My PCR products are showing mutations in the non-coding region of the gene. Do these mutations play any role in the unusual functioning of the gene? Or they are of no use?
Dear Arushdeep,
Answer of your question is yes, and non-coding regions may affect a gene function/expression.
Noncoding DNA sequences /Junk DNA (over 98% of the human genome) are components of an organism's DNA that do not encode protein sequences. Some noncoding DNA is transcribed into functional non-coding RNA molecules (e.g. transfer RNA, ribosomal RNA, and regulatory RNAs), while others are not transcribed or give rise to RNA transcripts of unknown function. These include genes for functional RNA molecules and sequences such as origins of replication, centromeres, and telomeres. Many types of non-coding DNA sequences do have important biological functions, including the transcriptional and translational regulation of protein-coding sequences, origins of DNA replication, centromeres, telomeres, scaffold attachment regions (SARs), genes for functional RNAs, and many others.
best
mehdi
Dear Jochen, thank you for rapid description. In total human DNA content only a small amount is related to genes (a distinct sequence of nucleotides, an information unit, which contains specific information e.g. protein sequence information.).
non coding sequences distribute inside (intron) and outside genes. Non coding sequences have effect on transcription and translational process and it means these large amount non coding sequence can affect expression and function of genes, in both inside and out side genes.
Finally I think as Dear Jochen told ( mutations in "intra-genic" non-coding regions can (and do) affect the expression and function of the gene.)
best
mehdi
Should've used a high-fidelity DNA polymerase for PCR to avoid or minimize errors
@ Jochen and Mehdi,
It is an interesting discussion...
Does that mean that using (cloning) a full-length of gene (introns + exons) is better than using a cDNA (no introns) version for overexpression experiment? What about if I use a strong promoter for both of them (such as 35S). Will the overexpression experiment make any difference?
Yes, for example , more than 15 diseases cause by intronic mutations. Some parts of introns (non coding sequence) play role in splicing like ISS and ISE. Hence mutation of them affects splicing machinery and gene expression.
@ Arushdeep Sidana: Jochen's answer is excellent +rep. I would like to add that it will be very difficult to prove the effect of intronic mutations except for those affecting 1.) splice sites or 2.) known regulatory sequences. So my approach to investigating the intronic mutations would be to make a map of the gene and then take a closer look at the mutations in those places. After that maybe look for "hot spots" where lots of mutations occur. *edit* as Rocio says below, of course focus on mutations in coding regions before everything else!*edit*
@ Yuan-Yeu Yau: There is no general rule to this and it strongly depends on which gene you are investigating. I have heard of quite a few cases were only one of both (cDNA or full-length) works well, and also of cases where the presence of intron 1 is needed for translation to work. Importantly, it will be more difficult to clone and manipulate (e.g. add EGFP tag to) the full-length sequence than the cDNA just due to the size. For example: I did overexpression of Dnmt1 in mouse cells - the cDNA is ~5kb (easy) , whereas the full-length is ~60kb (not so easy). Many labs won't even try full-length unless there is a really good reason to do so.
Apart from that it depends on what you are investigating. If you are analyzing the effects of intronic or splice site mutations on expression, you have no choice but to use the full-length. If you are analyzing the effects of the overexpression, full-length or cDNA should not make a difference as long as they both result in translation of the correct protein.
As Jochen explained thoroughly, non-coding mutations in a gene can alter the gene's function in many different ways. However, I wouldn't immediately assume that the non-coding mutations you see in your DNA are the cause of any alteration you may be seeing in gene function.
In general, as a consequence of less evolutionary pressure, non-coding regions of a gene allow for much more genetic variation than coding regions. This means that you have many more common and rare mutations in non-coding regions of a gene versus coding regions. Therefore, it's possible that the mutations you see in non-coding regions are just background genetic variation with no impact on gene function.
Mutations in non-coding areas are much more difficult to interpret than mutations in coding regions because we don't know as much about non-coding regions and we're rather bad at predicting the effect of variants in non-coding regions. Being pragmatic, when looking for the cause of alterations in gene function, I would start by looking for something in the coding region and only after that, open up to non-coding regions.
If you think that the non-coding mutations you see may be behind alterations in gene function, maybe the best is to follow this up with RNA studies (mRNA expression levels and sequencing to look for aberrant splicing).
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs