“If you know the process of sequencing, amplification by PCR is a dedicated step. Though PCR has its own importance, it is utilized in sequencing for various purposes. Let’s find out why PCR is used in the process of DNA sequencing.”
In a sentence, the answer to this question is to ‘get copies of a DNA, but it can be comprehensively explained which certainly opens new understanding opportunities for concepts in sequencing.
So let’s quickly go through the concept of PCR and sequencing.
Sequencing is an amazing technology and so PCR is! Both have their own advantages and limitations. PCR can investigate “known” alteration but can’t read it, technically; whereas sequencing can read it.
Sequencing actually tells us what the variation/alteration or the sequence is all about but unfortunately, the process is costly and tedious.
PCR is an amplification technique that was proposed in 1983 by Kary Mullis. Theoretically, the proposed definition of PCR is an in vitro process of DNA synthesis or replication. However, it’s notable that the actual process is totally different from the in vivo replication.
On the other hand, technically, the PCR is an enzyme-governed, temperature-dependent cyclic reaction. In a series of temperature alterations, a temperature stable polymerase performs the synthesis.
Three temperature steps in PCR are denaturation at 90 to 95°C, annealing at 55 to 65°C and extension at 72°C, followed by initial denaturation and final extension. Each step exhibits a specific function to denature or open the dsDNA, anneal the primers and extend the new strand using polymerase, respectively.
Furthermore, a typical PCR reaction contains Taq DNA polymerase, dNTPs (nucleotides), PCR primers, PCR buffer with reaction boosters, template DNA and ultra-pure nuclease-free water.
This is a brief overview, to know more you can follow our article on PCR: Polymerase Chain Reaction (PCR)- Definition, Principle, Steps, Procedure, Protocol, Applications and Types.
Contrary, sequencing is totally a separate state-of-art and high throughput technology to read every nucleotide. Keep in note that there are so many sequencing chemistries and platforms available nowadays, so we can’t accurately explain it with a single universal principle.
Nonetheless, a few steps are usually common in most of the techniques. Larger chinks of DNA that can’t be processed, are fragmented, ligated with a known adapter and sequenced in a machine.
The first sequencing platform was discovered by Sanger in 1973 using a chain termination method. Put simply, when a polymerase finds a ddNTP, instead of dNTP, the incorporation terminates the synthesis.
When the fragments are run on a PAGE or capillary electrophoresis, using the size-based separation, a sequence can be identified. To learn more about sequencing, follow this article: DNA Sequencing: History, Steps, Methods, Applications and Limitations.
Now the question is, where does PCR stand here, in the sequencing? And if so, why is it used in the process of sequencing?
Let us find out.
PCR in sequencing:
Firstly, the first generation sequencing techniques, as we described like the Sanger sequencing, rely on the amplification by polymerase and chain termination by ddNTPs. In such sequencing techniques, the sequencing is directly performed in a thermocycler (with minor modifications).
Note that, radio-labeling, fluoro-labeling or staining the PAGE gel with EtBr helps to investigate results. We can say the sequencing actually initiated using a PCR machine.
Now over the period and timely new chemistries in sequencing are introduced that actually do not use PCR directly in the process. For instance, In a Maxam-Gilbert method, chemical degradation occurs entirely using chemicals.
Whereas pyrosequencing measures the release of free pyrophosphate in the enzymatic reaction. It is important to understand that for any platform, to sequence a fragment or a gene, we need millions of copies of that particular fragment.
And here my friends, PCR is used to amplify a target gene, sequence or stand of DNA before sending it to the sequencer. Henceforth, PCR is a mandatory step in sequencing to get millions of copies of a target.
If we are talking about the most robust, advanced and rapid next-generation sequencing techniques, PCR remains irreplaceable here also. In this era of whole genome sequencing, the fragments of genomic DNA are amplified and stored in genomic DNA libraries.
See, the entire genome can’t be sequenced in a single or a few runs, thus researchers digest it and store it in a location called libraries. Genomic libraries are collections of known and similar-sized fragments.
Libraries are enriched by PCR amplification which can then be used anytime and anywhere.
Bridge amplification is yet another phenomenon in NGS, a crucial and important step in which the DNA is amplified vertically in a massively parallel fashion which generates clusters of DNA on a flow cell.
Bridge amplification, in a core, is a process of polymerase chain reaction (PCR).
It is not advisable to use genomic DNA directly in sequencing, it’s less in quantity, we have to isolate the region, a gene or target which we want to sequence. Using a sequence or target-specific primer set followed by amplification, our target can be enriched.
It’s easier for a machine to read a fragment than the entire genome. So PCR helps in actual target enrichment. These amplicons can be stored and used for future projects.
It is mandatory to use PCR before sequencing for low copy DNA samples such as rare fossils, old samples from mummies, samples from extinct species, environmental samples, and some cancer or fetal samples.
DNA from such a rare sample is carefully extracted, digested and amplified in a PCR to prepare libraries. Once we stored the DNA in the genomic library now we can use it anytime for any experiments.
Crime scene, criminal and rare body fluid samples are processed sequentially as above explained before sending it to sequencing.
In specialized sequencing platforms like whole-exome sequencing where we need cDNA, reverse transcription PCR helps to convert the mRNA into cDNA, thereby cDNA libraries are constructed.
cDNA library enrichment is even achieved by another round of PCR amplification.
Further to this, quantitative PCR techniques like real-time PCR, even have a post-processing role. Quantitative PCR can measure the sequenced fragment. Such assays help in the evaluation of sequencing accuracy and specificity.
As I said before, the answer in one line to the present question is to get copies of DNA, however, in-depth knowledge provided here shows that the PCR is used in the process of sequencing for creating genomic DNA library, library enrichment and storing precious biological material.
In addition, the bridge amplification-like step is actually a PCR step that provides a huge number of copies of a fragment and reads it in the same process as well, in NGS.