Week 1 HW: Week 2 Lecture Pep

“Homework Questions from Professor Jacobson, Dr. LeProust and George Church”

“Homework Questions from Professor Jacobson”

  1. Nature’s machinery for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the length of the human genome. How does biology deal with that discrepancy?

The polymerase makes approximately 1 error per 1.105 or 1.106 bases copied. Considering that the human genome has three million nucleotides, this would imply approximately 3,000 errors. However, the number of errors is reduced to 1 per 1 billion nucleotides due to its 3’-5’ exonuclease activity, which allows it to detect the error, backtrack, remove the incorrect nucleotide, and replace it with the correct one: Proofreading activity.

  1. How many different ways are there to code (DNA nucleotide code) for an average human protein? In practice what are some of the reasons that all of these different codes don’t work to code for the protein of interest?

The genetic code uses triplets (codons) of 3 nucleotides to encode amino acids. There are 64 possible codons, of which 61 encode amino acids, since the other 3 encode stop codons. There are 20 possible amino acids, which means that most amino acids are represented by more than one codon. This indicates that the same amino acid sequence can be encoded by different nucleotide sequences.

These differences between theory and practice may be due to various biological factors such as the structure of mRNA and its stability, the abundance of tRNAs that recognize these codons, which alters translation efficiency, among others.

“Homework Questions from Dr. LeProust”

  1. What’s the most commonly used method for oligo synthesis currently?

Currently, the most widely used method for oligo synthesis is chemical synthesis, specifically the phosphoramidite approach (Andrews et al., 2021). The phosphite triester approach to DNA synthesis using deoxynucleoside phosphoramidite as synthons has become the method of choice for the preparation of deoxy oligonucleotides. The general synthetic strategy involves adding mononucleotides sequentially to a deoxynucleoside, attached covalently to a silica-based insoluble polymeric support. Reagents, starting materials, and side products are then removed simply by filtration. At the conclusion of the synthesis, the deoxy oligonucleotide is chemically freed of blocking groups, hydrolyzed from the support, and purified to homogeneity by either polyacrylamide gel electrophoresis (PAGE) or high-performance liquid chromatography (HPLC)(Caruthers et al., 1987).

  1. Why is it difficult to make oligos longer than 200nt via direct synthesis?

While chemical synthesis has enabled the production of oligonucleotides with a high degree of control, the efficiency of chemical synthesis decreases as the length of the oligo increases, which often limits the practical length to about 200 bases (Gao et al., 2025). Depurination, particularly of adenosine, can occur during acidic detritylation and becomes particularly problematic in the production of long oligos. During the final removal of protecting groups from the bases and phosphate backbone, these abasic sites lead to cleavages that reduce the yield of long-length oligos. Finally, even successfully synthesized oligos contain appreciable errors. The dominant errors in purified oligos are single-base deletions that result from either failure to remove the DMT or combined inefficiencies in the coupling and capping steps. Newer chemistries and improved processes continue to arise and will further augment oligo length and quality (Korusi et al., 2014)

  1. Why can’t you make a 2000bp gene via direct oligo synthesis?

As mentioned above, a 2000 bp gene cannot be created by direct oligonucleotide synthesis because chemical DNA synthesis accumulates errors and losses exponentially as the length increases. There are studies, such as the one cited below (Yipeng et al., 2025) in which the direct synthesis of a protein gene was achieved in an automatic synthesizer. To do this, certain parameters of the traditional protocol were modified, such as the modification of the pores of traditional supports, for example. However, this has not yet been applied on a large scale.

“Homework Question from George Church”

What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”?

The essential amino acids are: Histidine, Leucine, Isoleucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine, Arginine. The “lysine contingency” is a fictional concept from Jurassic Park in which Henry Wu genetically modified the dinosaurs so that they could not produce lysine, an essential amino acid, forcing them to depend on supplements from the park to survive. This was intended to prevent them from escaping and surviving, although the films show that the animals could obtain lysine from their food. However, the strategy proposed in the film is absurd, given that dinosaurs never had the ability to produce lysine because it is an essential amino acid and these are animals.

References
  • Fijalkowska IJ, Schaaper RM, Jonczyk P. DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair. FEMS Microbiol Rev. 2012 Nov;36(6):1105-21. doi: 10.1111/j.1574-6976.2012.00338.x. Epub 2012 Apr 5. PMID: 22404288; PMCID: PMC3391330.
  • Gao N, Yu A, Yang W, Zhang X, Shen Y, Fu X. Enzymatic de novo oligonucleotide synthesis: Emerging techniques and advancements. Biotechnol Adv. 2025 Sep;82:108604. doi: 10.1016/j.biotechadv.2025.108604. Epub 2025 May 12. PMID: 40368114.
  • M.H. Caruthers, A.D. Barone, S.L. Beaucage, D.R. Dodds, E.F. Fisher, L.J. McBride, M. Matteucci, Z. Stabinsky, J.-Y. Tang. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods in Enzymology, Academic Press, 1987. doi: 10.1016/0076-6879(87)54081-2.
  • Kosuri S, Church GM. Large-scale de novo DNA synthesis: technologies and applications. Nat Methods. 2014 May;11(5):499-507. doi: 10.1038/nmeth.2918. PMID: 24781323; PMCID: PMC7098426.
  • Yin Y, Arneson R, Yuan Y, Fang S. Long oligos: direct chemical synthesis of genes with up to 1728 nucleotides. Chem Sci. 2024 Dec 18;16(4):1966-1973. doi: 10.1039/d4sc06958g. PMID: 39759933; PMCID: PMC11694485.