ASSIGNMENT

PREPARED BY:
MD IBRAHIM (65/BPH/DPSRU/17)
B. PHARM (5TH SEMESTER)

SUBJECT:
PHARMACEUTICAL BIOTECHNOLOGY

TOPIC:
ERROR-PRONE PCR
 Error-prone PCR
Error prone PCR is a method by which random mutants maybe inserted into any piece of
DNA.

If one is attempting to amplify a DNA with high fidelity, this is obviously a problem. On the
other hand, if the construction of a library of mutants of the target gene is the objective, then
this approach is a very powerful method for random mutagenesis.

Moreover, with DNA up to 10 kilobase pairs (kb) in size, it is possible to vary the number
of alterations per gene from about 1 to about 20 by modifying the DNA template
concentration.

PRINCIPLE
The technique is based on the well founded PCR (polymerase chain reaction), which is a
standard technique in many molecular biology laboratories.

Some of the temperature-stable DNA polymerases that are used to amplify target DNA by
PCR occasionally insert incorrect nucleotides into the replicating DNA.

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.

PROCESS
For the technique to work properly, it is important to use a Taq DNA polymerase which does
not have proof-reading ability. When error-prone PCR is performed using Taq DNA
polymerase, which lacks proofreading activity, the error rate may be increased by:

A) adding Mn2+

B) by increasing the concentration of Mg2+,

C) by adding unequal amounts of the four deoxynucleoside triphosphates to the reaction

buffer.

Alternatively, high error rates may be achieved with other temperature-stable DNA
polymerases in the absence of Mn2+ and with balanced amounts of the four deoxynucleoside
triphosphates. Following error-prone PCR, the randomly mutagenized DNA is cloned into
expression vectors and screened for altered or improved protein activity.

The DNA from those clones that encode the desired activity is isolated and sequenced so that
the relevant changes to the target DNA may be elaborated. Error-prone PCR has been used to
create enzymes with improved solvent and temperature stability and with enhanced
specific activity.
 Error-prone PCR of a target gene yields a variety of mutated forms of the gene.
Mutations are shown in blue. The horizontal arrows represent PCR primers.

The drawback of this approach is that size of the library is limited by the efficiency of the
cloning step. Although point mutations are the most common types of mutation in error prone
PCR, deletions and frameshift mutations are also possible. There are a number of commercial
error-prone PCR kits available, including the STRATAGENE and CLONTECH.

REFERENCE:
a) Glick R. Bernard, Pasternak J. Jack, Patten L. Cheryl, “Molecular Biotechnology” 4th
Edition, Page no.- 298.

b) https://www.slideshare.net/ifrahishaq/directed-evolution-62944148