EST (Expressed Sequence Tag) Primers - Where are they coming from? (Jul/03/2011 )
Hi,
I have a question on the EST sequencing approach.
If I understand the principle correctly: First you have to isolate mRNA and transcribe it with Reverse Transcriptase to cDNA.
The cDNA is made double-stranded then and now the EST can be generated from both ends of this dsDNA (3'-ESTs and 5'-ESTs).
But where are the primers for this sequences are coming from?
For the 3'-ESTs it would be obvious to take oligoT Primers. But what about the 5'-ESTs (especially when the gene is unknown)?
Best regards,
Ikar
-Ikar-
Ikar on Sun Jul 3 10:47:26 2011 said:
Hi,
I have a question on the EST sequencing approach.
If I understand the principle correctly: First you have to isolate mRNA and transcribe it with Reverse Transcriptase to cDNA.
The cDNA is made double-stranded then and now the EST can be generated from both ends of this dsDNA (3'-ESTs and 5'-ESTs).
But where are the primers for this sequences are coming from?
For the 3'-ESTs it would be obvious to take oligoT Primers. But what about the 5'-ESTs (especially when the gene is unknown)?
Best regards,
Ikar
Are you talking about EST or RACE?
If you are talking about EST, you will clone the cDNA (size fractionated) into a vector (depends on kit you are using). The you will use the vector primers for sequencing.
-biocrazy-
biocrazy on Sun Jul 3 11:19:22 2011 said:
Ikar on Sun Jul 3 10:47:26 2011 said:
Hi,
I have a question on the EST sequencing approach.
If I understand the principle correctly: First you have to isolate mRNA and transcribe it with Reverse Transcriptase to cDNA.
The cDNA is made double-stranded then and now the EST can be generated from both ends of this dsDNA (3'-ESTs and 5'-ESTs).
But where are the primers for this sequences are coming from?
For the 3'-ESTs it would be obvious to take oligoT Primers. But what about the 5'-ESTs (especially when the gene is unknown)?
Best regards,
Ikar
Are you talking about EST or RACE?
If you are talking about EST, you will clone the cDNA (size fractionated) into a vector (depends on kit you are using). The you will use the vector primers for sequencing.
I was thinking of the pyrosequencing of ESTs by 454 sequencing.
You say that before the cDNA is sequenced it is fractionated and cloned into a vector. How would this come together with the 454 approach (e.g, how would the emPCR work then?)?
-Ikar-