DNA Flashcards
Nucleosides and Nucleotides
- Nucleoside: Pentose + Nitrogenous Base (linked via covalent bond between base and C-1’ of sugar),
- Nucleotide: Nucleoside + 1-3 Phosphate Groups (linked via covalent bond between phosphate group and C-5’ of nucleoside).
- ATP is a nucleotide, and removing a terminal phosphate from it releases energy (exothermic/exergonic) due to repulsion between closely associated negative charges on its phosphate groups.
DNA and RNA Pentose
- If Pentose = Ribose, nucleic acid is RNA.
* If Pentose = Deoxyribose (ribose with 2’-OH replaced by -H), nucleic acid is DNA.
Purines & Pyrimidines
- Adenine (A), Thymine (T), Cytosine (C), Guanine (G) are Nitrogenous Bases in nucleotides.
- Purines: Two-ring structure; Adenine and Guanine (PURe As Gold).
- Pyrimidines: One-ring structure; Cytosine, Thymine, Uracil (CUT the PYe).
- Both are aromatic heterocycles (unusually stable ring structure).
Aromatic Structure
The compound: • is cyclic. • is planar. • is conjugated (alternating single and double/triple bonds). • has 4n+2 pi electrons (Hückel’s Rule).
Chargaff’s Rule
• In dsDNA, total purines = total pyrimidines.
DNA Denaturation
- Disrupts hydrogen bonding and base-pairing, separating double helix into two single strands; does not break covalent bonds between nucleotides in sugar-phosphate backbone.
- Heat, alkaline pH, and chemicals like formaldehyde and urea commonly used to denature DNA.
- DNA can be reannealed if denaturing condition is slowly removed.
Chromosome Organization
- 46 Chromosomes (23 Pairs) in Humans.
- DNA is wound around histone proteins to form nucleosomes (repeating structural unit of chromatin).
- DNA and its associated histones make up chromatin in nucleus.
Heterochromatin and Euchromatin
- Heterochromatin: Compacted, transcriptionally silent DNA that appears dark under light microscopy.
- Euchromatin: Dispersed, transcriptionally active DNA that appears light under light microscopy.
Telomeres and Centromeres
- Telomeres: Ends of chromosomes composed of heterochromatin; are slightly shortened during replication, but this can be partially reversed by telomerase; high GC-content to prevent unraveling of DNA.
- Centromeres: Middle of chromosomes composed of heterochromatin; hold sister chromatids together until separated during anaphase; high GC-content to maintain strong bond between chromatids.
DNA Replication
• DNA unwinds at (multiple) points of origin, and replisome creates replication forks; sister chromatids created via replication remain connected at centromere until mitosis.
• Helicase: Unwinds DNA.
• Single-Stranded Binding Proteins: Bind to unraveled strands to prevent reassociation of strands and degradation of DNA by nucleases.
• DNA Topoisomerase: Prevents supercoiling of strands by introducing negative supercoils; it nicks one or both strands ahead of helicase and removes positive supercoils to alleviate torsional stress, and then it reseals cut strands.
Primase: Synthesizes short RNA primer in 5’ to 3’ direction to start replication, as DNA cannot be synthesized de novo. Leading strand requires only one primer (or a few), but lagging strands requires new primer for each Okazaki fragment.
DNA Polymerase: Reads parental strand in 3’ to 5’ direction and synthesizes daughter strand in 5’ to 3’ direction (DNA polymerase a, d, e in eukaryotes and DNA polymerase III in prokaryotes). Leading strand is synthesized continuously, while lagging strand is made up of Okazaki fragments; incoming nucleotides are dNTPs.
• 5’-3’ Exonuclease: Removes RNA primers from 5’ end (RNase H in eukaryotes and DNA polymerase I in prokaryotes).
• DNA polymerase d (eukaryotes) or DNA polymerase I (prokaryotes) adds DNA nucleotides in place of RNA primer.
• DNA Ligase: Joins Okazaki fragments (or any unbound nucleotides) by catalyzing formation of phosphodiester bonds between them.
Oncogenes and Tumor Suppressor Genes
- Oncogene: Promotes rapid cell cycle advancement; mutation in only one allele of proto-oncogene sufficient to promote tumor growth (dominant mutation).
- Tumor Suppressor Gene: Inhibits cell cycle or participate in DNA repair processes; mutation in both alleles necessary for loss of tumor suppression activity (recessive mutation).
- Oncogenes promote cell cycle, while mutated tumor suppressors can no longer slow cell cycle.
Proofreading and Mismatch Repair
- DNA polymerase can excise incorrect base and replace it with correct base; DNA ligase (which closes gaps between Okazaki fragments) lacks proofreading ability, so likelyhood of mutation in lagging strand is significantly higher than leading strand. Daughter strand can be identified from its lack of methylation.
- Nucleotide Excision Repair: Fixes helix-deforming lesions (such as thymine dimers) via cut-and-patch process using excision endonuclease.
- Base Excision Repair: Fixes nondeforming lesions of DNA helix (such as cytosine deamination to uracil) by using a glycosylase enzyme to remove base and leave behind an apurinic/apyrimidinic (AP) site; damaged sequence is recognized and removed by AP endonuclease.
Genomic Library and cDNA Library
- Genomic Library: Made from chromosomal DNA and contain both coding (exon) and noncoding (intron) regions of genome; cannot be used to make recombinant proteins or for gene therapy.
- cDNA Library; Constructed by reverse-transcribing mRNA and contain only coding (exon) regions of genome; can be used to make recombinant proteins or for gene therapy.
Southern Blotting
• Used to detect presence and quantity of various DNA strands in sample; sample can be probed with single-stranded DNA molecules after electrophoresis to look for sequence of interest.
PCR
- Automated process that amplifies desired sequence by hybridization, granted that the sequences flanking the desired sequence are known.
- Requires Taq polymerase, primers that are complementary to DNA that flanks region of interest, and dNTPs.
- Primer stability determined by length, G-C content, and presence of 5’ and 3’ Gs and Cs.
