Molecular Biology BZO6T15

Syllabus topics (RTMNU)

• DNA Structure: Chemical composition of DNA
• Watson and Crick double helix model
• Forms of DNA (A-DNA, B-DNA, Z-DNA)
• DNA as genetic material (Griffith's Transformation Experiment, Avery, MacLeod and McCarty's Experiment, Hershey and Chase's Experiment)
• RNA Structure: Chemical composition of RNA
• Types of RNA (mRNA, tRNA, rRNA)
• Structural features of tRNA
• RNA as genetic material with reference to the Fraenkel–Conrat experiment on Tobacco Mosaic Virus (TMV)
• Gene and Genome: General organization of the genome in prokaryotes and eukaryotes
• Structural organization of a typical eukaryotic gene (exons, introns, promoter region)
• Overview of genome size variation and the C-value paradox (basic concept only)
• Introduction to repetitive and non-repetitive DNA sequences
• Gene families (single-copy and multigene families), and pseudogenes
• Chromatin Organization: Nucleosome as the basic structural unit of chromatin
• Role of histone and non-histone proteins
• Types of chromatin - Euchromatin and heterochromatin
• Packaging of DNA into Higher Order Structures (Beads-on-a-String, 30 nm Fibre, Higher Order Tertiary Structures, Looped Domains)

1.1 DNA Structure

Chemical composition: DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides. Each nucleotide has three parts: (1) a pentose sugar — 2′-deoxyribose; (2) a phosphate group; (3) a nitrogenous base — purines adenine (A) and guanine (G), pyrimidines cytosine (C) and thymine (T). Sugar + base = nucleoside; nucleoside + phosphate = nucleotide. Nucleotides are joined by 3′→5′ phosphodiester bonds. Chargaff's rule: A = T and G = C, so purines = pyrimidines.

Watson and Crick double helix model (1953), based on X-ray data of Rosalind Franklin and Maurice Wilkins:

1. Two polynucleotide strands wound round a common axis as a right-handed double helix.
2. Strands are antiparallel (one 5′→3′, the other 3′→5′).
3. Sugar–phosphate backbone outside; bases inside, stacked like steps of a ladder.
4. Complementary base pairing: A=T with 2 hydrogen bonds, G≡C with 3 hydrogen bonds.
5. Diameter 2 nm (20 Å); one turn = 3.4 nm with about 10 base pairs; distance between base pairs 0.34 nm.
6. Has a major groove and a minor groove where proteins bind.

Forms of DNA:

DNA as genetic material:

• Griffith (1928) transformation experiment: Used Streptococcus pneumoniae — virulent S-strain (smooth, capsule) and non-virulent R-strain (rough). Live S killed mice; live R did not; heat-killed S did not; but heat-killed S + live R killed mice and live S bacteria were recovered. Some "transforming principle" from dead S changed R into S.
• Avery, MacLeod and McCarty (1944): Purified the extract of S cells and treated it with enzymes. Protease and RNase did not stop transformation, but DNase destroyed it. So the transforming principle is DNA.
• Hershey and Chase (1952): Used T2 bacteriophage on E. coli. Phage protein was labelled with ³⁵S and DNA with ³²P. After infection and blending, ³²P (DNA) entered the bacteria and appeared in new phages, while ³⁵S (protein) stayed outside in the coats. So DNA is the genetic material.

1.2 RNA Structure

RNA is usually single-stranded; its sugar is ribose (has 2′-OH) and it has uracil (U) in place of thymine. It may fold into hairpins by internal base pairing.

Types: mRNA — carries the code from DNA to ribosome (eukaryotic mRNA has 5′ cap and poly-A tail); tRNA — brings amino acids, smallest (≈75–90 nucleotides, 4S); rRNA — structural and catalytic part of ribosome, most abundant (≈80%).

Structure of tRNA (Holley's clover-leaf model; L-shaped in 3-D):

• Acceptor arm: 3′ end ends in CCA; amino acid attaches to the terminal A.
• DHU (D) arm: has dihydrouridine; binds aminoacyl-tRNA synthetase.
• Anticodon arm: carries the three-base anticodon that pairs with mRNA codon.
• Variable arm (extra arm).
• TψC arm: has thymine and pseudouridine; binds ribosome.

