John M. S. Bartlett.pdf - Bio-Nica.info

468 Pearson and Stirling
into PCR products by having these sequences incorporated into the 5′ end of the PCR
primer(s). The short restriction site sequence on the 5′ end of the PCR primer will not
hybridize, but as long as the 3′ hybridizing region is long enough (i.e., its T m is high
enough; ~20 mer), the primer will specifically bind to the appropriate site. The PCR
product will thus have an additional DNA sequence at the 5′ end that will contain the
endonuclease restriction site. A similar or different restriction site sequence can be
added via the other PCR primer. If the other primer has a different restriction sequence,
then the PCR fragment can be inserted in a directional-dependent manner in a host
plasmid. There are a number of potential problems with this method that should be
considered. There is no easy way to prevent internal sites containing similar restriction
sequences from being cut when the end of the PCR product are cut. Care should
therefore be taken to use restriction sites that are not present in the fragment to be
amplified. Restriction sequences are inverse repeat sequences, thus the potential exists
for primer dimer association and resultant non-productive annealing. Finally, when
restriction sites are located very close to the end of an amplified fragment, the efficiency
of cleavage of those sites can be markedly impaired. It may therefore be necessary
to include not just the restriction site but an additional 5 to 10 bases to avoid this
problem.
3. Mutagenesis
In vitro site-directed mutagenesis is an invaluable technique for studying protein
structure–function relationships, gene expression, and vector modification. Several
methods have appeared in the literature, but many of these methods require singlestranded
DNA as the template. The reason for this, historically, has been the need
for separating the complementary strands to prevent reannealing. Use of PCR in
site-directed mutagenesis accomplishes strand separation by using a denaturing step
to separate the complementing strands and allowing efficient polymerization of the
PCR primers. PCR site-directed methods thus allow site-specific mutations to be
incorporated in virtually any double-stranded plasmid, eliminating the need for M13-
based vectors or single-stranded rescue.
Three divergent strategies for mutagenesis are outlined in the following chapters;
however, several points applicable to all three should be here. First, it is often desirable
to reduce the number of cycles during PCR when performing PCR-based site-directed
mutagenesis to prevent clonal expansion of any (undesired) second-site mutations.
Limited cycling, which would result in reduced product yield, can be offset by
increasing the starting template concentration. Second, a selection must be used to
reduce the number of parental molecules coming through the reaction. This is of
particular importance when the parental molecules are used in high concentrations.
Third, because of the tendency of some thermostable polymerases to add nontemplatedirected
nucleotides to the ends of double-stranded DNA fragments, it is often necessary
to incorporate an end-polishing step into the procedure prior to end-to-end ligation of
the PCR-generated product containing the incorporated mutations in one or both PCR
primers (see Subheading 2.1.).
Finally, even if the presence of the desired mutation is confirmed by restriction
digest or sequencing, it is essential to sequence the entire region of manipulated
DNA to ensure that there has been no undesirable mutation introduced by the PCR
processes.