Nested PCR

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Nested PCR

Nested PCR is one of the types of PCR designed to ensure specificity and accuracy of experiment. The main purpose of utilizing the Nested PCR is reduce the possibility of occurrence of non-specific bands.  In conventional types of PCR, there are more chances that we get non-specific bands, so Nested PCR is introduced to overcome the drawback. 
Two primers are used during the said reaction. First two set of primers are used for the amplification of initial product, whereas the product acquired from the first round is used to amplify the targeted sequence by using second set set of primers.
So that is quite less likely that we get any non-specific or unwanted bands contaminating our desired product. The primers of the first round are termed as the outer primers and are used to let the inner primers or nested primers to anneal to a specific DNA sequence. Outer primers are present upstream of the internal primers. 




Nested PCR requires smaller number of cycles around 15 to 20, this is why Nested PCR is more reliable as compared to conventional PCR. Apart from that, the Nested PCR is less prone to getting unwanted bands. 


Limitations of Nested PCR

More Costly: The procedure is more expensive as compared to other procedure have been used for the acquirement of specific DNA product. 

Apart from cost, another drawback associated with the use of Nested PCR is that sample is prone to getting contaminated as the products of first round are transferred to the other tube for the second round of amplification. 






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