RNA as genetic material — Fraenkel-Conrat (1956): Tobacco Mosaic Virus (TMV) has RNA and protein. He separated RNA and protein of two strains (TMV and HR/Holmes ribgrass) and made hybrid viruses (RNA of one + protein coat of the other). Progeny viruses always had the protein type of the RNA donor. So in TMV, RNA is the genetic material.

1.3 Gene and Genome

Prokaryotic genome: usually a single, circular, double-stranded DNA in the nucleoid, not enclosed by a membrane; little non-coding DNA; genes often in operons; extra plasmids. Eukaryotic genome: several linear chromosomes inside the nucleus, packed with histones; large amount of non-coding and repetitive DNA; also mitochondrial (and chloroplast) DNA.

Typical eukaryotic gene: promoter (TATA box about 25–30 bp upstream, CAAT box, GC box) → transcription start site → 5′ UTR → exons (coding) interrupted by introns (non-coding, removed by splicing) → 3′ UTR with poly-A signal (AAUAAA). Enhancers and silencers may lie far away.

C-value paradox: C-value is the amount of DNA in a haploid genome. Genome size does not match organism complexity — e.g. some amphibians and lungfish have far more DNA than humans. Explained mainly by differences in non-coding, repetitive DNA.

Repetitive and non-repetitive DNA: unique (single-copy) sequences contain most genes; moderately repetitive (e.g. rRNA genes, transposons like LINEs and SINEs/Alu); highly repetitive (satellite DNA at centromeres, minisatellites, microsatellites). Gene families: single-copy genes vs multigene families (globin family, histone genes). Pseudogenes: non-functional copies of genes that have lost the ability to make protein (e.g. processed pseudogenes without introns).

1.4 Chromatin Organization

Nucleosome (Kornberg, 1974) is the basic unit: about 146–147 bp of DNA wound 1.65 turns around a histone octamer (2 each of H2A, H2B, H3, H4); H1 binds linker DNA. Histones are small basic proteins rich in lysine and arginine; non-histone proteins include scaffold proteins, enzymes (DNA/RNA polymerases) and transcription factors.

Levels of packaging: (1) Beads-on-a-string (11 nm fibre — primary); (2) 30 nm fibre — solenoid formed with H1 (secondary); (3) looped domains of 30 nm fibre attached to a non-histone protein scaffold (about 300 nm, tertiary); (4) further coiling into the condensed metaphase chromosome (about 700 nm chromatid; 1400 nm chromosome) (quaternary).

Likely exam questions

1. (Long, 8 marks) Describe the Watson and Crick model of DNA. Add a note on the A, B and Z forms of DNA.
2. (Long, 8 marks) Describe the experiments that proved DNA is the genetic material (Griffith; Avery, MacLeod and McCarty; Hershey and Chase).
3. (Short, 4 marks) Draw and describe the clover-leaf structure of tRNA.
4. (Short, 4 marks) What is the C-value paradox?
5. (Short, 4 marks) Distinguish between euchromatin and heterochromatin.

Syllabus topics (RTMNU)

• DNA Replication: Semi-conservative model of DNA replication
• Basic steps of replication in prokaryotes—initiation, elongation, and termination
• Concept of origin of replication and replication fork
• Enzymes involved in DNA replication in E. coli, including DNA polymerases, helicase, primase, DNA ligase, topoisomerase, and single-strand binding proteins
• Overview of rolling circle replication (introductory concept)
• Mechanism of DNA replication in eukaryotes—initiation at multiple origins, major eukaryotic DNA polymerases (basic functions only), replication through chromatin, and the end-replication problem
• Concept of telomeres and role of telomerase in chromosome maintenance
• DNA Damage and Repair: Types and causes of DNA damage, including replication errors and damage due to physical and chemical agents
• Mechanisms of DNA repair, including proofreading during replication, direct repair, base excision repair, nucleotide excision repair, mismatch repair, and recombinational repair
• Recombination: Types of Recombination
• Holliday model for Homologous recombination
• Site-specific recombination in Eukaryotes and its Biological Significance
• Regulation of DNA Replication: origin recognition, initiation control mechanisms, and coordination with the cell cycle
• Role of telomeres and telomerase in maintaining chromosome stability
• Introduction to cell cycle checkpoints and their significance

2.1 DNA Replication

Semi-conservative model: each new DNA molecule has one old (parental) strand and one newly made strand. Proved by Meselson and Stahl (1958) using ¹⁵N and ¹⁴N and CsCl density gradient centrifugation in E. coli: after one generation all DNA was of hybrid (intermediate) density; after two generations, half hybrid and half light.

