Advertisement

PCR Protocols pp 467-468 | Cite as

Cloning and Mutagenesis

A Technical Overview
  • Helen Pearson
  • David Stirling
Part of the Methods in Molecular Biology™ book series (MIMB, volume 226)

Abstract

Strategies for cloning polymerase chain reaction (PCR) products and performing in vitro site-directed mutagenesis are legion, and the following chapters outline five robust and reliable protocols. Before embarking on such a strategy, however, it is worth considering if it is entirely necessary. Even high-fidelity, proofreading Taq polymerases carry the risk of misincorporated bases being included, especially late in the PCR, when dNTP concentrations may become limiting. Thus, if the product is cloned, there is a chance that the clone selected contains a misincorporated base. If the purpose of the exercise is simply to determine the sequence of the original template, it may be more appropriate to sequence the PCR product directly and so avoid such cloning bias. If the object is to produce a clone that can be further used in expression studies for instance, it is imperative that all cloned material is sequenced to verify its integrity, prior to expression.

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Product Polymerase Chain Reaction Primer Original Template Cloning Polymerase Chain Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Helen Pearson
    • 1
  • David Stirling
    • 2
  1. 1.Molecular Endocrinology UnitWestern General HospitalEdinburghUK
  2. 2.Department of HematologyRoyal Infirmary of EdinburghEdinburghUK

Personalised recommendations