B.Sc. 3rd Year Zoology – Section 3

1. Nucleic Acids: Structure and Composition

1.1 Components of Nucleic Acids

Nucleic acids are made up of repeating units called nucleotides. Each nucleotide consists of three components:

1. A nitrogenous base
2. A pentose sugar
3. One or more phosphate groups

1.2 Nitrogenous Bases

1.3 DNA Structure

DNA is the principal hereditary material in most organisms. It is composed of two complementary polynucleotide strands arranged in a double-helical structure.

Important structural features of DNA

• DNA contains deoxyribose sugar.
• The bases are adenine, guanine, cytosine and thymine.
• The two strands are antiparallel.
• One strand runs 5′ → 3′ while the other runs 3′ → 5′.
• A pairs with T through two hydrogen bonds.
• G pairs with C through three hydrogen bonds.
• The sugar-phosphate backbone lies on the outside.
• The nitrogenous bases project toward the interior.
• The strands are complementary.

1.4 Major Forms of DNA

1.5 DNA Packaging

In prokaryotes, DNA is usually present as a circular chromosome associated with proteins in a nucleoid region. In eukaryotes, DNA is associated with histone proteins to form chromatin.

2. DNA Replication

2.1 Important Characteristics of DNA Replication

• Semi-conservative: each daughter DNA molecule contains one parental and one newly synthesized strand.
• Semi-discontinuous: one strand is synthesized continuously while the other is synthesized discontinuously.
• Template-directed: each parental strand acts as a template.
• 5′ → 3′ synthesis: new DNA is synthesized by addition of nucleotides to the 3′-OH end.
• Primer-dependent: DNA polymerase requires a pre-existing primer with a free 3′-OH group.
• Replication may be unidirectional or bidirectional depending on the organism/system.

2.2 DNA Polymerases

DNA polymerases are enzymes that catalyse the synthesis of DNA using a template strand and complementary deoxyribonucleotides.

2.3 Mechanism of DNA Polymerase Action

1. DNA strands are separated.
2. A primer binds to the template.
3. The DNA polymerase recognizes the primer-template junction.
4. A complementary dNTP enters the active site.
5. The correct nucleotide is selected by base pairing.
6. The nucleotide is added to the 3′-OH of the growing strand.
7. Pyrophosphate is released during nucleotide incorporation.
8. The polymerase continues moving along the template.

2.4 Semi-discontinuous Replication

Because DNA polymerase can synthesize DNA only in the 5′ → 3′ direction, the two DNA strands cannot be copied in exactly the same manner.

2.5 Okazaki Fragments

Okazaki fragments are short DNA segments synthesized on the lagging strand during DNA replication. They are subsequently joined by DNA ligase to form a continuous DNA strand.

2.6 Major Enzymes of DNA Replication

3. DNA Replication in Prokaryotes

A classical example is replication of the circular chromosome of Escherichia coli. Replication begins at a specific origin called oriC.

3.1 Steps

1. Initiation: initiator proteins recognize the origin of replication.
2. DNA unwinding: helicase separates the two strands.
3. Stabilization: single-strand binding proteins stabilize the exposed strands.
4. Primer synthesis: primase synthesizes RNA primers.
5. Elongation: DNA polymerase synthesizes the new DNA.
6. Primer removal: RNA primers are removed and replaced with DNA.
7. Ligation: DNA ligase seals remaining nicks.
8. Termination: replication forks eventually meet in the termination region.

4. DNA Replication in Eukaryotes

Eukaryotic chromosomes are linear and much larger than bacterial chromosomes. Replication therefore begins at multiple origins along each chromosome, producing multiple replication bubbles and forks.

4.1 Important Features

• Multiple origins of replication.
• Replication occurs during the S phase of the cell cycle.
• Multiple replication forks are generated.
• DNA is associated with histones and must be repackaged after replication.
• Several DNA polymerases participate.
• Telomeres create a special end-replication problem.
• Telomerase helps maintain telomeric DNA in many eukaryotic cells.

4.2 Prokaryotic vs Eukaryotic DNA Replication

5. Structure and Composition of RNA

RNA is generally a single-stranded nucleic acid containing ribose sugar, phosphate and the bases adenine, guanine, cytosine and uracil.

6. RNA Processing

6.1 mRNA Processing in Eukaryotes

Primary eukaryotic transcripts are usually processed before becoming mature mRNA.

Major modifications

1. 5′ capping: a modified guanine nucleotide is added to the 5′ end.
2. 3′ polyadenylation: a poly(A) tail is added to the 3′ end.
3. Splicing: introns are removed and exons are joined.

