Why it is said that we must use purified PCR products for pyrosequencing and not the cDNA. what problems are going to happen if someone uses cDNA for sequencing
You can use pyrosequencing for cDNA. It is the basis for the Roche 454 sequencer . The problem for normal pyrosequencers ( old style not next generation) is signal strength and amplified pcr product gives a good strong signal but to get a stronger signal from mixed cDNAs it can be made into a library then sequenced
It is generally recommended to use purified PCR products for pyrosequencing, rather than cDNA (complementary DNA). This is because cDNA is synthesized from RNA, which may contain contaminants or artifacts that can interfere with the accuracy of the pyrosequencing results. In addition, cDNA may contain secondary structure or other factors that can affect the efficiency of the pyrosequencing reaction.
Using purified PCR products can help to ensure that the DNA template used for pyrosequencing is free of contaminants and is of high quality, which can improve the accuracy and reliability of the pyrosequencing results.
If someone were to use cDNA for pyrosequencing, it is possible that the results could be inaccurate or misleading due to the presence of contaminants or other factors in the cDNA template. It is therefore important to use purified PCR products for pyrosequencing to obtain the most accurate and reliable results.
Thank you soo much Bertrand Cornu for your valuable answer. it is really very helpful and made me to understand the reason very well. Thanks again.
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