Week 2 HW: Lecture Prep

Homework Questions from Professor Jacobson

What is the error rate of polymerase?
DNA polymerase copies DNA with very high accuracy. When proofreading is active, the error rate is approximately 1 in 10⁶ base pairs, meaning one mistake per million bases added. This is much more accurate than chemical DNA synthesis, which has an error rate of about 1 in 10². DNA polymerase is also fast, adding one base roughly every 10 ms.

How does this compare to the length of the human genome?
The human genome is approximately 3.2 billion base pairs long. At an error rate of 10⁻⁶, a single full replication would theoretically introduce around 3,200 errors across the genome.

How does biology deal with that discrepancy?
Biology uses multiple error-correction mechanisms. DNA polymerase corrects errors during synthesis through proofreading, and additional mistakes are fixed by mismatch repair systems (such as MutS, MutL, and MutH) that detect and replace incorrectly paired bases.

How many different ways are there to code for an average human protein?
Due to the redundancy of the genetic code, an average human protein—encoded by roughly 1,036 base pairs—can be specified by an extremely large number of different DNA sequences without changing the resulting amino acid sequence.

In practice, why don’t all of these different codes work?
Many DNA sequences fail in practice because they form secondary structures that block transcription or translation, have high GC content that makes them difficult to process, are cleaved by RNA-processing enzymes, or generate assembly errors that prevent proper protein expression.

Homework Questions from Dr. LeProust

What’s the most commonly used method for oligo synthesis currently?
The most widely used method is the phosphoramidite DNA synthesis cycle, which consists of four main steps: deprotection, base coupling, optional capping, and oxidation. While traditionally performed on solid-phase supports using acid-based deprotection, modern next-generation approaches apply the same chemistry on high-density DNA chips using technologies such as inkjet printing or photolithography.

Why is it difficult to make oligos longer than 200 nt via direct synthesis?
The main limitation is the cumulative loss of yield over many synthesis cycles. Even with high per-step efficiency, overall yield decreases exponentially as length increases. In addition, chemical synthesis has a relatively high error rate (approximately 1 in 10²), so as sequences grow longer, most molecules accumulate deletions or insertions, making the recovery of a correct full-length oligo highly inefficient beyond ~200 nt.

Why can’t you make a 2000 bp gene via direct oligo synthesis?
Direct chemical synthesis lacks the error-correction mechanisms present in biological systems. Unlike DNA polymerase, which achieves error rates of 1 in 10⁶ through proofreading, chemical synthesis accumulates errors rapidly. As a result, long genes must be built by assembling shorter oligos using methods such as PCR assembly, Gibson assembly, or PCA, with intermediate error-filtering steps to ensure sequence accuracy.

Homework Question from George Church

The ten essential amino acids for most animals are phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine, leucine, and lysine. These compounds are classified as essential because animals lack the metabolic pathways required for their endogenous synthesis and must therefore obtain them through diet (Wu, 2009). In humans and other vertebrates, the inability to synthesize lysine is a natural, preexisting biological trait rather than an engineered modification (Bender, 2014).

This biological reality undermines the premise of the Lysine Contingency presented in Jurassic Park. Dinosaurs, as vertebrates, would already have been incapable of producing lysine independently. As a result, the proposed safety mechanism was redundant and ineffective, particularly because lysine is abundant in natural food sources such as meat and legumes, enabling the animals to obtain it through normal foraging behavior (Church & Regis, 2012).

For effective biological containment, modern synthetic biology instead proposes the use of non-canonical amino acids that do not occur in nature. By engineering organisms to depend on these synthetic compounds, survival becomes strictly contingent on a controlled laboratory supply, providing a more robust and reliable containment strategy (Mandell et al., 2015).

Academic References

• Bender, D. A. (2014). Amino Acid Metabolism. Wiley-Blackwell.
• Church, G. M. (2012). Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves.
• Mandell, D. J., Lajoie, M. J., Mee, M. T., et al. (2015). Biocontainment of genetically modified organisms via episomal and genomic engineering. Nature, 518(7537), 55–60.
• Wu, G. (2009). Amino acids: metabolism, functions, and nutrition. Amino Acids, 37(1), 1–17.