PCR distribution

The distribution of mutations in an error prone library has been modelled in Sun (1995) based on the underlying principle of a PCR reaction. In the paper it is found that the distribution becomes more and more similar to a regular Possion and the two are virtually the same after 10 cycles. However, given that one most likely does not know what one's PCR efficiency is, using this formula may be dangerous. Whereas in reality getting a better estimate of the mean number of mutations may be more helpful.

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.

A note on Mutazyme and Manganese

Can Mutazyme be powered up?
Adding manganese works poorly, but stronger variants seem to be known.
(see also discussion about Mutazyme in Part I and II)

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...

Medical diagnosis with Mutazyme

A cornerstone enzyme for error-prone PCR is Mutazyme, an enzyme with an increase error rate, but less biased than manganese mutagenesis. The manual is very clear and the only major annoyance is that it implicitly says that it makes 1.3 mutations per kb per cycle —that is the log2 of the fold amplification of the target—, whereas it actually makes something around 0.9 mutations per kb per cycle —even with the assumption that no DNA is lost during spin column purification or that DNA cut out of an agarose gel is not shockingly dirty.
However, the biggest mystery is that it says "Not for medical diagnostics".

Mutagenesis calculator

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