Week 1 HW: Principles and Practices
Questions from Professor Jacobson:
- 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 standard polymerase error rate is 1:1 000 000. Human genome is around 3.2 Gbp = 3.2 x 10^9. Therefore, the copying of full human genome “at once” would yield around 3200 errors (mutations) - some silent and non-significant, some causing serious development and health issues. To avoid so many mistakes passing through the MutS repair system scans the DNA after replications, identifies the mismatched base pairs and trigger wrong sequence excision and re-copying of the fragment.
- 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 average human protein is around 1036 bp. So, if for the 100 amino acid sequence (with each amino acid being coded by 3 nucleotides) we face 3 ^ 100 options for a 1036 bp protein (around 345 amino acids) we face a possibility of 3 ^ 345 coding options. Not all of those sequences will yield a functional protein of interest due to the fact that some rare codons or their grouping can introduce more mistakes than others, act as unintentional stop signals or slow down the synthesis process [1].
Questions from Dr. LeProust:
- What’s the most commonly used method for oligo synthesis currently?
The most common method used for oligo synthesis is currently the solid-phase phosphoramidite chemistry, in which the nucleoside phosphoramidites are attachted to solid surface and grown in cycles of deprotection (taking off the protecting groups), coupling, capping, and oxidation.
- Why is it difficult to make oligos longer than 200nt via direct synthesis?
Diffuclty of making oligonucleotides longer than 200 nt with drect synthesis comes from depleeting the coupling efficiency with the lenght of disired synthesized product. For a fragment of 20 mer the coupling efficiency of 98% can yield about 68% of full-length product.Therefore, high coupling efficiency has to be preserved throught the process. This is particulary challenging due to the presence of water, which can dilute the concentrations of needed reagents (nuclotide derrivatives, activators, etc.). During the synthesis of longer fragments there is also a higher probability of errors and side reactions. Moreover, longer oligos are also more difficult to purify [1].
- Why can’t you make a 2000bp gene via direct oligo synthesis?
The direct oligo synthesis of a 2000 bp gene is not feasible due to several reasons, including those listed in the answer to Question 2. The accumulation of erros, decreasing coupling and termination of unreacted chains (capping) efficiency prevent practical use of direct synthesis for such long oligos.
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 10 essential amino acids (that animals can’t synthesize themselves) for all animals are phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, ariginine (though in some cases can be synthesized by animals), leucine and lysine. The “Lysine Contingency” plan that was introduced in the Jurrasic Park movies as a way of ensuring that none of the dinosaurs will be able to surive outside of the park as they lack the ability to synthesize the amino acid lysine was completely flawed concept. The animals, including dinosaurs and humans, cannot synthesieze lysine even without any molecular-biology-based intervention - we need to consume them within our diets (microbes, plants, other animals - that can be found anywhere, not just on an island with monitoring and lysine supplement facility).