Skewed mutational biases: English scrabble with Klingon tiles

Skewed mutational biases: English scrabble with Klingon tiles

In a game of Scrabble one draws seven tiles from a bag and the best results are found when a bag and the language used match. In library making with error-prone PCR one does not have that luxury, but different methods differing degrees of fairness. Mutazyme is like playing in English with a Latin tile set, while manganese Taq with equal nucleotides is like playing in English with a Klingon tile set.

In vivo shuffling via heteroduplex amplicons

Bluescreen transilluminator photo showing
different shades of green.

In a previous post I discuss the heteroduplicity of epPCR plasmids. Namely often colonies in a library will have bases that have two equal variants. The most likely cause being that the amplicons do not anneal perfect and that the transformed plasmids are actually heteroduplexes. These can either divide before being corrected or are corrected by mismatch repair.
I did a sneaky experiment to see if this could be used to make protocol to shuffle mutations between variants and got a positive result, but possibly not as effective as hoped.

On error rates of Mutazyme

On a previous jocular post about the 'not for diagnostic purposes' tag on Mutazyme I mention the mutational rate of Mutazyme II. The exact mutations per kb per doubling is never mentioned in the manual, but can be extrapolated. (see also part III: Mutazyme and Manganese)

The heteroduplicity of error prone PCR plasmids

A mix of wt and

mutant...

In an error prone PCR the ep-aDNA is ligated onto a plasmid backbone and transformed. When assessing the diversity from a naïve plasmid pool, something odd is seen: some bases are mutated but not to saturation. This is often just dismissed or simply overlooked, but I suspect it is actually something interesting...

Mutagenesis calculator

The calculator has been moved to www.mutanalyst.com.