6.2 Alternative Splicing

Alternative splicing allows a single primary transcript to produce different mRNA molecules by joining exons in different combinations. It increases protein diversity without requiring an equivalent increase in the number of genes.

6.3 rRNA Processing

In eukaryotes, precursor rRNA is synthesized mainly in the nucleolus. It undergoes cleavage, modification and assembly with ribosomal proteins to produce mature ribosomal subunits.

6.4 tRNA Processing

• Removal of leader sequences.
• Removal of trailer sequences.
• Base modification.
• Formation of the mature 3′ end.
• In many tRNAs, CCA is present at the 3′ end and serves as the amino-acid attachment site.

7. Genetic Code and Central Dogma

7.1 Genetic Code

7.2 Characteristics of Genetic Code

• Triplet: three nucleotides form one codon.
• 64 codons: 61 code for amino acids and three function as stop codons.
• Degenerate: most amino acids are represented by more than one codon.
• Unambiguous: a particular codon specifies only one amino acid.
• Nearly universal: the code is shared by most organisms, with some important exceptions.
• Non-overlapping: normally one nucleotide belongs to one codon in a given reading frame.
• Comma-less: codons are read continuously without punctuation.
• Start codon: AUG commonly initiates translation and codes for methionine.
• Stop codons: UAA, UAG and UGA terminate translation.

7.3 Wobble Hypothesis

The wobble hypothesis explains how a relatively small number of tRNA molecules can recognize multiple codons.

The third nucleotide of the codon and the corresponding first nucleotide of the anticodon can show greater flexibility in base pairing than the other positions.

7.4 Central Dogma

8. Transcription

8.1 Replication vs Transcription

9. Prokaryotic RNA Polymerase

The bacterial RNA polymerase is a multisubunit enzyme responsible for transcription of RNA.

9.1 Organization

The bacterial core enzyme is commonly represented as α₂ββ′ω. Association with a sigma factor forms the holoenzyme required for promoter recognition and initiation.

9.2 Bacterial Promoter

Important promoter elements in many bacteria include the -35 region and the -10 region. The -10 region is often called the Pribnow box.

10. Prokaryotic Transcription

10.1 Initiation

1. RNA polymerase holoenzyme recognizes the promoter.
2. The sigma factor helps identify the promoter.
3. DNA locally unwinds to form a transcription bubble.
4. The first RNA nucleotides are joined.
5. After promoter clearance, the sigma factor may dissociate or alter its interaction with the polymerase.

10.2 Elongation

RNA polymerase moves along the template DNA strand and synthesizes RNA in the 5′ → 3′ direction.

10.3 Termination

Termination may occur through intrinsic mechanisms or through factor-dependent mechanisms such as Rho-mediated termination.

11. Eukaryotic Transcription

Eukaryotes possess several RNA polymerases with specialized functions.

11.1 Major Steps of Eukaryotic Transcription

1. Chromatin must become accessible.
2. Transcription factors bind promoter and regulatory DNA.
3. RNA polymerase is recruited.
4. Transcription initiation occurs.
5. RNA chain elongates.
6. Transcription terminates.
7. The primary RNA transcript is processed.

11.2 Prokaryotic vs Eukaryotic Transcription

12. Translation and Protein Synthesis

12.1 Components Required for Translation

• mRNA
• Ribosomes
• tRNA
• Amino acids
• Aminoacyl-tRNA synthetases
• Initiation factors
• Elongation factors
• Release factors
• ATP and GTP

12.2 tRNA Structure and Function

tRNA acts as an adaptor molecule between the codon of mRNA and its corresponding amino acid.

12.3 Aminoacylation

Before translation, each tRNA must be attached to its appropriate amino acid. This process is catalysed by a specific aminoacyl-tRNA synthetase.

The process uses ATP and ensures accurate matching between amino acids and their corresponding tRNAs.

13. Translation in Prokaryotes

13.1 Initiation

1. The small ribosomal subunit binds to the mRNA.
2. The ribosome recognizes the Shine-Dalgarno sequence.
3. The start codon is positioned in the appropriate site.
4. Initiator tRNA carrying formylmethionine participates.
5. The large ribosomal subunit joins to form the complete ribosome.

13.2 Elongation

1. Aminoacyl-tRNA enters the A site.
2. Correct codon-anticodon pairing occurs.
3. Peptide bond formation takes place.
4. The ribosome moves along the mRNA.
5. The empty tRNA exits through the E site.
6. The growing peptide remains attached to the tRNA in the P site.

13.3 Termination

When a stop codon enters the decoding site, no normal tRNA recognizes it. Release factors interact with the ribosome and promote release of the completed polypeptide.

