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This method allows a scientist to make small mutations in a piece of DNA.  There are three types of mutations that can be made - substitution, deletion, or addition of nucleotides.  The alteration in nucleotide sequence then in turn alters the amino acid of the protein that it encodes for.  The mutation may or may not affect the function of the protein.


In this procedure one starts with miniprep plasmid DNA or cesium-chloride-purified DNA.  This isolates the desired DNA from others that may be in the "cocktail." The starting DNA is double stranded with the gene of interest inserted at some point.  This gene of interest will be changed in some fashion - by either inserting, deleting, or substituting, a few base pairs.  The starting DNA needs to be in the form of a vector so that it may be transformed into E. coli cells in the last step of the procedure.


Next, single stranded chains of DNA (oligonucleotides) are made complimentary to the opposite strands of the vector. These DNA strands contain the desired substitution, deletion, or insertion.  The oligonucleotides are called primers because they will anneal to the plasmid after the two strands have been denatured, or separated from each other.  The primers allow the Pfu Turbo polymerase to elongate the two DNA strands.

The parent DNA is elongated many times by temperature cycling. At a high temperature, the DNA is denatured so that there are two single strands upon which primers can anneal and the PfuTurbo DNA polymerase can elongate the entire strand of DNA.  There are dNTPs in the "cocktail" that the polymerase uses to elongate the DNA strand. At the ends of each DNA strand that is formed there are "staggered nicks" where the mutation lies.
 The new mutated DNA is treated and the parental DNA are treated with Dpn I.  Dpn I is an enzyme that digests methylated DNA. This simply means that the DNA has methyl groups (CH3) attached to it.   Most  bacterial strains do in fact methylate plasmid DNA. It is very important the parent DNA be methylated so that the enzyme can select for the new mutated DNA.  (The new DNA will not be mythylated.)
The nicked mutated DNA is then transformed into the E. coli cells.  Transformation is the uptake of foreign  DNA by a bacteria cell  from the surrounding environment.1  The foreign DNA is then incorporated into the genome of the bacteria and can be transcribed and translated and altered.1
After the E. coli cells are transformed, the XL1-Blue repairs the nicks and connects the ends of the DNA stands.

ï     This method involves 4 steps that can be completed in one day.  It does not require a single stranded oligonucleotide as a template for elongation.  The double stranded DNA is simply denatured via high temperatures in order to produce a template for the daughter cells.  No unique restriction sites are needed in the vector because the XL1-Blue E. coli repairs the nicks in the daughter DNA and connects the two ends of the DNA.

ï    This method is very efficient and accurate.  Pfu DNA polymerase is used to elongate the daughter DNA strands. It provides very accurate elongation with increased product. This method also provides an alternative to PCR (polymerase chain reaction).  Only the parent strands are amplified in this procedure.  The primers contain the deletion, insertion, or substitution and are incorporated into the daughter cell. In other words daughter DNA is never used as a template for "granddaughter" DNA.  This reduces the chances of unwanted mutations because the parent DNA and primers always have the correct sequence.  A mistake mutation in a daughter cell will not be amplified.  Additionally, more than 80% of the colonies are found to contain the selected DNA sequence when plated out.

ï    This method works best with small numbers of base pair deletions, insertions, or substitutions, usually between 1 and 12.  There are optimum numbers of base pair insertions, deletions, and substitutions and optimum numbers of base pairs in the vectors used. For more specific information on these numbers please click  here and scroll down to look at the chart for optimum number of base pairs in certain situations.

1 Campbell, Neil A. Biology. 4th Ed. New York: The Benjamin/Cummings Publishing Company, Inc., 1996. pg. 317 &338.

1999 February 11. PfuTurbo DNA Polymerase.<http://www.stratagene.com/pcr/pfuturbo.htm> Accessed 1999

February 15.
1999 February 11. QuikChange Site-Directed Mutagenesis Kit.<http://www.stratagene.com/mutagenesis/quikchng.

htmAccessed 1999 February 15.

Trajano R.1999 February 11.Stratagene Online Home Page.<http://www.stratagene.com> Accessed 1999 February

15 .

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