Replication in prokaryotes (E. coli):

1. Initiation: begins at a single origin of replication (oriC), rich in AT pairs. DnaA protein binds and opens the DNA; helicase (DnaB) unwinds it, forming two replication forks (bidirectional replication, forming a "theta" structure). Single-strand binding (SSB) proteins keep strands apart. Topoisomerase (DNA gyrase) removes supercoils ahead of the fork.
2. Elongation: Primase makes a short RNA primer. DNA polymerase III adds nucleotides only in the 5′→3′ direction. The leading strand is made continuously; the lagging strand is made discontinuously as Okazaki fragments. DNA polymerase I removes RNA primers (5′→3′ exonuclease) and fills gaps; DNA ligase joins the fragments.
3. Termination: forks meet at the ter region, where Tus protein stops helicase; topoisomerase IV separates the two daughter circles.

Rolling circle replication: seen in some phages (e.g. φX174, λ late stage) and plasmids during conjugation. One strand of the circular DNA is nicked; the free 3′-OH end is extended while the 5′ end is displaced, so the circle "rolls" and gives long linear copies.

Replication in eukaryotes: starts at many origins (replicons) on each chromosome, because DNA is long and the fork is slow. Main polymerases: Pol α (with primase — makes primer and short DNA), Pol δ (lagging strand), Pol ε (leading strand), Pol γ (mitochondrial DNA), Pol β (repair). Nucleosomes are removed ahead of the fork and re-assembled behind it with old and new histones. End-replication problem: after removal of the last primer at the end of the lagging strand, a gap remains that cannot be filled, so linear chromosomes get shorter at each division.

Telomeres and telomerase: telomeres are repeated sequences at chromosome ends (TTAGGG in humans) that protect ends. Telomerase is a ribonucleoprotein reverse transcriptase that carries its own RNA template and adds telomere repeats to the 3′ end. It is active in germ cells, stem cells and most cancer cells.

2.2 DNA Damage and Repair

Types and causes of damage: (1) Replication errors — wrong base insertion (mismatches); (2) physical agents — UV light forms thymine dimers; X-rays and gamma rays cause strand breaks; (3) chemical agents — deamination (C→U), alkylating agents, base analogues, intercalating dyes; spontaneous depurination.

1. Proofreading: 3′→5′ exonuclease of DNA polymerase removes a wrongly added nucleotide immediately.
2. Direct repair: damage is reversed without removing the base — photoreactivation by photolyase splits thymine dimers using visible light; methyltransferase removes alkyl groups.
3. Base excision repair (BER): DNA glycosylase removes the damaged base → AP endonuclease cuts → DNA polymerase fills → ligase seals.
4. Nucleotide excision repair (NER): a short stretch with bulky damage (thymine dimer) is cut out (UvrABC in E. coli) and resynthesised. Defect in humans causes xeroderma pigmentosum.
5. Mismatch repair: MutS, MutL, MutH recognise the newly made strand (unmethylated GATC in E. coli) and replace the wrong base.
6. Recombinational repair: double-strand breaks or gaps are repaired using the sister chromatid/homologous DNA as template.

2.3 Recombination

Recombination is the exchange of genetic material between DNA molecules. Types: homologous (between similar sequences, e.g. crossing over in meiosis), site-specific (at short specific sequences, by recombinase enzymes), and transposition (movement of transposable elements).

Holliday model (1964) of homologous recombination:

1. Homologous DNA duplexes line up.
2. A single-strand nick is made at the same place in one strand of each.
3. Free strands invade the other duplex and pair (strand exchange); ligation forms a cross-shaped Holliday junction.
4. Branch migration moves the junction, forming heteroduplex DNA.
5. The junction is resolved by cutting: cut in one plane gives patched (non-recombinant flanking) products; in the other plane gives spliced (recombinant) products.

Site-specific recombination in eukaryotes and significance: examples include V(D)J recombination of immunoglobulin and T-cell receptor genes (gives antibody diversity), yeast mating-type switching, and the FLP/FRT system of yeast (used, like Cre/lox of phage P1, as a genetic engineering tool). Significance: genetic variation, antibody diversity, gene regulation, integration of viruses, and tools for making transgenic animals.

2.4 Regulation of DNA Replication

DNA must be copied once and only once per cell cycle.

