Phosphatidylserine $\rightarrow$ purple/pink spots).

  • Dragendorff Reagent: Specific for choline-containing lipids (Phosphatidylcholine, Sphingomyelin $\rightarrow$ orange spots).

  • Dittmer-Lester (Molybdenum Blue) Reagent: Specific for phosphate esters (all phospholipids $\rightarrow$ blue spots).

  • Orcinol-Sulfuric Acid Reagent: Specific for glycolipids (cerebrosides, gangliosides $\rightarrow$ purple/violet spots).

Fatty Acid Methyl Esterification (FAME Analysis)

Fatty acids in complex lipids (TAGs, phospholipids) are non-volatile and must be converted into Fatty Acid Methyl Esters (FAMEs) prior to Gas Chromatography (GC-FID or GC-MS) analysis.

Complex Lipids (TAGs / Phospholipids)
       │
       ├─► Alkaline Transesterification: Methanolic NaOH or KOH (mild, preserves double bonds)
       │
       └─► Acidic Transesterification: Methanolic HCl, BF3, or H2SO4 (cleaves amide bonds in sphingolipids)
       │
       ▼
Fatty Acid Methyl Esters (FAMEs) ──► Volatilized & Separated via GC Column

• Alkaline Transesterification: Treatment with methanolic $\text{NaOH}$ or $\text{KOH}$ at $50\text{--}60^\circ\text{C}$. Cleaves ester bonds in glycerolipids without altering double bond positions or configurations (prevents isomerization).

• Acidic Transesterification: Treatment with methanolic $\text{BF}_3$ (boron trifluoride) or methanolic $\text{HCl}$. Required to hydrolyze amide bonds found in sphingolipids or esterify free fatty acids.

3. High-Yield Comparison Table: Carbohydrate vs. Lipid Extraction

 Definition

DNA fingerprinting (DNA profiling) is a molecular technique used to identify individuals based on unique patterns in their DNA. It analyzes highly variable regions called VNTRs (Variable Number Tandem Repeats) or STRs (Short Tandem Repeats). Developed by Sir Alec Jeffreys in 1984.

Principle

·        More than 99.9% of human DNA is identical, but repetitive DNA regions vary among individuals.

·        The number of repeat units differs from person to person.

·        These variations produce a unique DNA banding pattern for each individual (except identical twins).

Types of DNA Sequences Used

Modern forensic analysis mainly uses STR markers.

Steps of DNA Fingerprinting

1. Sample collection

DNA can be obtained from blood, hair root, saliva, semen, bone, teeth, and skin cells.

2. DNA isolation

DNA is extracted from the collected biological sample.

3. DNA amplification (PCR)

Specific STR/VNTR regions are amplified using the Polymerase Chain Reaction (PCR).

4. Restriction digestion (classical method)

Restriction enzymes cut DNA into fragments at specific sequences.

5. Gel electrophoresis

DNA fragments are separated according to size through an agarose gel.

6. Southern blotting

DNA fragments are transferred from the gel onto a nylon or nitrocellulose membrane.

7. Hybridization with labeled probes

Radioactive or fluorescent probes bind to specific repetitive DNA sequences.

8. Detection

Band patterns are visualized using autoradiography or fluorescence detection. The resulting pattern is the DNA fingerprint.

Workflow

Biological sample → DNA extraction → PCR amplification of STR/VNTR loci → Gel electrophoresis → Southern blotting → Probe hybridization → DNA fingerprint pattern

Applications

Forensic science: Identification of criminals, matching biological samples, and crime scene investigation.

Paternity and maternity testing: Establishing biological relationships and resolving inheritance disputes.

Identification of missing persons: Disaster victim identification and unknown body identification.

Medical genetics: Detection of inherited disorders and linkage analysis.

Wildlife conservation: Identification of endangered species and prevention of illegal wildlife trade.

Evolutionary and population studies: Genetic diversity analysis and human migration studies.

Advantages

·        Highly accurate

·        Requires a very small DNA sample

·        Useful even with degraded DNA

·        Fast analysis using PCR

·        High discriminatory power

Limitations

·        Identical twins have the same DNA fingerprint.

·        Contaminated samples may give misleading results.

·        Improper handling can affect accuracy.

·        Interpretation requires statistical analysis.

DNA Fingerprinting in India

The technique was developed in India by Dr. Lalji Singh, known as the Father of DNA Fingerprinting in India. He introduced DNA fingerprinting technology for forensic and paternity investigations in the country.

