Difficulty in Standardizing Long-Range PCR for CYP17A1 Gene in Congenital Adrenal Hyperplasia (CAH)
Hello everyone,
I am an intern at the Centre for Cellular and Molecular Biology (CCMB) working in the Molecular Diagnostics Lab. Currently, my project focuses on the comprehensive analysis and design of a gene panel for the rare genetic disorder Congenital Adrenal Hyperplasia (CAH). I am specifically investigating five genes known to be pathogenic to CAH:
- CYP21A2 (4599 bps)
- CYP11B1 (9391 bps)
- CYP11B2 (8052 bps)
- CYP17A2 (7649 bps)
- STAR (11067 bps)
I have successfully standardized PCR for all these genes using long-range PCR. For these genes, I have been using PrimeSTAR GXL Premix (by Takara Bio) and applying a 2-step touchdown PCR protocol, with varying annealing temperatures (Ta = 76, 74, 72, 70°C). However, I am facing difficulties in standardizing the CYP17A1 gene. Despite designing new primers, I am unable to obtain a single band in my PCR reactions.
Here are the key details:
- Tm = 60°C
- Annealing temperatures: 76, 74, 72, 70°C
- Extension time: 7 minutes
- Using PrimeSTAR GXL Premix (Takara Bio)
I have attached the gel image for reference, which shows the PCR results for CYP17A1 and might help in understanding the issue. I would greatly appreciate any advice or suggestions regarding troubleshooting this issue. Has anyone worked on CYP17A1 long-range PCR or encountered similar challenges? Any recommendations for improving the specificity of the PCR or troubleshooting primer design would be very helpful!
Looking forward to your suggestions.
I would try a gradient of 0 to 8% dmso and possibly increasing the length of the pcr primers to increase the annealing temperature ( and probably specificity). I have not worked with amplimers this long but it is possible that there are snps in the amplimer leading to heteroduplexes when the single stranded amplimers anneal on the last pcr cycle which run at a different speed from the homoduplex. This is testable by isolating the main band,heating and slowly reannealing it and running it on agarose gel and see if the smaller band are regenerated from the larger band
Actually I am using it in a gene panel so , as i discussed with my PI he is not suggesting for a gel elution, and these are the second set of new primers i have been using . Can i decrease the primer concentration and see for one last time .Will it be a better option to do ?
Decreasing the amunt of primer can often work as does increasing the annealing temperature (or adding dmso which works in much the same way as increasing Ta). Good luck.
Nandini Ravula Typically, we perform full PCR optimization (template DNA, primers, Mg2+ concentration, polymerase concentration, temperature profile) using the Taguchi method. This always yields good results.
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