• Origin recognition: in eukaryotes the Origin Recognition Complex (ORC) marks origins.
• Licensing in G1: Cdc6 and Cdt1 load the MCM helicase to form the pre-replicative complex — the origin is "licensed".
• Initiation control in S phase: S-phase CDKs and DDK activate MCM; replication starts. High CDK activity and the protein geminin then block re-licensing, preventing re-replication.
• In E. coli, initiation is controlled by the amount of DnaA-ATP and by SeqA, which binds newly made hemimethylated oriC to stop immediate re-initiation.
• Telomeres/telomerase protect chromosome ends and maintain stability.
• Cell-cycle checkpoints: G1/S (is DNA damaged? is the cell large enough?), intra-S, G2/M (is DNA fully and correctly replicated?) and spindle checkpoint. Proteins like p53, ATM/ATR kinases stop the cycle to allow repair or cause apoptosis — this prevents mutations and cancer.

Likely exam questions

1. (Long, 8 marks) Describe the mechanism of DNA replication in prokaryotes with the role of the enzymes involved.
2. (Long, 8 marks) Explain the different mechanisms of DNA repair.
3. (Short, 4 marks) What is the end-replication problem? How does telomerase solve it?
4. (Short, 4 marks) Explain the Holliday model of recombination.
5. (Short, 4 marks) What are cell-cycle checkpoints?

Syllabus topics (RTMNU)

• Transcription in Prokaryotes: Structure and function of RNA polymerase in prokaryotes
• Role of sigma (σ) factor in promoter recognition
• Promoter sequences and structural organization of genes
• Stages of transcription—initiation, elongation, and termination
• Basic concept of transcription bubble
• Overview of inhibitors of transcription and their applications
• Transcription in Eukaryotes: Types of eukaryotic RNA polymerases (RNA polymerase I, II, and III) and their functions
• Role of promoters and transcription factors in initiation of transcription
• Basic steps of transcription by RNA polymerase II—initiation, elongation, and termination
• Translation in Prokaryotes and Eukaryotes: Structure and function of ribosomes
• Role of mRNA, tRNA, and rRNA in protein synthesis
• Stages of translation—initiation, elongation, and termination in prokaryotes and eukaryotes (basic comparison)
• Overview of inhibitors of protein synthesis and their significance
• Genetic Code: Characteristics of genetic code
• Codon–anticodon interaction
• Wobble hypothesis
• Universality and known exceptions of the genetic code

3.1 Transcription in Prokaryotes

Transcription is the synthesis of RNA from a DNA template. Only one strand (the template / antisense strand, read 3′→5′) is copied; RNA grows 5′→3′. The other strand is the coding / sense strand.

RNA polymerase of E. coli: a single enzyme makes all RNAs. Core enzyme = α₂ββ′ω; holoenzyme = core + sigma (σ) factor. β and β′ form the catalytic site; α helps assembly and binds some activators. The σ factor (σ⁷⁰) recognises the promoter and helps start transcription, then leaves.

Promoter sequences: −10 box (Pribnow box, TATAAT) and −35 box (TTGACA) upstream of the start site (+1). A prokaryotic gene unit = promoter → transcription start → coding region → terminator.

1. Initiation: holoenzyme binds the promoter (closed complex), unwinds about 12–14 bp to form an open complex — the transcription bubble; first nucleotides join without a primer; after ~10 nucleotides σ is released (promoter clearance).
2. Elongation: core enzyme moves along, unwinding DNA ahead and rewinding behind; an RNA–DNA hybrid of about 8 bp exists inside the bubble.
3. Termination: (a) Rho-independent — a GC-rich inverted repeat forms a hairpin in RNA followed by a string of U's, causing release; (b) Rho-dependent — Rho protein (a helicase) binds RNA, moves to the polymerase and releases the transcript.

Inhibitors of transcription: rifampicin binds the β subunit of bacterial RNA polymerase and blocks initiation — used against tuberculosis and leprosy; actinomycin D intercalates in DNA and blocks elongation (used in cancer and research); α-amanitin (death cap mushroom toxin) blocks eukaryotic RNA pol II. They are used as drugs and as research tools to study gene expression.

3.2 Transcription in Eukaryotes

Promoters and transcription factors: RNA pol II promoters have a core promoter (TATA box at about −25 to −30, initiator) and upstream elements (CAAT box, GC box). RNA pol II cannot bind by itself; general transcription factors (TFIIA, B, D, E, F, H) are needed. TFIID, through its TATA-binding protein (TBP), binds the TATA box first.

