I isolated clones from a preconstructed cDNA library. After sequencing the clones, I found that they did not match known genes via BLAST as they should have, and that the sequence is characteristic of intronic regions. How could a cDNA library come to contain multiple clones of intronic DNA? How could this be avoided in future cDNA library construction? Is this a problem with impure mRNA?
Hi Cici,
All of your sequences are intronic regions or some of them? Maybe you are just seeing different isoforms of mRNA with intronic regions after alternative splicing. Have a look about alternative splicing in your organism of study.
And how did you isolate your RNA and construct the cDNA library?
Best,
Michele
Hi Daniel,
But how she would separate small fragments of intronic DNA when constructing cDNA library? E.g. If she used poly-A mRNA purification and poly-T por cDNA library construction, I do not think a lot of gDNA would be amplified. For random primers maybe, but I still don't know.
Best,
Michele
Thank you both very much for your responses.
The library was pre-constructed and provided to our lab (B73 strain of Zea mays). (Although I did not construct the library myself, I wanted to understand how this problem could happen and how the library construction technique could be troubleshooted.)
Some clones displayed exonic cDNA but multiple clones were intronic DNA.
Stable intronic RNA with important cellular functions have been described in several biological systems including plants. They are observed in the cytoplasm (and thus may be included in a cDNA library construction). See the following links :
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4138330/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4621838/
http://www.cell.com/current-biology/fulltext/S0960-9822(17)30209-9?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0960982217302099%3Fshowall%3Dtrue
http://science.sciencemag.org/content/331/6013/76
https://www.tandfonline.com/doi/full/10.1080/15476286.2018.1445404
Hi, dear. I am thinking that your cDNA most probably has some DNA contamination. Best way for checking DNA contamination is doing PCR with some House-keeping gene primers, It can show you if you have some DNA in your cDNA library.
Cheers,
Mahdi
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