“The PCR is a process employed to amplify the DNA and used in the DNA sequencing as well to get DNA copies, to reduce contamination, identify DNA mutations and recombinant clones.”
In the conventional PCR method, in order to get copies of DNA, our gene of interest or DNA is amplified enzymatically using the forward and reverse primer sets. While in the DNA sequencing process, the amplified DNA sequence is determined or the order of the sequence is determined computationally using a fluorescent dye or probe.
Both the processes are different and the purpose of both the technology is different, the immediately a question strikes in mind, what we are using the PCR in the DNA sequencing? In this short section, we are trying to answer this present question.
Lets, understand first, PCR and DNA sequencing separately.
The PCR is discovered in the year, 1983 by Kerry Mullis. A cyclic enzymatic temperature-dependent reaction- PCR amplifies or copies our DNA until a desired amount of amplicons are generated.
In the denaturation step, the DNA is denatured or break open into the two single-stranded DNA molecules. Genomic DNA, viral DNA, bacterial DNA or plant DNA is used as template DNA in the PCR reaction.
In the next step, an annealing step, The sequence-specific primers are bind to the complementary sequence of the DNA.
In the last step called an extension step, with the help of dNTPs, the Taq DNA polymerase adds nucleotides to the growing DNA strand.
The entire process of the PCR is shown in the figure below,
You can also read our article on PCR: Polymerase chain reaction.
Contrary, in DNA sequencing, the order of the nucleotides is determined. In the year 1977, Fredrick Sanger postulated the chain termination method of DNA sequencing, in the same year, Allan Maxam and Walter Gilbert, developed the chemical cleavage method of DNA sequencing.
Though both methods are different techniques, the central idea of both the methods are same to use the fluorescent molecules in determining DNA order.
Different fluorescent molecules are used for different dNTPs which emits light of different wavelengths. Each signal is recorded separately as a peak of the fluorescent. See the figure below,
The image shows different peaks of different dNTPs emits different fluorescent.
Now coming to our question,
Why is the PCR used in the process of DNA sequencing?
Why we use the PCR? To obtain multiple copies of DNA, obviously.
So, if the DNA sample is too small we can get millions of copies of DNA of our interest.
- Therefore, to sequence the sample having a low copy of DNA, PCR amplification facilitates additional advantages by multiplying DNA. For the investigation of the crime scene, crime samples such as hair, blood spot or any body fluid, PCR is used to amplify the DNA before DNA sequencing.
- For sequencing of some rare samples such as fossils, old samples of mummies and samples of extinct species, PCR is used for generating thousands of copies of DNA from such a sample. we can save the DNA of those precious samples.
- The chance of reannealing of single-stranded DNA is higher into the longer DNA templates (during the sequencing). In such cases, PCR helps in the generation of shorter DNA fragments and prevents the reannealing of DNA.
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- The PCR or amplification is one of the steps in DNA sequencing. Specifically, in the Sanger sequencing.
In the Sanger sequencing, the dNTPs and labeled ddNTPs are added to the growing DNA strand during the annealing steps of the PCR reaction.
Once the DNA is denatured, ddNTPs are added and the chain of the reaction is terminated. By doing this, the entire sequence of DNA is determined.
- Furthermore, in the bridge amplification step of the next-generation DNA sequencing, the PCR plays crucial roles by adding labeled nucleotides to the cluster of the single-stranded DNA on a surface. See the figure of bridge amplification,
In addition to this,
Before DNA sequencing, doing PCR is a wise decision because if our sample is too low or lost during transportation, the amplicons can be used. However, the sample DNA can directly be used for DNA sequencing as well.
The directly extracted DNA is generally not preferred for DNA sequencing because the extracted genomic DNA might be contaminated with other chemicals or DNA.
So the chance of the reaction failure in sequencing is high if we use directly the extracted genomic DNA. On the other hand, the amplified DNA or the amplicons are the pure forms of DNA. It does not have any contaminants in it.
Therefore use amplicon instead of direct genomic DNA for DNA sequencing.
- Amplicons of 16S rRNA and 18sRNA of bacteria are used in DNA sequencing instead of genomic DNA for the identification of bacterial species. We have covered an interesting article on this topic you can read it here: 16S rRNA: Gene, Sequencing and Importance.
- Interestingly, DNA sequencing is used to compare the PCR product with the template DNA to find out the mutation that occurred during the PCR reaction.
The activity of the Taq DNA polymerase is decreased over a period of time and it adds mismatched nucleotides into the growing DNA strand. These mismatches can be encountered using DNA sequencing.
PCR and DNA sequencing are also combinedly used to screen recombinant clone and identification of inserted sequence within the clone or plasmid.
- Furthermore, DNA sequencing is used in the confirmation of critical PCR results which can not be interpreted without the sequence information.
For more information of PCR and other components used in the PCR reaction and all about DNA sequencing read our additional resources enlisted below.
Additional resources related to this topic:
-History, definition, principle, procedure, steps and applications. Read more…..
-History, definition, principle, Different types of sequencing methods, application and limitations. Read more……..
PCR is one key technique in any of the genomic downstream applications, starting from the identification of SNP to DNA sequencing.
Further, the PCR is used in the diagnosis of several inherited diseases as well. The technique is used independently as well as in the combination of several other techniques. Amplification is one of the crucial steps in DNA sequencing.
We had covered some of the articles on the different types of PCR techniques. Read these articles here: Different types of PCR.