I'm trying to amplify the sequence of an Arabidopsis promoter, but I can't because other bands appear in the electrophosere gel that are not of interest to me. I believe this occurs because my sequence has many regions rich in A-T. So I would like to know how to improve the efficiency of PCR to amplify this sequence.
Please advise. Thank you for your time in advance.
Maria Eduarda Soares da Silva Look at the publication by Dhatterwal, P., Mehrotra, S. & Mehrotra, R. Optimization of PCR conditions for amplifying an AT-rich amino acid transporter promoter sequence with high number of tandem repeats from Arabidopsis thaliana . BMC Res Notes 10, 638 (2017). https://doi.org/10.1186/s13104-017-2982-1
Amplifying AT-rich sequences can indeed be challenging due to the lower melting temperature of AT base pairs compared to GC base pairs. However, there are several strategies you can employ to improve the efficiency of PCR for these sequences:
1. **Use a Specialized DNA Polymerase**: Some DNA polymerases, such as Phusion Plus DNA Polymerase¹ and Platinum SuperFi II DNA Polymerase², have been specifically designed to amplify AT-rich sequences. These enzymes can efficiently amplify DNA with AT content as high as 90% with minimal optimization¹².
2. **Optimize the Annealing Temperature**: For successful amplification of AT-rich sequences, the optimal annealing temperature should be determined via gradient PCR². Lowering the extension temperature to 60°C has also been recommended².
3. **Increase the Extension Time**: An increased extension period of 1.5 min/kb at a lower extension temperature of 65 °C has been shown to be effective³.
4. **Adjust the MgCl2 Concentration**: It may be necessary to adjust the final MgCl2 concentration in the PCR mix up to 4.25 mM, particularly if the AT content of the target sequence is extremely high (≥85%)².
5. **Optimize the DNA Concentration**: A DNA concentration of about 25–30 ng/µl was found to be essential to achieve successful amplification³.
Remember, PCR conditions can vary depending on the specific sequence you're trying to amplify and the reagents you're using. It's always a good idea to perform a few test runs with different conditions to determine what works best for your specific situation.
(1) Successful amplification of AT-rich DNA sequences with Phusion Plus DNA .... https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/at-rich-pcr-phusion-plus-app-note.pdf.
(2) Successful amplification of AT-rich DNA sequences with Platinum SuperFi .... https://assets.thermofisher.com/TFS-Assets/BID/Application-Notes/amplification-dna-sequences-platinum-superfi-ii-dna-polymerase-app-note.pdf.
(3) Optimization of PCR conditions for amplifying an AT-rich amino acid .... https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-017-2982-1.
Thanks Raghad Mouhamad and Alex Ignatov for your help!
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