Study sheet: BIO131 Nucleic Acids and Molecular Biology

📋 Course Outline

1. BIO131 Assessment and Practical Requirements
2. Nucleic Acid Discovery and Central Dogma
3. Nucleotide Chemistry and DNA Structure
4. Genomes Chromatin and RNA Diversity
5. DNA Melting and Spectrophotometry
6. DNA Replication and PCR
7. Transcription and Translation
8. Libraries and Molecular Cloning
9. Restriction Enzymes and Cloning Vectors
10. Electrophoresis Blotting and Sequencing

📅 Key Dates

1. 1869J. F. Miescher discovered nuclein, composed of DNA and associated proteins extracted from lymph-node cell nuclei — J.F. Miescher, 1869
2. 1952Avery, Hershey, and Chase established that DNA carries genetic information — Avery, Hershey and Chase, 1952
3. 1953Watson, Crick, Wilkins, with the contribution of Rosalind Franklin’s work, described the DNA double helix — D. Watson, F. Crick, M. Wilkind (R. Franklin), 1953

📖 1. BIO131 Assessment and Practical Requirements

★ Must-know

• BIO131 comprises 14 lectures, nine three-hour tutorial sessions, and two four-hour practical sessions on nucleic-acid and protein electrophoresis.

📌 Students must attend all lectures, tutorials, and practical sessions, and any unjustified absence prevents validation of the module, semester, and year.

• The practical sessions contribute 30% of the final module grade, the week-43 one-hour multiple-choice test contributes 20%, and the week-51 two-hour final examination contributes 50%.

Further detail

📌 Students must bring their practical booklet to each practical-session preparation and bring their own lab coat to practical sessions.

📖 2. Nucleic Acid Discovery and Central Dogma

📝 Essential Points

• 🔄 The information-flow sequence is (F. Crick, 1970):
  1. DNA replication
  2. DNA transcription into RNA
  3. RNA translation into protein

💡 Memory Hook

DNA replication → RNA transcription → protein translation

📖 3. Nucleotide Chemistry and DNA Structure

🔑 Key Concepts & Definitions

• Nucleotide : composed of a pentose sugar, a nitrogenous base, and one or more phosphate groups
• Nucleoside : formed by a pentose sugar covalently linked to a nitrogenous base through an N-glycosidic bond
• Phosphodiester bond : links the 3′ hydroxyl of one pentose to the 5′ hydroxyl of the following pentose and gives nucleic-acid strands their polarity

📝 Essential Points

• DNA contains adenine, thymine, guanine, and cytosine, whereas RNA contains adenine, uracil, guanine, and cytosine.

• DNA strands are antiparallel and DNA polymerization proceeds from 5′ to 3′ by adding nucleotides to a 3′ hydroxyl end.

💡 Memory Hook

Nucleoside = sugar + base; nucleotide = nucleoside + phosphate

📖 4. Genomes Chromatin and RNA Diversity

📝 Essential Points

• Human cells contain 23 chromosome pairs and approximately 3.3 × 10⁹ base pairs, whereas Escherichia coli has one circular chromosome of approximately 4 × 10⁶ base pairs and 4,288 genes.

• In animal eukaryotic cells, nuclear DNA is organized into linear chromosomes, whereas mitochondrial DNA is circular; plant cells also contain circular chloroplast DNA.

• Eukaryotic DNA is compacted by wrapping 146 base pairs around a histone octamer to form nucleosomes, which organize into chromatin fibers and chromosomes.

• The main cytoplasmic RNA categories are:

  • rRNA
  • tRNA
  • mRNA

💡 Memory Hook

A two-metre DNA thread folded into nucleosomes and chromosomes

📖 5. DNA Melting and Spectrophotometry

🔑 Key Concepts & Definitions

• Melting temperature : the temperature at which 50% of DNA molecules are single stranded

📝 Essential Points

📐 Formula — Beer–Lambert law relates absorbance to concentration as $A_\lambda = \varepsilon_\lambda l c$.

• Nucleic acids are quantified at 260 nm, while protein contamination is assessed using the A260/A280 ratio.

