I am working on molecular cloning of a gene we have already synthesized the 3'end of CDS now we want to clone 5' end and for the same I am following 5'RACE strategy. In final step with PCR anchor (forward) and Sp3 (gene specific reverse) I am getting good intensity band, but smearing is observed when I am re-amplifying the resultant product with same primer set.
1. Could anybody tell me reason and the solution for this type of problem?
2. I do have sufficient purified product, so with this product could I go for single pass DNA Sequencing. (As this product is giving smear on re-amplification) if yes please tell me which primer I should prefer (Forward or reverse).
I have gone through the following approaches to solve problem:
1. Reduced template concentration.
2. decreased the cycle no.
3. Gradient PCR.
4. Have re-diluted the Primers.
5. Have used both the direct PCR Product and the Purified product as template.
I am using 2x green taq from fermentas.
Thank you
Hi Prakesh, i will suggest you to do first sequencing of your PCR product and try to do re-amplify at higher temperature with low concentration of PCR product and low concentration of primer and decrease no. of the amplifing cycle.
Don't try simply re-amplifying your product with the same primer set. I would try a nested reverse primer that is closer to the 5'end than your SP3 primer. Your results indicate your PCR strategy is not yet specific enough to the target.
Thank you for your answers....
@ Ankita-- i have tried the things you are mentioning
@ Terry --- actually its the last step of my 5'RACE it the nearest nested reverse primer... i am using Roche kit .
If you already have a good product in the 1st PCR just sequence or clone it ;)
You can use the primers of the kit for sequencing as well.
have you tested your specific reverse primers with other specific forwards for instance with cDNA? Your nearest reverse primer may not be working or it may be out of the cDNA... another splicing, bad annotation etc?? double check sequence and quality in a gel.
For RACE I would recommend that you use a hot start taq... and without dyes on it in the first PCR
cheers.
If your gene has multiple initiation sites, which is very common, or is alternatively spliced, then you may be amplifying a mixture of products representing these sites and alternate forms. One strategy that I used for 5'-Race was to dilute the template for the final nested PCRs down to the point where you will actually get NO product in ~70% of the reactions. Thus you will effectively be amplifying the product of a single transcription initiation/elongation/splicing event. This approach is useful also if you want to determine the true "spectrum" of transcription initiation positions. An example of this is at:
https://www.researchgate.net/publication/8481161_Identification_of_the_major_promoter_and_non-coding_exons_of_the_human_arylamine_N-acetyltransferase_1_gene_(NAT1)?fulltextDialog=true&ev=prf_pub_xaddfulltext
Article Identification of the major promoter and non-coding exons of...
If you have a visible PCR product, you can directly sequence this. For sequencing you can best use the nested reverse primer that you already have. Sequencing with the forward primer usely does not work, for several reasons as mentioned by others already. If a terminal transferase step is included in the 5' RACE protocol, this will give different 5' anchor lenghts.
Thank You Martin I was also thinking to sequence my PCR product with Nearest Reverse Primer(as you mentioning)... but one of my Senior confused me...... then I put down The Question to the Resarchgate members... And thanks again to all the followers and contributors of the question ....
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