Molecular biology Study Guide

📖 Core Concepts Molecular biology – study of DNA, RNA, and proteins and how they interact to store, transmit, and express genetic information. Central Dogma – DNA → RNA → Protein (replication, transcription, translation). DNA structure – double‑helix; complementary base pairing (A↔T, G↔C) obeys Chargaff’s rule (A + G = C + T). Genetic code – triplet codons; 64 possible codons encode 20 amino acids + 3 stop signals; non‑overlapping, read 5’→3’. Semiconservative replication – each daughter DNA contains one parental strand and one newly synthesized strand. Horizontal gene transfer – movement of genetic material between contemporaneous organisms (e.g., transformation). Gene – DNA segment that encodes a functional RNA or protein; mutations alter phenotype. 📌 Must Remember Chargaff’s rule: $[\text{A}] + [\text{G}] = [\text{C}] + [\text{T}]$. Semiconservative replication proven by Meselson–Stahl (density gradient shows hybrid DNA after one round). Key experiments: Griffith (1928): “transforming principle” → DNA candidate. Avery–MacLeod–McCarty (1944): purified DNA transforms non‑virulent strain. Hershey–Chase (1952): $^{32}$P‑DNA enters bacteria, not $^{35}$S‑protein. Codon facts: 3 nucleotides per codon; start codon = AUG (Met); stop codons = UAA, UAG, UGA. PCR amplification: $N = N0 \times 2^{n}$ (exponential; $n$ = number of cycles). Gel electrophoresis: DNA moves toward positive electrode; smaller fragments run faster. Blotting purposes: Southern – detect specific DNA. Northern – detect specific RNA. Western – detect specific protein. CRISPR‑Cas9: requires a protospacer adjacent motif (PAM) (e.g., NGG) next to target. 🔄 Key Processes DNA Replication Initiation: origin → helicase unwinds; primase adds RNA primer. Elongation: DNA polymerase III adds nucleotides 5’→3’; leading strand continuous, lagging strand Okazaki fragments. Termination: DNA ligase joins fragments; proof‑reading exonuclease corrects errors. Transcription RNA polymerase binds promoter → DNA unwinds → RNA strand synthesized 5’→3’. Terminator signals end; RNA is processed (5’ cap, poly‑A tail, splicing). Translation mRNA binds ribosome (A, P, E sites). tRNA anticodon pairs with codon; peptide bond formed; ribosome translocates. PCR Cycle Denaturation (≈95 °C, DNA strands separate). Annealing (≈50‑65 °C, primers bind). Extension (≈72 °C, Taq polymerase synthesizes new DNA). Repeat 25‑35×. Agarose Gel Electrophoresis Prepare % agarose → melt → cast with comb. Load samples + loading dye, run constant voltage. Visualize with ethidium bromide or SYBR‑Safe. Southern Blot Digest DNA → run gel → denature → transfer (capillary/electroblot). Hybridize with labeled probe → detect (autoradiography/chemiluminescence). CRISPR‑Cas9 Editing Design sgRNA complementary to target. Cas9‑sgRNA complex binds PAM‑adjacent site → double‑strand break. Repair via NHEJ (knock‑out) or HDR (knock‑in with donor template). 🔍 Key Comparisons DNA vs. RNA Sugar: deoxyribose vs. ribose. Bases: DNA has T, RNA has U. DNA is double‑stranded; RNA usually single‑stranded. PCR vs. RT‑PCR PCR → DNA template. RT‑PCR → RNA → reverse transcription → cDNA → PCR. Southern vs. Northern vs. Western Southern → DNA detection. Northern → RNA detection. Western → Protein detection (antibody based). Transformation vs. Transfection Transformation: bacteria uptake naked DNA. Transfection: eukaryotic cells uptake DNA (chemical, electroporation, liposome). Semiconservative vs. Conservative replication Semiconservative: each daughter DNA gets one old + one new strand (true). Conservative: one daughter DNA identical to parent, other entirely new (disproved). ⚠️ Common Misunderstandings “DNA is positively charged.” DNA is negatively charged (phosphate backbone). “PCR needs only one primer.” Two primers (forward & reverse) are required for exponential amplification. “All genes code for proteins.” Some genes encode functional RNAs (tRNA, rRNA, miRNA). “Semiconservative means both strands are new.” Only one strand per daughter is newly synthesized. “CRISPR works without a PAM.” PAM is essential for Cas9 binding and cleavage. 🧠 Mental Models / Intuition DNA as a zipper: helicase “unzips”; each half acts as a template for its own copy. PCR as a photocopier: each cycle doubles the number of copies – like repeatedly making copies of a document. Blotting as a “photocopy onto paper”: gel separates molecules, then they are transferred (copied) onto a membrane for probing. CRISPR as a molecular GPS: sgRNA guides Cas9 to the exact address (target sequence) next to a required “landmark” (PAM). 🚩 Exceptions & Edge Cases Mitochondrial genetic code: a few codons differ (e.g., UGA codes for Trp, not stop). High‑GC templates: need higher annealing temperature and additives (DMSO, betaine) for PCR. Non‑canonical start codons: rare organisms use alternatives (e.g., GUG, UUG). Off‑target effects in CRISPR: mismatches tolerated in seed region → unintended cuts. RNA‑dependent DNA polymerases: reverse transcriptases (used in RT‑PCR) not present in standard PCR. 📍 When to Use Which Detect a specific DNA fragment? → Southern blot (high specificity) or PCR (rapid). Measure gene expression (RNA)? → Northern blot (size) or RT‑qPCR (quantitative). Confirm protein size/abundance? → Western blot. Quick size check of PCR product? → Agarose gel electrophoresis. Introduce a point mutation? → Site‑directed mutagenesis via PCR with mutant primers. Knock‑out a gene in cells? → CRISPR‑Cas9 with NHEJ repair. Large‑scale gene expression profiling? → DNA microarray or RNA‑seq. 👀 Patterns to Recognize Band ladder pattern on gel → indicates fragment size; evenly spaced bands = correct ladder. “ATG” at 5’ end of coding region → start codon. Multiple restriction sites flanking insert in cloning vectors → easy ligation. High background on blot → insufficient blocking or excessive antibody. PCR “smear” → degraded template or too many cycles. CRISPR design: target sequence followed by NGG PAM on genomic DNA. 🗂️ Exam Traps Distractor: “DNA replication is conservative.” – Wrong; Meselson–Stahl proved semiconservative. Distractor: “Southern blot detects proteins.” – Wrong; Western blot does that. Distractor: “PCR works without primers.” – Wrong; primers are essential. Distractor: “All stop codons are UAA only.” – Wrong; UAG and UGA also stop. Distractor: “CRISPR can edit without a PAM sequence.” – Wrong; PAM is required for Cas9 activity. Distractor: “RNA polymerase can transcribe DNA without a promoter.” – Wrong; promoter is needed for initiation. --- Use this guide for rapid recall right before the exam – focus on the bolded keywords and the step‑wise processes!