📌 A pure DNA solution has an A260/A280 ratio of 1.8–2; a ratio below 1.7 indicates protein contamination, whereas a ratio above 2 indicates RNA contamination.

💡 Memory Hook

Heat breaks hydrogen bonds → DNA strands separate → absorbance increases

📖 6. DNA Replication and PCR

📝 Essential Points

• DNA replication is semiconservative and bidirectional from one origin of replication, producing two replication forks.

📌 DNA polymerases add complementary nucleotides only to a primer-bound template at a 3′ hydroxyl end, so newly synthesized DNA grows from 5′ to 3′.

• The lagging strand is synthesized as 150–200-nucleotide Okazaki fragments that begin with short RNA primers and are joined by DNA ligase.

• PCR uses a thermostable Taq polymerase, two primers, and repeated thermal cycles to amplify a target DNA sequence; theoretically, 20 cycles produce 2²⁰, or approximately 10⁶, copies of the initial duplex.

💡 Memory Hook

Origin → fork → primer → polymerase → Okazaki fragments → ligation

📖 7. Transcription and Translation

📝 Essential Points

• Transcription copies the DNA template strand into RNA in the 5′ to 3′ direction, producing an RNA sequence identical to the coding strand except that RNA contains U instead of T.

• Eukaryotes use RNA polymerase I for rRNA genes, RNA polymerase II for mRNA genes, and RNA polymerase III for tRNA genes.

• Eukaryotic pre-mRNA is stabilized by addition of a 5′ cap, cleavage and addition of an approximately 200-adenine poly(A) tail, and splicing of introns when present.

• Translation occurs in the cytoplasm in three major stages: initiation at an AUG codon, elongation by codon reading and amino-acid addition, and termination at a stop codon.

💡 Memory Hook

Promoter → RNA → mRNA processing → ribosome → polypeptide

📖 8. Libraries and Molecular Cloning

★ Must-know

📌 A genomic library represents the entire genome, whereas a cDNA library represents only expressed transcripts converted from fully transcribed mRNAs.

• To clone a gene, the gene is isolated, inserted into a cloning vector, replicated in a host organism, and analyzed.

Further detail

• Genetic engineering methods allow:
  • Creation of genetically modified organisms
  • Study of genes and their products
  • Diagnosis of genetic illnesses
  • Gene therapy

💡 Memory Hook

Genomic library represents the whole genome; cDNA library represents transcripts

📖 9. Restriction Enzymes and Cloning Vectors

🔑 Key Concepts & Definitions

• Restriction enzyme : a restriction endonuclease that cleaves both DNA strands at or near a specific recognition site
• Plasmid : a small circular, double-stranded, autonomous, extrachromosomal bacterial DNA molecule that can be used as a cloning vector

★ Must-know

• 🔄 The main cloning sequence is:
  1. Restriction digestion
  2. Ligation
  3. Bacterial transformation
  4. Selection
  5. Amplification

Further detail

• Mandatory vector components include:
  • An origin of DNA replication
  • One or two resistance genes
  • A multiple cloning site

💡 Memory Hook

Cut → ligate → transform → select → amplify

📖 10. Electrophoresis Blotting and Sequencing

★ Must-know

• Agarose-gel electrophoresis separates DNA fragments primarily according to their size, and a molecular-weight marker provides fragments of defined lengths for calibration.

• Southern blotting transfers separated DNA fragments to a membrane and detects a complementary sequence by hybridization with a labeled probe.

• Sanger sequencing verifies a cloned insert by detecting fluorescence signals from incorporated chain-terminating nucleotides.

Further detail

• The principal enzyme activities are:
  • Polymerase
  • Exonuclease
  • Endonuclease
  • Ligase
  • Phosphatase
  • Kinase

📊 Synthesis Tables

Genomic and cDNA Libraries

Feature	Genomic library	cDNA library
Source	Genomic DNA	Fully transcribed mRNAs
Content	Whole genome, including non-transcribed regions	Expressed genes only
Preparation	Genomic DNA fragmentation and cloning	Reverse transcription followed by cDNA cloning