Important NEET Points

·        Inventor: Alec Jeffreys (1984)

·        Indian pioneer: Dr. Lalji Singh

·        Main markers used today: STRs

·        Technique involved: PCR + Gel electrophoresis

·        Classical method: Southern blotting

·        Used in: Forensics, paternity testing, and identification

Academic References

1.      Jeffreys, A. J., Wilson, V., & Thein, S. L. (1985). Hypervariable "minisatellite" regions in human DNA. Nature, 314, 67–73.

2.      Alberts, B., et al. (2022). Molecular Biology of the Cell (7th ed.). Garland Science.

3.      NCERT. (2025). Biology, Class XII. Chapter: Biotechnology and its Applications.

4.      Butler, J. M. (2015). Advanced Topics in Forensic DNA Typing: Interpretation. Academic Press.

5.      Jobling, M. A., & Gill, P. (2004). Encoded evidence: DNA in forensic analysis. Nature Reviews Genetics, 5, 739–751.

The five kingdom system was developed by Robert H. Whittaker in 1969 and was built on the work of previous biologists such as Carolus Linnaeus.

Living things can be classified into five major kingdoms. 

1. Kingdom prokaryote : Prokaryote is a kingdom or division in the classification organization developed for all life on Earth. This kingdom which is also elected as Monera contains all bacteria and blue-green algae (also known as Cyanobacteria). 

2. Kingdom Protista or Protoctista :   It is a kingdom of simple eukaryotic organisms usually made from a single cell or a colony of similar cells. Protists live in water, in moist terrestrial habitats, as parasites and other symbiotic in the bodies of multicellular eukaryotes. 

 3. Kingdom Fungi : A fungus is any member of the group of eukaryotic organisms that comprises microorganisms such as yeasts and molds as well as the more familiar mushrooms. These organisms are classified as a kingdom which is separate from the other eukaryotic life kingdoms of plants and animals. 

 4. Kingdom Plantae : This kingdom contains all land plants such as mosses, ferns, conifers and flowering plants etc. an amazing range of diverse forms. With more than 250,000 species they are second in size only to the arthropods. Plants have been around for a very long time. 

5. Kingdom Animalia : All animals are members of the Kingdom Animalia, also called Metazoa. This Kingdom does not contain prokaryotes (Kingdom Monera, includes bacteria, blue-green algae) or protists (Kingdom Protista, includes unicellular eukaryotic organisms).

Some branches of biology are below: 

1. Botany: The study of Plants and their features. 

 2. Zoology: The study of animals and their futures. 

 3. Microbiology: The study of Microorganisms and their features. 

 4. Taxonomy: It is the science of identification, nomenclature and classification of organisms. 

 5. Morphology: It is the study of external form, size, shape, color, structure and relative position of several living organ of living organisms. 

 6. Anatomy: It is the study of internal structure. 

 7. Histology: It is the study of tissue organization and structure. 

 8. Cytology: It is the study of form and structure of cells containing the behavior of nucleus and other organelles. 

 9. Cell Biology: It is the study of morphological, organizational, biochemical, physiological, genetic, developmental, pathological and evolutionary features of cell and its components. 

 10. Molecular Biology: It is the study of the nature of physicochemical organization, synthesis working and interaction of bio-molecules. 

11. Physiology: It is the study of different types of body functions and procedures. 

 12. Embryology: It is the study of fertilization, growth, division and distinction of the zygote into embryo. 

13. Ecology: It is the study of living organisms is relative to other organism and their environment. 

 14. Genetics: It is the study of inheritance of characters or heredity and variations. 

15. Evolution: It is the study of the origin of life as well as new types of organism from the previous ones by modifications involving genetic changes and adaptations. 

 16. Paleontology: It deals with the study of fossils or remains and impressions of past organisms. 

 17. Virology: It is the study of viruses and all their aspects.

 Introduction

Phylum Platyhelminthes (flatworms) characteristics :

Class 1- Turbellaria (L., turbella= a little string) 

• Mostly free-living but some ectocommensals and endocommensals or parasitic called.
• Terrestrial marine or freshwater.
• Body unsegmented and covered with ciliated cellular or syncytial epidermis, containing mucus-secreting cells and rod-shaped body called.
• Mouth ventral. intestine preceded by the muscular pharynx.
• Adhesive organs(suckers) abundantly present.
• Sense organ i.e. Tango, chemo, and photoreceptors common in free living forms.
• The excretory system consists of protonephridia, the flame cells.
• Mostly reproduction sexual, asexual and by regeneration.
• Life cycle simple. 

Class 2- Trematoda (Gr., trematodes= having pore)