Biology Reference
In-Depth Information
mutations are not targeted to only the gene of interest with these methods, subsequent
cloning steps are necessary before screening. Mutations accumulating in host DNA cause
growth deficiencies, so several rounds of growth, plasmid purification, transformation,
and regrowth are often required.
The most widely used method for creating random mutation gene libraries is error-prone
polymerase chain reaction (error-prone PCR).
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This technique involves PCR-based gene
amplification using a low-fidelity DNA polymerase (e.g.
Taq
polymerase, which lacks 3
0
to 5
0
exonuclease proofreading activity), and any number of PCR conditions that promote
a relatively controlled frequency of deoxynucleotide misinsertion during DNA replication.
The primer sequences selected dictate the gene region to be amplified and therefore mutated.
Elevated concentrations of MgCl
2
(e.g.
1.5 mM helps to stabilize non-
complementary pairs), addition of Mn
2
1
(which replaces Mg
2
1
as a mutagenic cofactor
during DNA replication), or a deoxynucleotide analogue such as 8-oxo-dGTP or dITP, and
using unequal concentrations of deoxynucleotides all increase frequency of mutation during
template amplification.
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Other parameters such as the template concentration and number
of PCR cycles can also be used to adjust mutation frequency. Depending on the conditions
used, mutation rates can be varied from one to
7 mM instead of
B
B
20 mutations per kb. It is important to
remember that many nucleotide substitutions result in silent mutations (no change in
protein amino acid sequence), and amino acid changes requiring more than one nucleotide
substitution within a single codon occur with low probability.
B
While a powerful and essentially universally effective protein engineering technique,
there are several limitations associated with random mutagenesis via error-prone PCR.
For example, often the distribution of mutations appearing during PCR amplification is not
even, and in some cases mutation
'
hot spots
'
arise.
Taq
polymerase also preferentially
introduces A
-
T, T
-
A, A
-
G, and T
-
C mutations, and rarely incorporates G
-
C and
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C
G transversions. Many reports describe optimized protocols and reagents to help
alleviate such limitations, and commercial error-prone PCR kits featuring these
improvements are available. For example, DNA polymerases have been engineered to
exhibit altered mutation biases. Fuji and coworkers developed error-prone rolling circle
amplification, allowing for direct transformation of the product.
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This method requires no
ligation step, but mutations are distributed throughout the entire expression vector.
-
Recombination
Gene recombination involves the exchange or sharing of genetic information between
multiple parental sequences to create new
genes. A key benefit of using
recombination to generate genetic diversity is the fact that the parental sequence fragments
being combined correspond to pieces of related and functional proteins (the homologues
provide
'
progeny
'
). Compared to random mutagenesis, where increasing the
number of mutations drastically reduces the probability of obtaining a folded or functional
protein, recombination allows for a higher probability of a greater number of amino acid
changes (relative to a given parent sequence) to retain the protein
'
functional diversity
'
s ability to fold and carry
traits from both parents, as well as potentially new properties. In vitro recombination for
the sake of creating gene libraries is classified as either homologous, when the parent genes
share a high level of sequence similarity, or non-homologous, when the parents are not
homologues or share low sequence similarity.
In Vitro Homologous Recombination
DNA shuffling is the first reported method of
in vitro recombination.
24
Here, DNAse I is first used to randomly fragment the parental genes.
Then, provided there is sufficient sequence similarity between overlapping regions of DNA
fragments from different parents, the fragments can anneal to one another and reassemble into
a full-length gene using PCR. Depending on the total gene size, the degree of sequence
similarity between parents, and desired number of crossovers (regions where different parental
'
