Molecular biology tricks that make the central dogma stick

Central dogma: DNA → RNA → Protein (replication, transcription, translation)

Central Dogma — Replication: DNA → DNA. Transcription: DNA → mRNA (nucleus). Translation: mRNA → protein (ribosomes). Reverse transcriptase (HIV) breaks the rule: RNA → DNA.

DNA base pairs: A-T (2 H-bonds), G-C (3 H-bonds). RNA uses U instead of T.

DNA Base Pairing — A pairs with T (or U in RNA). G pairs with C. G-C has 3 hydrogen bonds (stronger). A-T has 2. Higher G-C content → more thermally stable DNA.

Codon = 3 bases = 1 amino acid. AUG = start. UAA UAG UGA = stop.

The Genetic Code — 64 possible codons (4³). 61 encode amino acids. 3 are stop codons. AUG = start (methionine). Code is degenerate — most AAs have multiple codons.

Mutation types: missense (wrong AA), nonsense (stop codon), silent (same AA), frameshift (insertion/deletion)

MissenseWrong amino acid

NonsensePremature stop codon

SilentSame amino acid — synonymous

FrameshiftInsertion/deletion shifts reading frame

PCR: denature → anneal primers → extend with Taq polymerase. Amplifies DNA exponentially.

Polymerase Chain Reaction — Three steps: (1) Denaturation: heat to 95°C, separate DNA strands. (2) Annealing: cool to ~55°C, primers bind. (3) Extension: 72°C, Taq polymerase builds new strand. Each cycle doubles the DNA. 30 cycles = ~1 billion copies.

DNA replication: semiconservative — each new DNA has one old strand and one new strand

Semiconservative Replication — Each daughter DNA molecule retains one parental strand and one newly synthesized strand. Meselson-Stahl (1958): used nitrogen isotope labeling to prove this. DNA polymerase: synthesizes new strand 5'→3'. Needs a primer (RNA) to start. Leading strand: continuous synthesis. Lagging strand: Okazaki fragments.

RNA types: mRNA (messenger), tRNA (transfer), rRNA (ribosomal). Each has a distinct function in protein synthesis.

mRNAMessenger — carries instructions

tRNATransfer — brings amino acids

rRNARibosomal — forms the ribosome

Transcription: RNA polymerase reads template strand 3'→5', synthesizes mRNA 5'→3'

Transcription Details — Promoter: where RNA polymerase binds to start. Template strand: read 3'→5'. mRNA synthesized 5'→3' (complementary to template, same sequence as coding strand but with U instead of T). Terminator: signals end of transcription. In eukaryotes: pre-mRNA processed → 5' cap, poly-A tail, introns spliced out.

Translation: ribosome reads mRNA codon by codon. tRNA anticodon pairs with codon. Peptide bond forms.

A siteAminoacyl — incoming tRNA

P sitePeptidyl — growing chain

E siteExit — leaving tRNA

Gene expression regulation: promoters, enhancers, transcription factors. Epigenetics: DNA methylation, histone modification.

Gene Regulation — Prokaryotes: operons — lac operon (repressor blocks transcription when lactose absent). Eukaryotes: more complex. Transcription factors bind specific DNA sequences near genes. Enhancers: regulatory sequences far from gene, loop to promoter. Epigenetics: heritable changes in gene expression without DNA sequence change. Methylation silences genes, histone acetylation activates.

CRISPR-Cas9: guide RNA directs Cas9 protein to cut specific DNA sequence — gene editing tool

CRISPR Gene Editing — CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): naturally occurring bacterial immune system. Cas9: a DNA-cutting protein. Guide RNA: designed to match the target DNA sequence. Cas9 cuts both strands at target. Cell repairs cut: can introduce mutations (knockout) or insert new sequence. Applications: disease research, potential genetic therapies.

Restriction enzymes: cut DNA at specific recognition sequences — molecular scissors for biotechnology

Restriction Enzymes — Restriction endonucleases: bacterial defense against foreign DNA. Cut at specific palindromic sequences (EcoRI cuts GAATTC). Create 'sticky ends' (overhanging single strands) or blunt ends. Sticky ends facilitate ligation (joining) with complementary pieces. Foundation of recombinant DNA, cloning, genetic engineering, PCR.