I have designed a set of primers with the amplicon length of 137bp, I have done my qRT-PCR but it is not specific . I have done the sequencing but the result is only showing the 23bp which is the same location as the reverse primer (refer to figure Sequencing Result.jpg). May I know what does this mean? I am using Qiagen one step RT-PCR Quantifast SYBR Green. The primers sequence and pcr protocol are as below:
Forward: 5'-tcattttgacggaaatggcaacc
Reverse: 5'-aaggaacatagcttcaaccgcag
42C, 10 min
95C, 5 min
95C, 10s ; 62C, 10s (50 cycles)
95C, 1 min
55C, 1 min
65C-95C, 10s
This is the graphical layout of the primers and the highlighted region is the sequencing blast result which is matched to my gene of interest.
Shin Wei, your reverse primer is homologous to highly conservative part of nuclear receptor subfamily 1, and it could amplify non-specific fragment of 100 bp (200 bp can be dimer of one). Make more specific reverse primer for your target sequence.
I feel your primers are not specific for the region you want to amplify. What did you use as template? Genomic DNA? If yes, Did you BLAST your primers against the genome of the organism you are working on? I am unable to understand your PCR conditions, Could you please describe them: what was your initiation temp, denaturing temp, annealing temp, and extension temp and time. 50 seconds are enough for extension of an amplicon of 137 bp. First of all, I highly recommend that you check if your primers are specific to the region, it is very common to design primers which can anneal at many places of the genome. Hope that makes sense.
Additionally your Tm of the Primers are > 2°C away from each other and the fe primer is able to form a HP of 5bp and your rev has a lot of GC at the 3´end, better check your Primers using a free tool such as Primer 3 or others ! I which you good luck!
Dear Shin Wei
Hello,
Please check you primer with oligo analyzer (https://eu.idtdna.com/calc/analyzer). Annealing temperature is low and tis seemed your forward primer have a high hairpin Tm. However, based on your positive control ,which is worked appropriately, you don't have problem with your primers. I think you should focus on you DNA sample.
42C, 10 min
95C, 5 min
95C, 10s ; 62C, 10s (30 cycles) dcreasing the annealing temperature
95C, 1 min
55C, 1 min
65C-95C, 10s
Forward:
5'-tcattt t g acggaaatggcaacc
| | | |
3’Gacgcca a c ttcgatacaaggaa
Product is fragment of Human farnesol receptor HRR-1 (HRR-1) mRNA?complete cdsSequence ID: gb|U68233.1|HSU68233Length: 2218Number of Matches: 1
Hi Shin Wei,
It seem that you are working on NR1H4 gene. BLAST of your primers showed that it can detect this gene on various organism with various variant. I think your primer do not have too much of problem since your positive control is quite good. You should focus on the template that you use for your qRT-PCR and the protocol.
As for your protocol, i just screen through the manual.... do you follow the cycling protocol suggested by the manufacturer? There is one-step and two-step protocol. Check which protocol suitable for your sample, read through the troubleshooting part.
As for your sequencing result that you get only a few basepair... i believed it is because your sample is low concentration. From what you write here, the PCR is fail, thus i can assume, the concentration of your PCR product sure not high enough for the sequencing. That's why your result is truncated.
Dear Alex, Thorben, Naghmeh & Hadi, thank you for the opinion.
Dear Bikiran, my template is RNA.
Dear Dr Huai, I am focusing on human FXR (NR1H4).
Dear Aries, you are right. I am following the manufacturer's protocol and use one step qRT-PCR. Ya, the sample concentration is claimed to be low for sequencing.
Dear all,
May I know how to know what is the acceptable value of energy for the hairpin, homodimer & heterodimer? And how to design a region which is specific to human only and can detect all variants? What is the software that you recommend?
Actually I have tried another primer but it gave 2 melt peaks (confirmed by agarose gel-2 bands). After I blast, only forward primer is working (amplifying one exon) for one of the 6 variants. Reverse primer is amplifying another region but that particular variant does not have that particular exon. Will it be the reason that I saw 2 peaks in my melt peaks?
Really appreciated your kind advice and all the best in your work also. Have a nice day. :)
There are 3 guidelines that you should follow :
1) You must use primers with a random base distribution and with a content of GC similar to the fragment being amplified. You should avoid stretches of polypurines and polypyrimidines (there are some of them in your forward primer ).
2) You must ckeck the primers against eah other for complementarity, particularly, avoiding primers with 3' overlaps will reduce the incidence of "primer dimer" (perhaps the problem you have (only products of 23 bp).
3) You must avoid primers with sequences that can form secondary structures.
Hello Shin,
This short sequencing product may have been arisen because of not fulfilling some parameters: The appropriate GC content, similar annealing temperature for both F and R primers, and avoiding loops and primer-dimer formation. In short sequences, there are more chances of forming dimers. So, it is advisable in this case to try design primers from different location so as to have ideally no loop and no dimer forming primers along with similar annealing temperature for F and R primer. I hope this helps.
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