1. Initiation: TFIID binds TATA → TFIIA and TFIIB join → RNA pol II with TFIIF → TFIIE and TFIIH join to make the pre-initiation complex. TFIIH unwinds DNA and phosphorylates the C-terminal domain (CTD) of pol II, releasing it to start.
2. Elongation: phosphorylated pol II moves on with elongation factors; the RNA is processed at the same time — 5′ capping (7-methylguanosine cap), splicing of introns by spliceosome.
3. Termination: after the poly-A signal (AAUAAA) is copied, the RNA is cut and poly-A tail (≈200 A) is added; pol II stops some distance later.

3.3 Translation in Prokaryotes and Eukaryotes

Ribosomes: prokaryotic 70S (50S + 30S; rRNAs 23S, 5S and 16S); eukaryotic 80S (60S + 40S; rRNAs 28S, 5.8S, 5S and 18S). Each ribosome has three tRNA sites: A (aminoacyl), P (peptidyl) and E (exit). The large subunit has peptidyl transferase activity (a ribozyme — the rRNA catalyses it).

Roles: mRNA carries codons; tRNA brings specific amino acids (charged by aminoacyl-tRNA synthetase using ATP) and reads codons with anticodons; rRNA forms the ribosome and catalyses peptide bonds.

1. Initiation: prokaryotes — 30S subunit binds mRNA at the Shine–Dalgarno sequence (pairs with 16S rRNA); initiator tRNA carries N-formylmethionine (fMet); factors IF1, IF2, IF3. Eukaryotes — 40S binds the 5′ cap and scans to the first AUG (Kozak sequence); initiator tRNA carries methionine; many eIFs.
2. Elongation: aminoacyl-tRNA enters A site (EF-Tu / eEF1) → peptide bond formed by peptidyl transferase → translocation by one codon (EF-G / eEF2), moving tRNAs from A→P→E.
3. Termination: a stop codon (UAA, UAG, UGA) enters A site; release factors (RF1, RF2, RF3 in prokaryotes; eRF1, eRF3 in eukaryotes) free the polypeptide; subunits separate.

Inhibitors of protein synthesis: streptomycin (binds 30S, causes misreading), tetracycline (blocks A site in 30S), chloramphenicol (inhibits peptidyl transferase of 50S), erythromycin (blocks translocation on 50S), puromycin (looks like aminoacyl-tRNA, causes early chain release — both types), cycloheximide (blocks eukaryotic 80S), diphtheria toxin (inactivates eEF2). Significance: many are antibiotics that selectively kill bacteria because their ribosomes differ from ours; they are also research tools.

3.4 Genetic Code

Genetic code is the relationship between the base sequence of mRNA and the amino acid sequence of protein. Deciphered by Nirenberg, Matthaei, Khorana and others.

Characteristics:

1. Triplet: three bases = one codon; 64 codons.
2. Degenerate: most amino acids have more than one codon (61 sense codons for 20 amino acids).
3. Non-overlapping and comma-less: read continuously, one codon after another.
4. Unambiguous: one codon codes for only one amino acid.
5. Start codon: AUG (methionine); stop codons: UAA (ochre), UAG (amber), UGA (opal).
6. Polarity: read 5′→3′.
7. Nearly universal in all organisms.

Codon–anticodon interaction: anticodon of tRNA pairs antiparallel with the codon (e.g. codon 5′-GCU-3′ pairs with anticodon 3′-CGA-5′).

Wobble hypothesis (Crick, 1966): the first two bases of a codon pair strictly, but the third base (3′ end of codon) pairs loosely with the first base of the anticodon. E.g. G in anticodon can pair with C or U; inosine (I) can pair with U, C or A. So fewer tRNAs (about 32 minimum) can read all 61 codons, explaining degeneracy.

Exceptions to universality: in mitochondria UGA codes for tryptophan (not stop), AUA for methionine in human mitochondria, AGA/AGG are stop codons in vertebrate mitochondria; in some ciliates (e.g. Paramecium, Tetrahymena) UAA and UAG code for glutamine; in Mycoplasma UGA codes for tryptophan.

Likely exam questions

1. (Long, 8 marks) Describe the process of transcription in prokaryotes. Add a note on inhibitors of transcription.
2. (Long, 8 marks) Describe the mechanism of translation and compare it in prokaryotes and eukaryotes.
3. (Short, 4 marks) Write the characteristics of the genetic code.
4. (Short, 4 marks) Explain the wobble hypothesis.
5. (Short, 4 marks) Name the three eukaryotic RNA polymerases and their products.

