The PCR is used in the process of DNA sequencing to reduce the chance of contamination, identification of mutation and identification of recombinant clones.
In the conventional PCR, a defined specific DNA sequence of interest amplified using forward and reverse primers enzymatically and produces millions of copies of DNA.
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.
Both the processes are different and the purpose of both the technology is different, then how PCR is applicable in the DNA sequencing or why is PCR used in the process of DNA sequencing?
In this short FAQs section, we are trying to answer this present question.
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The PCR is discovered in the year, 1983 by Kerry Mullis.
The PCR is a cyclic enzymatic reaction in which the DNA of our interset amplified many times until the 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 binds 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: The complete guide of polymerase chain reaction
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.
There are different methods of DNA sequencing, however, the central idea of the DNA sequencing depends on the use of the fluorescent molecules.
Different fluorescent molecules are used for different dNTPs which emits light of different wavelength. Each signal is recorded separately into the peak of the fluorescent. See the figure below,
Image shows different peaks of different dNTPs emits different fluorescent.
Now coming to our question,
Why is 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.
- The chance of reannealing of single-stranded DNA is higher into the longer DNA templates. In such cases, PCR helps in the generation of shorter DNA fragments and prevents reannealing of DNA.
- What is a multiplex PCR?
- What is Restriction Digestion and how to do it?
- What is gene editing and CRISPR-CAS9?
- The PCR is also one of the steps in DNA sequencing. Specifically, in the Sanger sequencing.
In the Sanger sequencing, the dNTPs and labelled 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 labelled nucleotides to the cluster the single-stranded DNA into on the surface. See the figure of bridge amplification,
Before DNA sequencing, doing PCR is the wise decision because if our sample is too low or lost during transportation, the amplicons can be used. However, the sample DNA is directly 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 contaminant in it.
Therefore use amplicon instead of direct genomic DNA used into the DNA sequencing.
- Amplicons of 16S rRNA and 18sRNA of bacteria are used in the DNA sequencing instead of genomic DNA for identification of bacterial species.
- Interestingly, DNA sequencing is used to compare the PCR product with the template DNA to find any mutation occurred during the PCR reaction or not.
The activity of the Taq DNA polymerase is decreased over a period of time and it adds mismatched nucleotides into the growing DNA strand.
PCR and DNA sequencing are also combinedly used to screen recombinant clone and identification of inserted sequence within the clone.
- Furthermore, the 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 into 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 identification of SNP to DNA sequencing. Further, the PCR is used in the diagnosis of several inherited diseases as well. PCR 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.