Week 2 Lecture Prep Assignments

Professor Jacobson:

  1. Biological DNA polymerase is an enzyme that couples a 5’-3’ polymerization domain with a 3’-5’ exonuclease proofreading domain. As this enzyme moves along the template DNA strand, it adds deoxynucleoside-triphosphates (dNTPs) complementary to the exposed base, forming a phosphodiester bond at the primer’s 3’-OH. This enzyme has an error rate of 1:106 (one error for every million base additions). If an incorrect nucleotide is incorporated, the resulting mismatch destabilizes the nascent strand, the polymerase pauses, and the mismatched base is transferred into an exonuclease pocket, where the 3’-5’ exonuclease clives it off, inserting then the correct base. The human genome contains about 3.2 billion base pairs, so without further correction, a single replication of the genome would result in approximately 3200 errors. To deal with this discrepancy, biology uses error-correcting mechanisms to mitigate this mismatch: Polymerase proofreading that removes misincorporated nucleotides, Post-replication mismatch repair that scans the newly synthesized strand for remaining errors, such as the MutS Repair System, and Redundancy from having two homologous chromosome sets, allowing cellular quality-control checkpoints to detect and eliminate damaged cells.

  2. An average human protein is encoded by about 1036 bp of coding DNA (≈345 amino acids). Since the genetic code is degenerate, 62 codons specify the same 20 amino acids, where each amino acid is encoded by 2 to 6 different codons. When synthesizing or expressing these proteins, only a small fraction of these sequences are usable because the DNA and its transcript prevent synonymous codes from being equally effective through several factors like: Secondary structure interference, where certain DNA or mRNA sequences may fold into stable minimum free energy “hairpins”, blocking the cellular machinery from translating the code, Codon-bias and tRNA availability: cells preferentially use codons that match abundant tRNAs, Regulatory motifs: accidental creation of splice sites, ribosome-binding sites or motifs recognized by cellular enzymes which target the mRNA for destruction, GC-content and stability: extreme GC-rich or AT-rich regions affect DNA stability, replication and transcription efficiency, and many more factors are the reasons why all of these different codes don’t work for a single protein of interest.

Dr. LeProust

  1. The most used method for oligo synthesis currently is the phosphoramidite method, a chemical process that involves a four-step cycle repeated for each nucleotide added: coupling, capping, oxidation and deblocking.

  2. Direct synthesis of oligonucleotides longer than 200 nt is difficult due to the accumulation of chemical errors and truncated/mutated sequences with each cycle, which significantly reduces the yield of full-length sequences.

  3. Due to the inefficiencies mentioned above, it is not possible to make a 2000 bp gene via direct oligo synthesis, because the yield for a single strand of that length would be effectively zero. Because of the 1:100 error rate, this long sequence would likely contain at least 20 error, making it biologically non-functional. Besides, direct chemical synthesis is generally limited to around 200-300 nt. Instead, genes of this length are created through assembly, using techniques like PCR assembly or Gibson assembly, in order to assemble shorter oligos.

Dr. Church

  1. The 10 amino acids generally considered essential for animals are:
  • Arginine
  • Histidine
  • Isoleucine
  • Leucine
  • Lysine
  • Methionine
  • Phenylalanine
  • Threonine
  • Valine
  • Threonine

The “Lysine Contingency” in Jurassic Park movies was a genetic alteration developed by Dr. Henry Wu where the dinosaurs were engineered to be unable to produce the essential amino acid Lysine. The idea was that the animals would die if they escaped the park because they wouldn’t have access to the lysine supplements provided by their carers. Knowing that Lysine is already an essential amino acid this breaks the logic of this contingency because animals (including dinosaurs) generally can not produce lysine naturally, so the genetic modification to “remove” this ability was redundant, because they already had it. Therefore, all animals obtain lysine by eating plants or other animals, like red or white meat, cheese, eggs, soy, etc. If the dinosaurs escaped, they would not die from a lack of supplements, they would simply survive by eating standard protein-rich food sources found in the wild.