“Explore 7 key roles of PCR in DNA sequencing— beyond just amplification! Learn how PCR is applied in sequencing in this insightful article.
If someone asks this question, the straightforward answer would be to “amplify the target DNA and generate multiple copies for analysis,” right?
That’s partially true!
PCR, often referred to as the “DNA Xerox machine,” plays a crucial role in various types of DNA sequencing techniques by amplifying target DNA, enriching sequencing libraries, enabling cycle sequencing, facilitating reverse transcription, and quantifying as well as enhancing the detection of rare or low-abundance DNA samples.
Curious!
Let’s explore how PCR is used in DNA sequencing, in detail.
7 Ways PCR is Used in DNA Sequencing:
The very first and most common application of PCR in DNA sequencing is to specify the target. Meaning, which region do we want to sequence? That particular region is first covered, amplified and enriched using the PCR.
Techniques like Sanger sequencing highly rely on PCR as it has a limited sequencing range (<1000bp). Furthermore, in the automated Sanger sequencing as well, target amplification is a dedicated step.
Here, a set of primers (forward and reverse) has been used to cover and amplify the target region or gene. The reason is, that a complete genome can’t be amplified using Sanger sequencing, in a single run– technically!
Second, again PCR amplification has been used for cycle sequencing in the same method. After amplification and PCR purification, the product is ready for sequencing. Now, the amplified fragments are our template for sequencing.
With a sequencing mix and a single primer, again the template DNA is amplified and sent for fragment analysis by capillary gel electrophoresis.
So in the same sequencing technique (in Sanger sequencing) PCR is used twice!
Third, it is used for library preparation for downstream analysis such as NGS. Libraries are collections of similar-sized fragments. Once the DNA fragmentation has been completed, all the fragments are equally amplified in the PCR.
This process is known as library enrichment and is so important in the case of Next-generation sequencing. Excellently enriched libraries give excellent results.
Both genomic DNA libraries and cDNA libraries are enriched with the same PCR method and then used for various applications like whole-genome sequencing, whole-exome sequencing or RNA sequencing.
Notedly, bridge amplification which is a form of amplification (just like the PCR!) is used in the sequencing-by-synthesis process in NGS to generate copies of DNA fragments on a solid surface.
The fourth and most crucial application is to amplify the low-abundant DNA or samples.
Low-copy DNA samples, such as those from ancient fossils, mummies, extinct species, environmental sources, or rare medical specimens (e.g., cancer or fetal DNA), require careful amplification to ensure sufficient DNA for sequencing.
Because these samples often contain degraded or minimal genetic material, highly sensitive techniques like PCR-based enrichment, whole genome amplification, or targeted capture are used to enhance sequencing success while minimizing contamination and errors.
DNA from such rare samples is carefully extracted, amplified and stored in the libraries. Crime scene samples such as blood or semen spots, hair or other body fluid have been processed using the same scheme before investigation.
Fifth, RNA samples are difficult to sequence directly. Here also, PCR is used to solve the problem. The technique known as reverse transcription PCR is employed to convert the RNA into cDNA.
The reaction of RNA to cDNA conversion is governed by the enzyme reverse transcription. Both reverse transcription and amplification have been performed using PCR. Afterward, the cDNA is ready for sequencing.
The same process is followed for cDNA library preparation for the NGS.
Next, quantitative PCR is used for library quantification and post-sequencing quantitative gene expression analysis.
Lastly, PCR becomes a crucial player when scientists have to sequence the low abundant gene variant. Meaning, that a variant, present in the very least or trace amount in the sample has been first amplified and then sequenced. This will increase the detection sensitivity and accuracy.
Wrapping up:
In conclusion, PCR is used in sequencing to increase the accuracy, sensitivity and detection range. I hope now you understand why we use PCR in the sequencing. Share this article or link back and subscribe to Genetic Education.
If you want to learn more about DNA sequencing and/or PCR, you can check out our courses at Genetic Education Academy.
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