Weaknesses of Polymerase Chain Reaction

There are some drawbacks of using PCR that one should be aware of as well. First, if you are wanting to amplify a specific gene, you will need some knowledge of the gene’s DNA sequence in order to properly design some primers. In this case, specific primers are used so that amplificationof only the gene takes place, rather than random areas of the chromosome. There may also be a need to optimize concentrations of each chemical component. For example changing the amount of DNA template, MgCl₂ and Taq polymerase can affect both the quantity and quality of bands produced. Some studies have shown that even the brand of Taq polymerase can affect results (Holden et al. 2003). Likewise the temperature cycles may need adjusted. 

When PCR was originally developed, DNA fragments in the range of 100 - 500 bp could be copied. Now there are modifications which allow successful PCR for DNA fragments up to 5 kb, but any larger and the reaction becomes less efficient (Barnes 1994; Cheng et al. 1994). There are reports of long extension PCR with fragments of as large as 42 kb being amplified, but these types of reactions are not yet routine (Barnes 1994; Cheng et al. 1994). Therefore, PCR is currently limited to copying only parts of genes or other DNA clones.