Editing Polymerase chain reaction (section)

==Limitations==
One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification.<ref name="Garibyan, Avashia 1–4"/> This means that, typically, PCR users must know the precise sequence(s) upstream of the target region on each of the two single-stranded templates in order to ensure that the DNA polymerase properly binds to the primer-template hybrids and subsequently generates the entire target region during DNA synthesis.{{<ref>{{cite journal |pmc=4102308 |date=2013 |title=Research Techniques Made Simple: Polymerase Chain Reaction (PCR) |journal=The Journal of Investigative Dermatology |volume=133 |issue=3 |pages=1–4 |doi=10.1038/jid.2013.1 |pmid=23399825 | vauthors = Garibyan L, Avashia N }}</ref><ref>{{cite journal |url=https://pubmed.ncbi.nlm.nih.gov/23794048/ |pmid=23794048 |date=2013 |title=Specific primer design for the polymerase chain reaction |journal=Biotechnology Letters |volume=35 |issue=10 |pages=1541–1549 |doi=10.1007/s10529-013-1249-8 | vauthors = Chuang LY, Cheng YH, Yang CH }}</ref>}}

Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR fragments that are generated.<ref>{{cite journal | vauthors = Zhou YH, Zhang XP, Ebright RH | title = Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase | journal = Nucleic Acids Research | volume = 19 | issue = 21 | pages = 6052 | date = November 1991 | pmid = 1658751 | pmc = 329070 | doi = 10.1093/nar/19.21.6052 }}</ref>

Another limitation of PCR is that even the smallest amount of contaminating DNA can be amplified, resulting in misleading or ambiguous results. To minimize the chance of contamination, investigators should reserve separate rooms for reagent preparation, the PCR, and analysis of product. Reagents should be dispensed into single-use [[Sample (material)|aliquots]]. Pipettors with disposable plungers and extra-long pipette tips should be routinely used.<ref name="Schochetman 1988 1154–1157"/> It is moreover recommended to ensure that the lab set-up follows a unidirectional workflow. No materials or reagents used in the PCR and analysis rooms should ever be taken into the PCR preparation room without thorough decontamination.<ref>{{Cite web|last=Stursberg|first=Stephanie|date=2021|title=Setting up a PCR lab from scratch|url=https://www.integra-biosciences.com/en/blog/article/setting-pcr-lab-scratch|website=INTEGRA Biosciences}}</ref>

Environmental samples that contain humic acids may inhibit PCR amplification and lead to inaccurate results.{{citation needed|date=August 2024}}