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Rapid Amplification of cDNA Ends question?

Rapid Amplification of cDNA Ends question? - PCR - Polymerase Chain Reaction Forum

Rapid Amplification of cDNA Ends question? - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 01-05-2010, 12:38 AM
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Default Rapid Amplification of cDNA Ends question?



Hi, I am new to pcr and i need some explaination on it..
In 5' RACE, after the production of 1st strand cDNA using gene-specific-primer 1 and addition of polyT tail, another gsp 2 is used together with anchor primer to amplify that cDNA. My question is, why don't we use gsp1 for amplification instead of gsp2?
2nd is, if gsp1 & 2 are parts of known sequence (designated as X) which i obtain from 3'RACE, will X have overlapping sequence with cDNA obtain from 5'RACE? If yes, can i assume that the overlapping sequence is the sequence of gsp2+a few nucleotide, since i get gsp1 from X?
3rd, can i directly use cDNA generated from 3'RACE as primer for 5'RACE, instead of using some part of it to design gsp1? If not, y?
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Old 02-14-2010, 10:47 AM
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Default Re: Rapid Amplification of cDNA Ends question?

Hi,

Your post is a little bit unclear, but here are my replies:

1. On why you use a GSP 2 after an amplification.
Technically, you can use GSP1. But RACE is a complicated and non-specific process. By using GS1 and then GSP2, it helps to increase specificity and likelihood that the gene you are targetting will be amplified.

As an analogy, you are looking red-coloured zebrafishes in large swimming pool. In your first search, you manage to narrow it down to zebrafishes - blue, red, green, etc. Now among the pool of zebrafishes, the chances of catching red zebrafishes are higher.

2. Your 2nd question is unclear. You only get overlapping sequences if you design the GSP1 and GSP 2 accordingly, eg:

...ATCGATCGATCGATCGATCGATCGATCGATCGATC...
GSP1--> <---GSP2

GSP1 being the primer for 3'-RACE and GSP2 being the primer for 5'-RACE. Then if your RACE was performed successfully, you get an overlapping 5'- and 3'-RACE product. However, if your primers are :

...ATCGATCGATCGATCGATCGATCGATCGATCGATC...
< --GSP1 GSP2-->

GSP1 is the primer for 5'-RACE and GSP 2 from 3'-RACE. And the both the RACE products would NOT be overlapping.

3. I assume you meant using the 3'-RACE product as the primer for 5'-RACE. Technically possible, but extremely difficult. This is due to the conditions required for a good primer, e.g. 21-28 nucleotides, sufficiently high TM, GC content, and GC ends. And not forgetting that the primer is a conserved region of the gene as many genes have non-specific regions eg repeats etc. If your 3'-RACE product can fulfill all those criteria, well then you can use it as a 5'-RACE primer.

Last edited by jonoave; 02-16-2010 at 02:49 PM.
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Old 02-17-2010, 05:34 AM
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Default Re: Rapid Amplification of cDNA Ends question?

Quote:
Originally Posted by jonoave View Post
2. Your 2nd question is unclear. You only get overlapping sequences if you design the GSP1 and GSP 2 accordingly, eg:

...ATCGATCGATCGATCGATCGATCGATCGATCGATC...
GSP1--> <---GSP2

GSP1 being the primer for 3'-RACE and GSP2 being the primer for 5'-RACE. Then if your RACE was performed successfully, you get an overlapping 5'- and 3'-RACE product. However, if your primers are :

...ATCGATCGATCGATCGATCGATCGATCGATCGATC...
< --GSP1 GSP2-->

GSP1 is the primer for 5'-RACE and GSP 2 from 3'-RACE. And the both the RACE products would NOT be overlapping.
Hmm I see... But about the GSPs, i thought both GSPs are used in 5'RACE. Cuz from what i read in a 5'RACE manual, it seems that it uses 2 GSPs, both towards the same direction. ie
...ATCGATCGATCGATCGATCGATCGATCGATCGATC...
.................................................. ...< --GSP1
.......................................... <--GSP2
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Old 02-21-2010, 04:13 PM
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Default Re: Rapid Amplification of cDNA Ends question?

Quote:
Originally Posted by hianghao View Post
Hmm I see... But about the GSPs, i thought both GSPs are used in 5'RACE. Cuz from what i read in a 5'RACE manual, it seems that it uses 2 GSPs, both towards the same direction. ie
...ATCGATCGATCGATCGATCGATCGATCGATCGATC...
.................................................. ...< --GSP1
.......................................... <--GSP2

GSP (Gene Specific Primer) is just a term used in RACE. For me, it's most commonly used as a the primer in combination with another non-specific primer in a RACE Reaction. Eg: GSP1 with the manufacturer provided 5'-RACE primer. Depending on what kit you used, maybe the instructions used the terms differently. Ok, from now on I won't use GSG1 or GSP2, but 5'-GSP and 3'-GSP to make things clearer.

What you're referring to in your example, is more commonly known as nested-RACE. Nested here refers to a subsequent amplification within an amplifcation. Based on your example:
GSP1 --> 5'-GSP
GSP2 --> nested 5'-GSP

Nested RACE is good to improve yield, as generally yield by 5'-RACE is low (lower than 3'-RACE). And it also helps to improve specificity (a concept which is similar to your 1st question).

However, I'm still not quite sure what your second question is. 5'-RACE and 3'-RACE is 2 separate processes. Here's what an ideal RACE reaction consist of:

------ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCG----
3'-GSP--> nested 3'-GSP--> <--nested 5'-GSP <--5'-GSP

Notice that the I design the primers so that the 5'-RACE GSP is further than the 3'-GSP, and how the nested-GSPs are designed. Therefore, if everything proceeds well, you will get an overlapping 5'- and 3'-RACE product which you can combine to get the complete sequence.

Last edited by jonoave; 02-21-2010 at 04:18 PM.
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