Molecular Genetics Basics

Cheatsheet Content

### Genetic Material - **DNA (Deoxyribonucleic Acid):** Double-stranded helix, primary genetic material in most organisms. Contains deoxyribose sugar. - **RNA (Ribonucleic Acid):** Single-stranded (mostly), involved in gene expression. Contains ribose sugar. - **mRNA:** Messenger RNA, carries genetic code from DNA to ribosomes. - **tRNA:** Transfer RNA, carries amino acids to ribosomes during protein synthesis. - **rRNA:** Ribosomal RNA, a component of ribosomes. - **Other RNAs:** e.g., siRNA, miRNA, snRNA, involved in gene regulation. ### DNA Denaturation & Renaturation - **Denaturation (Melting):** Separation of DNA double helix into single strands. - Caused by heat (thermal denaturation), extreme pH, or chemical agents. - Breaks hydrogen bonds between base pairs, but not phosphodiester bonds. - Monitored by increased UV absorbance (hyperchromic effect). - **Renaturation (Annealing):** Re-association of complementary single DNA strands to form a double helix. - Occurs when denaturing conditions are removed (e.g., cooling). - Rate depends on DNA concentration, temperature, and ionic strength. ### Cot Curves (DNA Reassociation Kinetics) - **Purpose:** Measures the rate of DNA renaturation to determine the complexity and proportion of repetitive sequences in a genome. - **Cot Value:** $C_0 t$ = initial DNA concentration ($C_0$) multiplied by time ($t$). - **Interpretation:** - **Fast renaturation:** Highly repetitive DNA (low complexity). - **Intermediate renaturation:** Moderately repetitive DNA. - **Slow renaturation:** Unique/single-copy DNA (high complexity). - **Formula:** Fraction of single-stranded DNA remaining = $1 / (1 + kC_0 t)$ - $k$ is the reassociation rate constant. ### DNA Topology - **Linking Number (Lk):** An invariant topological property of a closed circular DNA molecule. - $Lk = Tw + Wr$ - **Twist (Tw):** Number of helical turns. - **Writhe (Wr):** Number of supercoils. - For relaxed B-DNA, $Tw \approx$ total base pairs / 10.5 bp/turn. - **Supercoiling:** Coiling of a DNA helix upon itself. - **Negative Supercoiling:** Most common in vivo; facilitates DNA unwinding, replication, and transcription. - **Positive Supercoiling:** DNA is overwound. - **Topoisomerases:** Enzymes that change DNA topology by cutting and rejoining DNA strands. - **Type I Topoisomerases:** Cut one DNA strand, pass the other strand through the break, then re-ligate. Change Lk by 1. - **Type II Topoisomerases:** Cut both DNA strands, pass another DNA segment through the break, then re-ligate. Change Lk by 2. (e.g., DNA gyrase in bacteria, eukaryotic topoisomerase II). ### Organization of DNA - **Prokaryotes (e.g., Bacteria):** - Circular chromosome, typically one per cell. - Located in the nucleoid region, not membrane-bound. - Supercoiled and condensed by DNA-binding proteins (not histones). - Plasmids (small, circular DNA) often present. - **Eukaryotes:** - Multiple linear chromosomes. - Located within the membrane-bound nucleus. - Tightly packed with **histone proteins** to form **chromatin** (nucleosomes, 30nm fiber, etc.). - Chromatin can be **euchromatin** (less condensed, active genes) or **heterochromatin** (highly condensed, inactive genes). - **Viruses:** - Can have DNA or RNA genomes. - Genomes can be linear or circular, single or double-stranded. - Packaged within a protein capsid. - **Mitochondria & Chloroplasts:** - Own circular DNA genomes (similar to prokaryotic DNA). - Replicate independently of nuclear DNA. - Encode some of their own proteins (e.g., rRNAs, tRNAs, some respiratory/photosynthetic proteins). - Exhibit maternal inheritance. ### DNA Replication - **Semi-conservative:** Each new DNA molecule consists of one original (template) strand and one newly synthesized strand. - **Bidirectional Replication:** Replication forks move in opposite directions from a single origin of replication (common in circular and linear chromosomes). - **Unidirectional Replication:** Replication proceeds in one direction from the origin (less common). - **Semi-discontinuous Replication:** - **Leading Strand:** Synthesized continuously in the 5' to 3' direction, following the replication fork. - **Lagging Strand:** Synthesized discontinuously in short fragments (Okazaki fragments) in the 5' to 3' direction, away from the replication fork. These fragments are later joined by DNA ligase. - **Key Enzymes:** - **Helicase:** Unwinds DNA double helix. - **Primase:** Synthesizes RNA primers. - **DNA Polymerase:** Synthesizes new DNA strands (e.g., DNA Pol III in prokaryotes, Pol delta/epsilon in eukaryotes). - **DNA Ligase:** Joins Okazaki fragments. - **Topoisomerase:** Relieves supercoiling ahead of the fork. - **SSBPs (Single-Strand Binding Proteins):** Stabilize unwound DNA.