GeneMorph Random Mutagenesis Instruction Manual
GeneMorph II Random Mutagenesis Kit
Instruction Manual
Catalog #200550
Revision B
Research Use Only. Not for Use in Diagnostic Procedures.
200550-12
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GeneMorph II Random Mutagenesis Kit
CONTENTS
Materials Provided .............................................................................................................................. 1
Storage Conditions .............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 1
Notice to Purchaser ............................................................................................................................. 1
Introduction ......................................................................................................................................... 2
Random Mutagenesis with the GeneMorph II Random Mutagenesis Kit ............................. 2
Mutational Spectrum of the GeneMorph II Kit ..................................................................... 4
Preprotocol Considerations ................................................................................................................ 6
Amplification Targets ............................................................................................................ 6
Initial Amount of Target DNA .............................................................................................. 6
Cycle Number ........................................................................................................................ 7
Primer Design ........................................................................................................................ 7
PCR Product Yield ................................................................................................................ 7
Achieving High Mutation Frequencies.................................................................................. 7
Protocol ................................................................................................................................................ 8
Troubleshooting ................................................................................................................................ 10
Appendix: How to Calculate Mutation Frequency ........................................................................ 11
References .......................................................................................................................................... 13
MSDS Information ............................................................................................................................ 13
Quick-Reference Protocol ................................................................................................................ 14
GeneMorph II Random Mutagenesis Kit
M
ATERIALS PROVIDED
Materials provideda Concentration Quantity
Mutazyme II DNA polymeraseb 2.5 U/μl 30 μl
10× Mutazyme II reaction buffer 10× 150 μl
40 mM dNTP mix 10 mM each dNTP 30 μl
1.1-kb Gel standard 20 ng/μl 150 μl
a
Sufficient reagents are provided for 30 reactions.
b
Mutazyme II DNA polymerase is not sold separately.
STORAGE CONDITIONS
All Materials: –20°C
ADDITIONAL MATERIALS REQUIRED
Temperature cycler
PCR tubes
PCR primers
NOTICE TO PURCHASER
Notice to Purchaser: Limited License
Purchase of this product includes an immunity from suit under patents specified in the product
insert to use only the amount purchased for the purchaser’s own internal research. No other patent
rights (such as 5’ Nuclease Process patent rights) are conveyed expressly, by implication, or by
estoppel. Further information on purchasing licenses may be obtained by contacting the Director
of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Revision B © Agilent Technologies, Inc. 2012.
GeneMorph II Random Mutagenesis Kit 1
INTRODUCTION
Random mutagenesis is a powerful tool for elucidating protein structurefunction relationships and for modifying proteins to improve or alter their
characteristics. Error prone PCR is a random mutagenesis technique for
generating amino acid substitutions in proteins by introducing mutations
into a gene during PCR. Mutations are deliberately introduced through the
use of error prone DNA polymerases and/or reaction conditions. The
mutated PCR products are then cloned into an expression vector and the
resulting mutant library can be screened for changes in protein activity.
Random mutagenesis allows researchers to identify beneficial mutations in
the absence of structural information, or when such mutations are difficult to
predict from protein structure.
Random Mutagenesis with the GeneMorph II Random Mutagenesis Kit
The mutational bias exhibited by error prone PCR enzymes undoubtedly
skews representation of random mutant libraries, diminishing the effective
size of the collection produced by error prone PCR. Mutazyme II DNA
polymerase is a novel error prone PCR enzyme blend, formulated to provide
useful mutation rates with minimal mutational bias. Mutazyme II is a blend
of two error prone DNA polymerases—Mutazyme I DNA polymerase (from
the original GeneMorph Random Mutagenesis Kit) and a novel Taq DNA
polymerase mutant that exhibits increased misinsertion and misextension
frequencies compared to wild type Taq. For the Mutazyme II polymerase
formulation, the Mutazyme I polymerase and the Taq polymerase mutant
have been combined to produce a less biased mutational spectrum with
equivalent mutation rates at A’s and T’s vs. G’s and C’s. Therefore, libraries
created with Mutazyme II should exhibit greater mutant representation
compared to libraries generated with other enzymes. However, the original
GeneMorph I kit favors mutations at G’s and C’s which in some cases may
be desirable.
With the GeneMorph II random mutagenesis kit*, mutation rates of
1–16 mutations per kb can be achieved using a single set of buffer
conditions (MgCl
The desired mutation rate can be controlled simply by varying the initial
amount of target DNA in the reaction or the number of amplification cycles
performed.
* U.S. Patent Nos. 6,803,216; 6,734,293; 6,444,428; 6,183,997; 5,489,523.
, balanced dNTPs) optimized for high product yield.
2
1
2 GeneMorph II Random Mutagenesis Kit
How Mutation Frequency is Controlled
Mutation frequency is the product of DNA polymerase error rate and
number of duplications (see Appendix). In the GeneMorph II kit, a
sufficiently high error rate is achieved through use of Mutazyme II DNA
polymerase. A low, medium or high mutation frequency is produced by
adjusting the initial target DNA amounts in the amplification reactions. For
the same PCR yield, targets amplified from low amounts of target DNA
undergo more duplications than targets amplified from high concentrations
of DNA. The more times a target is replicated, the more errors accumulate.
Therefore, higher mutation frequencies are achieved simply by lowering
input DNA template concentration. Conversely, lower PCR mutation
frequencies can be achieved by using higher DNA template concentrations
to limit the number of target duplications. Mutation rates can also be
decreased by lowering the number of cycles to achieve fewer target
duplications. For targets that produce high product yields after 30 cycles,
lower mutation rates can be achieved by amplifying lower target amounts
for 20–25 cycles.
Selecting the Appropriate Mutation Frequency
The GeneMorph II kit allows researchers to choose the mutation frequency
that is most appropriate for a particular application. For analyzing protein
structure-function relationships, the desired mutation frequency is one
amino acid change (1–2 nucleotide changes) per gene.
studies, mutation frequencies of 1–4 amino acid changes (2–7 nucleotide
changes) per gene are commonly employed.
activities have also been isolated from highly mutagenized libraries
exhibiting 20 mutations per gene.
1
3, 4, 5, 6
2
In directed evolution
Proteins with improved
Achieving the Desired Mutation Frequency
Table I presents the initial amount of target DNA required to produce low,
medium, or high mutation frequencies. An initial target amount of
500–1000 ng is recommended to achieve low mutation frequencies of
0–4.5 mutations/kb. Low mutation frequencies can also be achieved by
using 100–500 ng of target DNA with a lower number of PCR cycles (see
Cycle Number in Preprotocol Considerations). Initial target amounts
ranging from 100–500 ng are recommended for producing mutation
frequencies of 4.5–9 mutations/kb (medium mutation frequency range).
High mutation frequencies (>9 mutations/kb) are obtained by using
0.1–100 ng of input target DNA, where the highest mutation rates can be
achieved using the lowest recommended target amounts. Mutation rates up
to 16 mutations per kb have been achieved using 0.01 ng of target DNA,
although PCR product yields tend to decrease at amounts below 0.1 ng.
The predicted mutation frequencies shown in Table I are accurate for
amplification reactions producing the indicated approximate fold
amplification. The actual number of mutations in individual clones may
differ as the values in Table I represent the average mutation frequency for
the entire pool of clones.
GeneMorph II Random Mutagenesis Kit 3
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