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Error Prone PCR

Error prone PCR is a random mutagenesis technique that introduces mutations into genes during PCR by using error-prone DNA polymerases and reaction conditions. This results in amino acid substitutions in proteins. The mutated PCR products are cloned and screened to find changes in protein activity. Careful control of the buffer composition regulates the frequency of base pair substitutions introduced, typically around 1-3 substitutions per kilobase. A Taq DNA polymerase without proofreading ability must be used so introduced mutations are not corrected.

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Takwa Shlaki
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84 views3 pages

Error Prone PCR

Error prone PCR is a random mutagenesis technique that introduces mutations into genes during PCR by using error-prone DNA polymerases and reaction conditions. This results in amino acid substitutions in proteins. The mutated PCR products are cloned and screened to find changes in protein activity. Careful control of the buffer composition regulates the frequency of base pair substitutions introduced, typically around 1-3 substitutions per kilobase. A Taq DNA polymerase without proofreading ability must be used so introduced mutations are not corrected.

Uploaded by

Takwa Shlaki
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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introduction

Error prone PCR


Random mutagenesis is a powerful tool for elucidating protein structure function
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.
Error prone PCR is a method by which random mutants may be inserted into any
piece of DNA.
The technique is based on the well founded PCR (polymerase chain reaction),
which is a standard technique in many molecular biology laboratories.
Normally the replication of DNA by the polymerase is extremely specific ,The
difference in error prone PCR is that the fidelity of the Taq DNA polymerase is
modulated by alteration of the composition of the reaction buffer.
In these conditions, the polymerase makes mistakes in the base paring during
DNA synthesis that results in the introduction of errors in the newly synthesized
complementary DNA strand.
By carefully controlling the buffer composition the frequency of mis-
incorporation of nucleotide bases, and therefore the number of errors introduced
into the sequence may be regulated.
In directed evolution experiments, the substitution frequency is normally
controlled at around 1 - 3 base pair substitutions per kilobase of DNA.
For the technique to work properly, it is important to use a Taq DNA
polymerase which does not have proof-reading ability.
This proof-reading, or auto-correction of nucleotide sequence, is a property that
is found in many commercially available Taq DNA polymerases.
However, use of a proof-reading DNA polymerase is used in a error prone PCR
reaction will result in the automatic correction of the mismatched nucleotides,
and any mutations that were introduced during the reaction will be lost.
to induce favorable mutations we plan to employ directed mutagenesis on the
aforementioned global transcription factors to increase current output. The
mutations would be introduced by error prone PCR, which is a technique used to
generate randomized genomic libraries. Error-prone PCR will allow us to
initiate DNA amplification starting with tiny amounts of parent molecule to
produce considerable amounts of the mutated gene.
This technique is based on the principle that Taq polymerase is capable of
annealing incompatible base-pairs to each other during amplification under
imperfect PCR conditions. The figure below contrasts the external conditions
between PCR and error prone PCR.

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