Applications and Alternatives
With the basic PCR technique well established, the world turned to adapting PCR for a broad range of purposes. These range from forensics and authentication of works of art, to molecular computation, total amplification of all DNA in a sample, production of recombinant genetic constructs, and routine clinical diagnostics.
Another activity spurred by the success of PCR was inventing alternative techniques. This was done either with the hope of avoiding broad patents or for convenience.
Examples of such alternative amplification methods are nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), and loop-mediated isothermal amplification (LAMP).
In practice, however, PCR easily maintains grounds as the dominant approach for sensitive and specific gene detection.
Is there any room for alternatives to PCR for future genetic analyses? Well, there certainly are situations where PCR may not represent the ultimate solution.
First, PCR’s requirement for target recognition by pairs of oligos carries with it a rapidly increasing risk of cross-reactive detection as more sequences are targeted in the same reaction. The problem has been addressed, for example, by compartmentalizing many individual amplification reactions, something that can also allow digital counts of sequence copy numbers when applied with limiting amounts of sample DNA.
An alternative approach to achieving multiplex gene detection is that of using linear probes that incorporate pairs of target-complementary sequences at either ends, and substituting the polymerase with a DNA ligase that can connect the ends and thus circularize the probe strands when properly hybridized to their targets. Unlike PCR, these so-called padlock probes have been shown by us and others to permit tens of thousands target sequences to be targeted in the same reaction.
Incidentally, the circularized probes also lend themselves to localized amplification via a rolling circle amplification mechanism, resulting in easily detectable individual reaction products, as an attractive alternative to digital PCR for counting target molecules with no requirement for compartmentalization of reaction products and limiting dilution of samples.
One shortcoming of PCR is the restriction to amplification of DNA molecules, and of RNA molecules only after reverse transcription to DNA. Surely proteins represent another class of macromolecules in great need of amplified detection, but neither PCR nor any other known process is capable of amplifying protein sequences in convenient in vitro reactions.
The proximity ligation reaction developed in our lab meets this challenge by detecting target protein via binding by pairs of oligo-modified antibodies, resulting in the production through DNA ligation of amplifiable reporter DNA strands, as a replacement for amplifying the actual protein molecules.
A radically different approach to PCR and other affinity-based molecular detection reactions is that of simply analyzing all molecules in order to identify them via the sequence of building blocks they are composed of.
For protein analysis this is the approach taken via mass spectrometry. For nucleic acids, next-generation sequencing now affords a means of recording the nucleotide sequence of all molecules in a sample, so that the analysis of specific sequences in the sample can subsequently be undertaken entirely in silico.
So, will next-generation sequencing and its successors replace PCR for DNA detection? For my money, this will only be the case to a limited extent. Future molecular tools for DNA analysis are likely to remain dependent on affinity reactions to seek out molecules of interest. They are likely to exhibit greater molecular complexity than current techniques, so as to further improve specificity, efficiency, and convenience of reporting, but they will also be far simpler to use.