Syllabus topics (RTMNU)

• Regulation of Gene Expression in Prokaryotes and Eukaryotes: Organization of bacterial operons with reference to the Lac operon and Tryptophan operon
• Concept of attenuation
• Regulation of gene expression in eukaryotes, including transcription factors, activators and repressors, enhancers and silencers, and chromatin remodeling
• Basic Techniques in Molecular Biology: Principle and applications of spectrophotometry
• Types of centrifugation (differential and density gradient centrifugation)
• Introduction to ultracentrifugation
• Basic concept of X-ray diffraction (DNA structure determination) and agarose gel electrophoresis for separation of nucleic acids
• Nucleic Acid and Protein Analysis Techniques: Principle and applications of blotting techniques—Southern, Northern, and Western blotting
• Basic concept of ELISA and immunodetection methods
• Introduction to hybridoma technology and production of monoclonal antibodies
• Modern Molecular Techniques and Applications: Principle and applications of PCR
• Basic concept of DNA sequencing and molecular DNA markers (RFLP, RAPD, SNPs)
• Introduction to DNA fingerprinting and its applications

4.1 Regulation of Gene Expression in Prokaryotes

An operon (Jacob and Monod, 1961) is a group of structural genes under the control of a single promoter and operator, transcribed together as one polycistronic mRNA. Parts: regulator gene (makes repressor), promoter, operator (where repressor binds) and structural genes.

Lac operon of E. coli (inducible, negative control): structural genes lacZ (β-galactosidase — splits lactose into glucose and galactose), lacY (permease — lactose entry) and lacA (transacetylase); regulator lacI makes the repressor.

• No lactose: active repressor binds the operator → RNA polymerase is blocked → genes "off".
• Lactose present: allolactose (inducer) binds the repressor, changing its shape so it leaves the operator → transcription "on".
• Positive control (catabolite repression): when glucose is low, cAMP rises and binds CAP (CRP); the cAMP–CAP complex binds near the promoter and strongly increases transcription. If glucose is present, cAMP is low and the operon stays weak even with lactose.

Trp operon (repressible): five genes (trpE, D, C, B, A) make enzymes for tryptophan synthesis. The repressor made by trpR is inactive alone. When tryptophan is high, tryptophan acts as a co-repressor, binds the repressor, and the complex binds the operator → genes "off". When tryptophan is low, genes are "on".

Attenuation (Yanofsky): a second control in the trp operon. The leader region (trpL) has two Trp codons and four segments that can form different hairpins. When tryptophan is plentiful, the ribosome moves quickly past the Trp codons and a 3–4 terminator hairpin forms → transcription stops early. When tryptophan is scarce, the ribosome stalls at the Trp codons, a 2–3 anti-terminator forms → transcription continues. This works because transcription and translation are coupled in bacteria.

Regulation of Gene Expression in Eukaryotes

• Transcription factors: general factors (TFII family) plus specific factors that bind DNA through motifs like zinc finger, helix-turn-helix, leucine zipper.
• Activators and repressors: activators bind DNA and recruit the transcription machinery or co-activators; repressors block activators or recruit co-repressors.
• Enhancers: DNA sequences that increase transcription; they can work far away (thousands of bp), upstream or downstream, in either orientation, by DNA looping (via Mediator complex). Silencers reduce transcription.
• Chromatin remodeling: ATP-dependent complexes (SWI/SNF) slide or remove nucleosomes; histone acetylation (by HATs) loosens chromatin and activates genes, deacetylation (HDACs) closes it; DNA methylation at CpG islands usually silences genes.
• Other levels: RNA processing (alternative splicing), mRNA stability, microRNAs, translation and protein modification.

4.2 Basic Techniques in Molecular Biology

Spectrophotometry: based on the Beer–Lambert law — absorbance (A) = ε·c·l, i.e. absorbance is proportional to concentration. Nucleic acids absorb at 260 nm, proteins at 280 nm; A₂₆₀/A₂₈₀ ≈ 1.8 shows pure DNA. Uses: measuring concentration and purity of DNA/protein, enzyme kinetics, bacterial growth (OD at 600 nm). Hyperchromic effect: absorbance rises when DNA denatures.