14. Translation in Eukaryotes

Eukaryotic translation follows the same fundamental principle but uses different initiation factors, ribosomal subunits and regulatory mechanisms. The cytoplasmic ribosome is generally 80S, consisting of 40S and 60S subunits.

14.1 Initiation

1. The 40S ribosomal subunit associates with initiation factors.
2. The initiator methionyl-tRNA associates with the small subunit.
3. The complex interacts with the 5′ end of mRNA.
4. The ribosome scans the mRNA to identify an appropriate AUG start codon.
5. The 60S subunit joins.
6. The functional 80S ribosome is formed.

14.2 Elongation

Elongation involves repeated aminoacyl-tRNA entry, codon recognition, peptide bond formation and ribosomal translocation.

14.3 Termination

Translation terminates when a stop codon is encountered. Release factors promote hydrolysis of the bond between the polypeptide and tRNA, releasing the newly synthesized protein.

14.4 Prokaryotic vs Eukaryotic Translation

15. Post-translational Modification

The newly synthesized polypeptide may not immediately possess its final functional form. It may undergo structural folding, cleavage or chemical modifications.

16. Gene Regulation

16.1 Gene Expression

Gene expression involves the use of genetic information to produce a functional RNA or protein.

17. Regulation in Prokaryotes

Bacteria often regulate genes according to environmental conditions and metabolic requirements.

17.1 Lac Operon

The lac operon of Escherichia coli is a classical example of inducible gene regulation.

Major components

• Regulatory gene
• Promoter
• Operator
• Structural genes: lacZ, lacY and lacA

17.2 Regulation Without Lactose

In the absence of lactose, the repressor can bind to the operator and reduce transcription of the structural genes.

17.3 Regulation With Lactose

When lactose or an appropriate inducer is available, the repressor is inactivated or its binding to the operator is reduced, allowing transcription of genes involved in lactose utilization.

18. Regulation in Eukaryotes

Eukaryotic gene regulation is more complex and can occur at several levels.

18.1 Transcriptional Regulation

Transcription factors can bind promoters, enhancers and other regulatory DNA sequences to increase or decrease transcription.

18.2 Translational Regulation

Cells may regulate whether a particular mRNA is translated efficiently. Regulatory proteins and small RNAs can influence translation.

18.3 Post-translational Regulation

Protein activity may be controlled through phosphorylation, cleavage, localization, degradation and other modifications.

18.4 Hormonal Control of Gene Expression

Some hormones regulate gene expression by binding intracellular receptors that influence transcription. Steroid hormones are important examples.

19. Techniques of Molecular Biology

Molecular biology techniques are used to isolate, amplify, identify, analyze, sequence and manipulate DNA, RNA and proteins.

20. Polymerase Chain Reaction (PCR)

20.1 Basic Components

• Template DNA
• Forward primer
• Reverse primer
• Thermostable DNA polymerase such as Taq polymerase
• dNTPs
• Reaction buffer
• Mg²⁺ ions
• Water

20.2 Steps of PCR

1. Denaturation

The double-stranded DNA is heated so that the two strands separate.

2. Annealing

The temperature is lowered to allow primers to hybridize with their complementary target sequences.

3. Extension

The thermostable DNA polymerase extends the primers by adding complementary nucleotides.

20.3 PCR Cycle

20.4 Types of PCR

20.5 Applications of PCR

• Detection of infectious organisms.
• Detection of genetic mutations.
• Forensic DNA analysis.
• DNA fingerprinting workflows.
• Medical diagnostics.
• Research and cloning.
• Ancient DNA analysis.
• Genetic identification.
• Food and environmental testing.
• Detection of specific genes.

21. DNA Fingerprinting

21.1 Principle

Many regions of the human genome contain highly variable repetitive DNA sequences. These regions can generate characteristic DNA profiles. Modern forensic DNA profiling commonly uses short tandem repeats (STRs).

21.2 General Steps

1. Collection of biological sample.
2. DNA extraction.
3. Quantification and quality assessment.
4. Amplification of selected STR loci using PCR.
5. Separation and detection of amplified fragments.
6. Generation of a DNA profile.
7. Comparison of profiles.
8. Statistical interpretation of the match.

21.3 Types / Approaches

21.4 Applications

• Forensic identification.
• Parentage testing.
• Identification of missing persons.
• Identification of disaster victims.
• Wildlife and conservation genetics.
• Population genetic studies.

