Can it use CDS (coding domain sequence) clones for expression in RRL reactions (from transcribed RNA) and in cells (transfection)? Due to using CDS DNA, this RNA will be deprived of 5'-UTR and 3'-UTR. Will it be stable and translatable in vitro and in vivo?
0
3 replies to this topic
#2
perneseblue
-
- Global Moderators
-
- 530 posts
Unlimited ligation works!
8
Neutral
Neutral
Posted 07 November 2010 - 04:10 PM
The short answer is unknown.
The long answer....is complicated. There is much evidence that the 5' and 3' UTR contains structural elements that regulate the stability (or lack off) of the mRNA transcript. Found there too are element required in the control of the transcription and translation of the mRNA to produce a normal pattern protein expression in vivo.
Recent evidence also indicate that the introns also regulate the mRNA transcription and perhaps selection of splice sites. Transcription factor binding sites have been detected in intron. Lastly there are genes which will only express when intron are present in their native location. cDNA do not express protein.
However given the limited ability to manipulate large DNA sequences, it is likely there is not much choice other than to use cDNA.
The long answer....is complicated. There is much evidence that the 5' and 3' UTR contains structural elements that regulate the stability (or lack off) of the mRNA transcript. Found there too are element required in the control of the transcription and translation of the mRNA to produce a normal pattern protein expression in vivo.
Recent evidence also indicate that the introns also regulate the mRNA transcription and perhaps selection of splice sites. Transcription factor binding sites have been detected in intron. Lastly there are genes which will only express when intron are present in their native location. cDNA do not express protein.
However given the limited ability to manipulate large DNA sequences, it is likely there is not much choice other than to use cDNA.
May your PCR products be long, your protocols short and your boss on holiday
#3
Oleg
-
- Active Members
-
- 8 posts
member
0
Neutral
Neutral
Posted 07 November 2010 - 04:15 PM
perneseblue, on 07 November 2010 - 04:10 PM, said:
The short answer is unknown.
The long answer....is complicated. There is much evidence that the 5' and 3' UTR contains structural elements that regulate the stability (or lack off) of the mRNA transcript. Found there too are element required in the control of the transcription and translation of the mRNA to produce a normal pattern protein expression in vivo.
Recent evidence also indicate that the introns also regulate the mRNA transcription and perhaps selection of splice sites. Transcription factor binding sites have been detected in intron. Lastly there are genes which will only express when intron are present in their native location. cDNA do not express protein.
However given the limited ability to manipulate large DNA sequences, it is likely there is not much choice other than to use cDNA.
The long answer....is complicated. There is much evidence that the 5' and 3' UTR contains structural elements that regulate the stability (or lack off) of the mRNA transcript. Found there too are element required in the control of the transcription and translation of the mRNA to produce a normal pattern protein expression in vivo.
Recent evidence also indicate that the introns also regulate the mRNA transcription and perhaps selection of splice sites. Transcription factor binding sites have been detected in intron. Lastly there are genes which will only express when intron are present in their native location. cDNA do not express protein.
However given the limited ability to manipulate large DNA sequences, it is likely there is not much choice other than to use cDNA.
Thanks for your reply. Actually, there is a choice between a full-length cDNA (including 5'-UTR and 3'-UTR) and CDS cDNA (only coding ORF). But CDS DNA is much more accessible than full-length one.
Edited by Oleg, 07 November 2010 - 04:18 PM.
#4
perneseblue
-
- Global Moderators
-
- 530 posts
Unlimited ligation works!
8
Neutral
Neutral
Posted 07 November 2010 - 06:33 PM
If possible, I would try both to see if there is any difference.
May your PCR products be long, your protocols short and your boss on holiday
