DNA Revision
& Central Dogma

DNA (deoxyribonucleic acid) is the molecule that stores genetic information in all living cells. Understanding its structure is the foundation for everything in molecular biology. Think of DNA as a twisted ladder — the sides are the sugar-phosphate backbone, and the rungs are pairs of nitrogenous bases held together by hydrogen bonds.

The Nucleotide — The Building Block

DNA is a polymer of repeating units called nucleotides. Each nucleotide has three components: (1) a phosphate group (PO₄), (2) a 5-carbon sugar called deoxyribose, and (3) a nitrogenous base. The four bases are: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). Nucleotides are linked by alternating sugar-phosphate bonds to form a long chain.

Figure 1: Structure of nucleic acids — each nucleotide = phosphate (P) + sugar + base. Nucleotides are linked by sugar-phosphate bonds to form a polymer chain.

Base Pairing Rules (Chargaff's Rules)

The two strands of DNA are held together by hydrogen bonds between complementary bases: A pairs with T (2 hydrogen bonds) and G pairs with C (3 hydrogen bonds). The strands run in opposite directions (5'→3' and 3'→5') — they are antiparallel.

DNA vs RNA — Key Differences

The Central Dogma describes the flow of genetic information: DNA is transcribed into mRNA (in the nucleus), and mRNA is translated into protein (at ribosomes in the cytoplasm). Every protein your body makes — enzymes, structural proteins, hormones, antibodies — is produced by this pipeline.

Figure 2: The Central Dogma — DNA is transcribed into mRNA in the nucleus; mRNA exits to the cytoplasm where it is translated into protein.

Before a cell divides, it must copy all of its DNA so each daughter cell gets a complete set. Replication is semi-conservative — each new double helix contains one original (parent) strand and one newly synthesised (daughter) strand.

Key Enzymes and Their Roles

Leading vs Lagging Strand

• Leading strand: Synthesised continuously in the same direction as fork movement. One primer needed.
• Lagging strand: Synthesised discontinuously as Okazaki fragments (opposite to fork). Each fragment needs its own primer. Ligase joins them.

Transcription copies a gene's DNA sequence into mRNA. It occurs in the nucleus and is carried out by RNA polymerase.

Figure 3: Transcription — RNA polymerase reads the template strand (3'→5') and synthesises mRNA (5'→3'). The coding strand has the same sequence as mRNA (T→U).

Key Concepts

• Template strand (antisense): Read by RNA polymerase, runs 3'→5'. Complementary to mRNA.
• Coding strand (sense): Same sequence as mRNA (except T not U), runs 5'→3'. NOT read by polymerase.
• Base pairing: A(DNA)→U(RNA), T(DNA)→A(RNA), G→C, C→G. T in DNA becomes U in RNA.
• Three Stages: Initiation (polymerase binds promoter), Elongation (mRNA built 5'→3'), Termination (reaches terminator, detaches).

In eukaryotes, pre-mRNA must be processed before leaving the nucleus. Eukaryotic genes contain exons (coding, EXpressed) and introns (non-coding, INterruptions).

Figure 4: mRNA splicing — introns removed, exons joined to form mature mRNA that exits to ribosomes.

Three Modifications to Pre-mRNA

• 5' Cap: Modified guanine added to 5' end. Protects from degradation; required for ribosome recognition.
• 3' Poly-A Tail: ~100-250 adenines added to 3' end. Protects from degradation; aids nuclear export.
• Splicing: Spliceosome removes introns, joins exons. Mature mRNA = exons only.

Translation reads mRNA and assembles amino acids into a polypeptide at ribosomes in the cytoplasm. mRNA is read in codons (3-nucleotide groups), each specifying one amino acid.

Figure 5: Translation stages — Initiation (ribosome binds at AUG), Elongation (amino acids added), Termination (stop codon reached, polypeptide released).

Key Players

Three Stages

Initiation: Ribosome finds AUG, first tRNA binds. Elongation: Ribosome moves 5'→3', tRNAs deliver amino acids, peptide bonds form. Termination: Stop codon → release factors → polypeptide released.

Example 1 — MARGARITA (from lecture)

DNA coding strand: 5'-ATGGCCCGAGGGGCTCGCATAACAGCG-3'
Step 1 — Transcribe (T→U): 5'-AUGGCCCGAGGGGCUCGCAUAACAGCG-3'
Step 2 — Codons: AUG-GCC-CGA-GGG-GCU-CGC-AUA-ACA-GCG
Step 3 — Translate: Met-Ala-Arg-Gly-Ala-Arg-Ile-Thr-Ala
One-letter: M-A-R-G-A-R-I-T-A

Example 2

DNA coding strand: 5'-ATGGTTCCAATTGCGATA-3'
Transcribe: 5'-AUGGUUCCAAUUGCGAUA-3'
Codons: AUG-GUU-CCA-AUU-GCG-AUA
Translate: Met-Val-Pro-Ile-Ala-Ile = MVPIAI

The genetic code maps each 3-nucleotide codon to an amino acid. 64 codons but only 20 amino acids = degenerate (redundant). Key features: universal, non-overlapping, one start codon (AUG = Met), three stop codons (UAA, UAG, UGA).

Figure 6: Circular codon table — read from centre outward (5'→3'). Inner ring = 1st base, middle = 2nd, outer = 3rd.

In Practical 1, you perform a miniprep — extracting plasmid DNA using alkaline lysis.

Figure 7: Phenol extraction — aqueous phase (top) = DNA; interphase = proteins; organic phase (bottom) = RNA, lipids.

Alkaline Lysis Steps

• Solution 1 (Resuspend): Tris + EDTA + RNase. Resuspends cells, chelates Mg²⁺, degrades RNA.
• Solution 2 (Lyse): NaOH + SDS. Denatures DNA/proteins, dissolves membrane.
• Solution 3 (Neutralise): K-acetate. Chromosomal DNA tangles and precipitates; small plasmid renatures and stays in solution.

Supernatant purified by phenol/chloroform or silica column, then ethanol precipitation (DNA is less soluble in alcohol).

Template Strand vs Coding Strand

Central Dogma

Mnemonic: DNA→RNA→Protein = "Don't Really Party"
Meaning: Replication=DNA, Transcription=RNA, Translation=Protein

Base Pairing

Mnemonic: A-T (Apple Tree, 2 bonds), G-C (Good Car, 3 bonds)
Meaning: Purines (A,G) pair with pyrimidines (C,T/U). PURe As Gold.

Exons vs Introns

Mnemonic: EXons = EXpressed, INtrons = INterruptions
Meaning: Exons stay; introns are spliced out.

mRNA Processing: CPS

Mnemonic: Cap, Poly-A tail, Splicing
Meaning: Three modifications before mRNA leaves the nucleus.

Start/Stop Codons

Mnemonic: AUG = Always Use for Go! Stops: UAA, UAG, UGA
Meaning: AUG=Met=start. Three stops code for nothing.

Replication Strands

Mnemonic: Leading=continuous, Lagging=fragments(Okazaki)
Meaning: Both synthesised 5'→3' but in different patterns.

Miniprep: R-L-N

Mnemonic: Resuspend, Lyse, Neutralise
Meaning: Plasmid survives because it is small and circular.

Transcription Direction

Mnemonic: Template read 3'→5'; mRNA built 5'→3'
Meaning: Coding strand = same as mRNA.