What is the Difference Between Conventional Nested and Real-time PCR Assays?

The main differences between conventional nested and real-time PCR assays are as follows:

Conventional PCR (C-PCR):

• Developed to amplify specific fragments of DNA.
• Single amplification reaction with a single set of primers.
• Less sensitive compared to nested and real-time PCR.

Nested PCR (N-PCR):

• Developed to improve the sensitivity of conventional PCR.
• Requires two successive amplification reactions with two separate sets of primers.
• The first round of amplification is performed with the outer primers, and the product is then used as a template for the second round of amplification with the inner primers.
• More sensitive than conventional PCR but can give erroneous positive results due to DNA contamination.

Real-time PCR (Q-PCR):

• Developed to quantify the amplified PCR product.
• Able to provide results within 2 hours from genomic DNA extraction to data analysis.
• Highly sensitive and specific.
• Potential for automation and high-throughput analysis.

In summary, conventional PCR is the original technique for amplifying specific DNA fragments, while nested PCR is a variation that improves sensitivity by using two successive amplification reactions with two sets of primers. Real-time PCR, on the other hand, is a more advanced technique that allows for the quantification of amplified PCR products and offers higher sensitivity and specificity compared to conventional and nested PCR assays.

Comparative Table: Conventional Nested vs Real-time PCR Assays

Here is a table comparing the differences between conventional nested and real-time PCR assays:

Both conventional nested and real-time PCR assays are modifications of conventional PCR and are used to amplify DNA samples for various genetic analyses. However, real-time PCR is a more sensitive and specific technique that allows for the quantification of amplified products, which is not possible with conventional nested PCR. Real-time PCR also has the advantage of shorter processing time and being less labor-intensive compared to conventional nested PCR.