Centrifugation: separation of particles by spinning, based on size, shape and density (sedimentation coefficient in Svedberg units, S). Differential centrifugation: successive spins at increasing speed separate nuclei, then mitochondria, then microsomes. Density gradient centrifugation: (a) rate-zonal (sucrose gradient, by size); (b) isopycnic (CsCl gradient, by buoyant density — used by Meselson and Stahl). Ultracentrifugation: very high speeds (up to about 1,00,000 rpm or more), in vacuum and cooled chambers (Svedberg) — used to separate ribosomes, macromolecules and to find molecular weight.

X-ray diffraction: X-rays passing through a crystal or fibre are scattered into a pattern that reveals atomic arrangement. Rosalind Franklin's Photo 51 of B-DNA showed an X-shaped pattern, indicating a helix with 3.4 Å base spacing and 34 Å repeat — basis of the Watson–Crick model.

Agarose gel electrophoresis: DNA is negatively charged (phosphate groups), so in an electric field it moves towards the anode (+) through agarose gel; small fragments move faster. A DNA ladder (marker) gives sizes; DNA is seen with ethidium bromide or safer dyes under UV. Uses: checking PCR products, restriction digests, DNA fingerprinting.

4.3 Nucleic Acid and Protein Analysis Techniques

Steps (Southern): digest DNA with restriction enzymes → agarose gel electrophoresis → denature to single strands → transfer (blot) onto a nitrocellulose/nylon membrane → hybridise with a labelled probe → detect by autoradiography or chemiluminescence. Uses: gene mapping, RFLP, DNA fingerprinting; Northern — gene expression; Western — detection of viral proteins (e.g. confirmation tests).

ELISA (Enzyme-Linked Immunosorbent Assay): antigen or antibody is detected by an enzyme-linked antibody that gives a coloured product with its substrate. Types: direct, indirect, sandwich. Uses: HIV, hepatitis, dengue tests, pregnancy tests. Immunodetection methods use specific antibodies to locate proteins in tissues (immunohistochemistry) or on blots.

Hybridoma technology (Köhler and Milstein, 1975): immunise a mouse with antigen → take antibody-producing B cells from spleen → fuse with myeloma cells using PEG → select hybrids in HAT medium (unfused myeloma cells die, B cells die naturally) → screen clones for the desired antibody → grow the selected clone to get monoclonal antibodies. Uses: diagnosis, cancer therapy, blood typing, research.

4.4 Modern Molecular Techniques and Applications

PCR (Kary Mullis, 1983): makes millions of copies of a DNA segment in a test tube. Needs template DNA, two primers, dNTPs, Mg²⁺ and heat-stable Taq polymerase (from Thermus aquaticus). Each cycle: denaturation (≈94–95 °C), annealing of primers (≈50–65 °C), extension (≈72 °C). DNA doubles every cycle (2ⁿ; 30 cycles ≈ a billion copies). Uses: diagnosis (COVID-19 RT-PCR), forensics, cloning, paternity tests.

DNA sequencing: finding the order of bases. Sanger's dideoxy (chain termination) method uses ddNTPs that lack 3′-OH and stop the chain; fragments are separated by size to read the sequence. Next-generation sequencing reads millions of fragments together (Human Genome Project completed 2003).

Molecular DNA markers: RFLP — differences in fragment lengths after restriction digestion, detected by Southern blot; RAPD — PCR with short random primers giving polymorphic bands; SNPs — single-base differences between individuals, the most common markers. Uses: genetic mapping, disease diagnosis, breeding, evolution studies.

DNA fingerprinting (Alec Jeffreys, 1984): based on variable number tandem repeats (VNTRs/minisatellites) and STRs that differ between individuals. Steps: DNA isolation → restriction digestion (or PCR of STRs) → electrophoresis → Southern blotting → hybridisation with VNTR probe → autoradiography → band pattern. In India, Dr. Lalji Singh (CCMB, Hyderabad) pioneered it. Applications: crime investigation, paternity disputes, identifying disaster victims, wildlife forensics, pedigree analysis.

Likely exam questions

1. (Long, 8 marks) Describe the lac operon model of gene regulation in E. coli.
2. (Long, 8 marks) Explain the principle, steps and applications of PCR and DNA fingerprinting.
3. (Short, 4 marks) What is attenuation in the trp operon?
4. (Short, 4 marks) Distinguish between Southern, Northern and Western blotting.
5. (Short, 4 marks) How are monoclonal antibodies produced by hybridoma technology?