Since its introduction in the 1980s, polymerase chain reaction (PCR) has become a standard tool in biomedical research. The PCR equipment (a thermal cycler) and reagents (thermostable polymerases, oligonucleotides, and so on) are widely available and reasonably priced. One advantage of PCR is its high sensitivity, which allows it to detect and analyse low abundance DNAs. This is especially useful when starting material is scarce or only a few copies of the target sequence are present.
All molecular biology products and procedures, including PCR, have advanced over time. We have listed some of the applications of PCR that most people are unaware of in this blog. Let’s get started!
Applications Of PCR Technology That Most People Don’t Know
PCR Based Cloning And Sequencing Methods
PCR has been used to facilitate the cloning of known DNAs and to identify novel DNAs. It can be performed on genomic DNA as well as cDNA derived from mRNA (reverse transcription PCR (RT-PCR)). This eliminates the need for the creation and testing of DNA libraries. When the target DNA sequence is known, it is simple to amplify the target DNA using oligonucleotide primers based on the sequence.
When only a portion of a target cDNA sequence is known, rapid amplification of cDNA ends (RACE) can be used to amplify unknown sequences at the 5′ or 3′ ends of the known region. A technique known as homology PCR can also be used to identify novel homologues of known proteins. The degenerate mixtures of oligonucleotide primers designed to recognise conserved motifs are used in this method. Homology PCR can be used to clone novel protein family members or protein homologues from other species. (Robinson)
To detect genetic polymorphisms in large populations, PCR-based techniques such as allele-specific PCR and PCR restriction length polymorphism (PCR-RFLP) can be used. PCR fragments can also be sequenced directly, allowing for population screening for novel mutations. PCR has been directly incorporated into DNA sequencing technologies, allowing for the sequencing of small (nanogram) amounts of target DNA as well as the sequencing of unpurified DNA— Direct sequencing of DNA from bacterial colonies or phage plaques, for example.
Construction Of Mutant And Chimeric DNA Using PCR
Mutant and chimeric DNA sequences are frequently used in studies of gene and protein structure and function. PCR primers containing one or more point mutations, deletions, or insertions can be used to introduce mutations. Splice overlap extension PCR can join two or more distinct DNA sequences. This method greatly simplifies the production of cDNAs encoding chimeric and fusion proteins because it does not require the presence of restriction sites or other specific sequences. (Robinson)
Quantification Of mRNA Using PCR
PCR has been widely used to assess mRNA expression levels. The main advantage of PCR-based mRNA quantification methods is their high sensitivity. If the amount of starting material is limited (as is often the case when analysing mRNA expression in primary cells) or the target sequence is expressed at a very low copy number, PCR-based methods may be the only option. However, when RT-PCR is performed in a standard manner (reverse transcription of RNA followed by a fixed number of cycles of cDNA amplification), the amount of product DNA does not correlate with the amount of input RNA in a consistent or predictable manner.
PCR has been widely used to assess mRNA expression levels. The main advantage of PCR-based mRNA quantification methods is their high sensitivity. If the amount of starting material is limited (as is often the case when analysing mRNA expression in primary cells) or the target sequence is expressed at a very low copy number, PCR-based methods may be the only option. However, when RT-PCR is performed in a standard manner (reverse transcription of RNA followed by a fixed number of cycles of cDNA amplification), the amount of product DNA does not correlate with the amount of input RNA in a consistent or predictable manner. (Robinson)
To Wrap Up:
PCR is a very helpful technique and its applications have undoubtedly brought about a revolution in the field of biological testing. With that said, if you want to order PCR plates and the best quality filter tips like the Hamilton filter tips, Molecular Biology Products has them available for you.
Work Cited
Robinson, Professor B W S. “Recent advances in molecular biological techniques and their relevance to pulmonary research.” BMJ Journals, Thorax.bmj, 09 02 2018, https://thorax.bmj.com/content/55/4/329. Accessed 26 05 2022.
