Hi! I am using nested PCR approach to detect mucosal HPV in cervical specimens as well as from FFPE tissues. This is to increase the sensitivity. But in doing so, I am getting a same similar size band of 150 bp which in some samples on sequencing gives non HPV match or no match in NCBI. What could be the reason? However, the blood negative control shows no band & cell line positive samples show a good seq of the GP band. Can anyone explain me the spurious amplification in these cervical samples?
Presumably your primers are amplifying another virus not found in your positive or negative controls but found in some of your samples. In this case there may be a sample that amplifies both virus sequences which will give an unreadable sequence being a mixture of both sequences. BLASTing the sequence should give an indication as to which organism is giving you the second sequence if you are lucky and your sequence has been deposited online.
formalin fixation cause cross linking between nucleic acid and protein and hence during DNA extraction of FFPE specimen, some other DNA may be fragmented. These fragmented DNA can somehow act like a primer amplifying unspecific sequences and giving you spurious band.
Thanks Paul. Thanks Yong.
The blast is maximum times a fungal type or no significant match.I wd look at the clinical characters of such females where I get fungal match definitely. But still my question is if GP nested is prone to false positivity ?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line