Molecular Biology — Biotech & Common Comparisons

1.PCR, polymerase chain reaction, amplifies a specific DNA fragment from tiny starting material. Three step cycle: One, denaturation at ninety-four to ninety-eight degrees — separates double-stranded DNA. Two, annealing at fifty to sixty-five degrees — primers bind the template. Three, extension at seventy-two degrees — Taq DNA polymerase synthesizes new strands.

2.After n cycles, DNA quantity equals starting amount times two to the power of n. Taq polymerase comes from the thermophilic bacterium Thermus aquaticus — heat-resistant, survives ninety-four degree denaturation across cycles.

3.Applications: COVID-19 testing uses RT-PCR — reverse transcribes RNA to cDNA first, then amplifies. Forensics amplifies trace crime scene DNA. Medicine uses it for prenatal diagnosis of genetic diseases.

4.Restriction enzymes, also called restriction endonucleases, come from bacterial defense systems. They recognize specific palindromic sequences and cleave them. Sticky ends have staggered cuts with single-stranded overhangs — easy to recombine via complementary pairing. Blunt ends cut evenly — harder to ligate but more versatile.

5.Gel electrophoresis: DNA carries negative charge from phosphates, migrates toward the positive electrode in an electric field. Smaller fragments migrate faster. Agarose gel for DNA; polyacrylamide PAGE for proteins.

6.Gene cloning steps: One, cut target gene and vector with restriction enzymes. Two, insert gene into vector using DNA ligase. Three, transform recombinant vector into host bacteria. Four, screen for bacteria containing the target gene, often using antibiotic resistance.

7.CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It originated as a bacterial adaptive immune system against viruses. Bacteria store fragments of past viral DNA between palindromic repeats and use them to recognize and cut returning invaders.

8.Cas9 is the molecular scissors protein. Guide RNA, abbreviated gRNA, directs Cas9 to a specific target DNA sequence by complementary base pairing. After binding, Cas9 cleaves the double-stranded DNA at that exact location.

9.Two repair pathways after cutting. NHEJ, non-homologous end joining, produces insertion or deletion mutations — used for gene knockout to study gene function. HDR, homology-directed repair, allows precise insertion or replacement of a sequence — used for gene therapy and correcting mutations.

10.Clinical use: in twenty-twenty, CRISPR inventors Jennifer Doudna and Emmanuelle Charpentier received the Nobel Prize in Chemistry. CRISPR has entered clinical trials for treating sickle cell anemia and beta-thalassemia.

11.RNA interference, or RNAi, is a separate gene-silencing technology often paired with CRISPR in essay questions. Small interfering RNAs, siRNAs, bind to complementary mRNA and trigger degradation by the RISC complex — silencing the gene without altering DNA. CRISPR edits the gene; RNAi knocks down its expression.

12.DNA uses deoxyribose sugar; RNA uses ribose. DNA bases A T G C; RNA bases A U G C — uracil replaces thymine. DNA is usually double-stranded; RNA usually single-stranded. DNA is mostly in the nucleus; RNA is in nucleus and cytoplasm. DNA is more stable because deoxyribose lacks the two-prime hydroxyl.

13.DNA replication uses both strands as templates and produces DNA via DNA polymerase, requiring an RNA primer, with three-prime to five-prime proofreading. Transcription uses only the template strand and produces RNA via RNA polymerase, needs no primer, has no proofreading.

14.Prokaryotes have one type of RNA polymerase; eukaryotes have three types — Pol one, two, three. Prokaryotic mRNA is not processed and is translated immediately — transcription and translation are coupled. Eukaryotic mRNA undergoes five-prime cap, poly-A tail, and splicing — transcription happens in nucleus, translation in cytoplasm. Prokaryote mRNA is polycistronic; eukaryote mRNA is monocistronic. Prokaryote initiator amino acid is fMet; eukaryote is Met.

15.lac operon handles lactose catabolism — inducible, default OFF, induced by allolactose, with cAMP-CAP positive regulation. trp operon handles tryptophan synthesis (anabolism) — repressible, default ON, corepressed by tryptophan, with attenuation as additional regulation.