22. Gene Cloning

22.1 Major Components

• Target DNA
• Restriction enzyme
• DNA ligase
• Cloning vector
• Host cell
• Selectable marker

22.2 General Steps

1. Isolation of target DNA.
2. Selection of a suitable vector.
3. Cutting of insert and vector using appropriate restriction enzymes.
4. Joining of DNA insert and vector using DNA ligase.
5. Introduction of recombinant DNA into host cells.
6. Selection of recombinant cells.
7. Screening and confirmation of clones.
8. Amplification or expression of the cloned gene.

22.3 Applications

• Production of recombinant proteins.
• Research on gene function.
• Production of medically important proteins.
• Construction of DNA libraries.
• Study of mutations.
• Biotechnology and agricultural research.

23. DNA Sequencing

23.1 Sanger Sequencing

The classical Sanger method uses chain-terminating dideoxynucleotides (ddNTPs). Incorporation of a ddNTP terminates DNA chain extension because it lacks the appropriate 3′-OH required for further elongation.

23.2 General Principle

1. DNA template is prepared.
2. Primer binds to the template.
3. DNA polymerase extends the primer.
4. Occasional incorporation of a ddNTP terminates a strand.
5. Fragments of different lengths are generated.
6. Fragments are separated according to size.
7. The sequence is read from the resulting pattern.

23.3 Next-Generation Sequencing

Next-generation sequencing technologies can process very large numbers of DNA fragments in parallel, allowing rapid sequencing of genomes, transcriptomes and targeted gene panels.

23.4 Applications

• Genome sequencing.
• Mutation detection.
• Cancer genetics.
• Pathogen genome analysis.
• Population genetics.
• Evolutionary studies.
• Personalized medicine research.

24. Blotting Techniques

Blotting techniques are used to identify specific DNA, RNA or protein molecules after separation.

24.1 Southern Blotting

Southern blotting is used to detect a specific DNA sequence.

Basic steps

1. DNA isolation.
2. Restriction digestion.
3. Gel electrophoresis.
4. Denaturation of DNA.
5. Transfer to membrane.
6. Hybridization with a labelled probe.
7. Detection of the hybridized probe.

24.2 Northern Blotting

Northern blotting detects specific RNA molecules and can therefore be used to investigate gene expression.

24.3 Western Blotting

Western blotting detects specific proteins using antibodies. Proteins are separated by electrophoresis, transferred to a membrane and then detected using specific antibodies.

24.4 Comparison

25. Enzyme-Linked Immunosorbent Assay (ELISA)

25.1 Principle of ELISA

ELISA depends on the highly specific interaction between an antigen and its corresponding antibody. An enzyme attached to an antibody or another assay component acts on a substrate to produce a detectable signal.

25.2 Major Components

• Antigen or antibody
• Specific antibody
• Enzyme-labelled conjugate
• Solid surface, usually a microtiter plate
• Blocking reagent
• Washing buffer
• Enzyme substrate

25.3 General ELISA Procedure

1. Coat the well with antigen or antibody.
2. Block nonspecific binding sites.
3. Add the test sample.
4. Allow antigen-antibody interaction.
5. Wash away unbound components.
6. Add enzyme-labelled conjugate.
7. Wash again.
8. Add enzyme substrate.
9. Observe or measure the colour reaction.

25.4 Types of ELISA

25.5 Direct ELISA

25.6 Indirect ELISA

25.7 Sandwich ELISA

25.8 Competitive ELISA

In a competitive assay, sample antigen competes with a reference or labelled antigen for limited antibody binding sites. Depending on the specific assay design, the signal may be inversely related to the concentration of the analyte.

25.9 Applications of ELISA

• Detection of antibodies against infectious agents.
• Detection of microbial or viral antigens.
• Hormone measurement.
• Allergy testing.
• Research immunoassays.
• Detection of specific proteins.
• Food allergen testing.
• Veterinary diagnosis.

26. Important 10-Mark Questions

27. Important 5-Mark Questions

• Explain the semi-discontinuous nature of DNA replication.
• Write a note on DNA polymerase.
• Differentiate leading and lagging strands.
• Explain Okazaki fragments.
• Write a note on RNA processing.
• Explain the wobble hypothesis.
• Describe the central dogma.
• Write a note on prokaryotic promoter.
• Explain bacterial RNA polymerase.
• Describe initiation of translation.
• Write a note on post-translational modification.
• Explain lac operon.
• Explain conventional PCR.
• Write a note on qPCR.
• Explain DNA fingerprinting.
• Write a note on gene cloning.
• Differentiate Southern, Northern and Western blotting.
• Explain indirect ELISA.
• Explain sandwich ELISA.

28. Important 2-Mark Questions

29. Quick Revision