Hello Sir Thank you for the video. One thing I do not understand is that you mention @3:32 that this is an mRNA sequence. Why does it contain thymine nucleotides rather than uracil? Thanks!
Please upload more tutorials on biological data bases. Bioinformatics and also biotechnology. It was really delightful to see this video.. Thank you sir.
Thanks for this fantastic video. I've tried this way, but I got a reverse primer with score of 1.90 Self Complementarity, could I still use this primer? How could I get both primer without self complementarity? Thanks in advance.
Yes you can use this primer. Primer 3 settings take care of this so as to avoid primer forming secondary structure. I have used 1.9 score and never had an issue. If at all you have an issues, self complimentarity of 1.9 score is an unlikely reason. All the best.
Yes, you can use CDS. If you align CDS and mRNA sequence, you will see a major portion aligned. Reverse transcription of mRNA generates cDNA, and such cDNA contains the 5' and 3' untranslated regions (UTRs). UTRs are not part of the coding DNA sequence (CDS). With exceptions aside, CDS starts with an AUG codon and stops with one of the stop codons. While using Oligo dTs for cDNA synthesis, you have better chance of designing primers close to polyA tail for PCR if using chosen transcript variant (or complete mRNA).
If one of the primers does not 'solely' match with the sequence of your interest in BLAST search, that may affect the efficiency of your primer pair. If you see amplification, you may isolate the amplicon and sequence it to confirm your target sequence amplification.
Thank you for the fantastic video, I want to know if it will work for dengue viral RNA? and also exons are not mentioned in viral rna data(but mat_peptide is given). Also, how do i design genus and species-specific probes?
Viral RNA is not my expertise. One needs exon information to avoid genomic DNA contamination in cDNA analysis. You may ignore exon information for your purpose. Please note that Taq DNA polymerase will not amplify RNA in PCR. To design genus or species specific probes you need to align all the sequences of the genus and species you are concerned with and identify your unique sequence in your species of interest and design probe. I hope I am clear.
I want to design primers for qpcr.my gene have 5 mRNA variants.2 of them are predicted variants.do i have to take them in to account when i am designing primers for all variant? Moreover, i want to design primers for exon-exon junctions but when i aligne the sequences , the junctions are different in every variants, what should i do?
Option 1: Design primer pairs for all the five mRNA variants and see which works for your study model; andOption 2: Identify the conserved region by aligning all the variants, ignoring the exon-exon junction, and design primer pair.Remember, if you are facing this problem, there will be many facing the same problem working with your gene of interest. Your success here will be appreciated by the community and your article will be well cited. All the best. By the way, did you subscribe to my channel? More videos are coming:)
Hi im looking for primers and probes to work on the sequence with accession number AY151287.1. But i cant find anything on where the exons are in this sequence on ncbi. How do i go about making sure that its spanning an exon exon junction?
Some genes are single exon genes (SEGs), which means they have only one exon. Complete coding sequence (CDS) of your gene of interest AY151287.1 (Cavia porcellus interferon gamma (Ifng)) is 500 nucleotides long (start at nucleotide 69 and end at nucleotide 569) and the protein has 166 amino acids, which means 498 nucleotides are coding for this 166 amino acid protein. This confirms your CDS has no introns, unless I am missing some critical information about your gene of interest. Design your primer and probe sequences and check the efficiency of your pair. All the best.
Rani Andaleeb Thanks for viewing the video. Please do subscribe too. The Primer 3 is an open access online program. Please go to the link I mentioned and use it. You don’t have to download anything. Best.
I don't think I understood your question. If your working concentration of primers in 20 uM and you add 0.25 ul primers in a 25 ul reaction mix, your final concentration of primers is 0.2 uM.
You need to align all of your mRNA variants and look for conserved sequences. For example, if you align three variants of CD25 (IL-2RA) mRNA (NM_000417.2, NM_001308242.1, and NM_001308243.1), you will notice that 1-586 nucleotides are perfectly aligned or same in all the three variants. So, you should design primer sequences between 220 (start codon site for this mRNA) and 586, that will cover all three variants. I hope this helps.
very useful.. but would i have to choose the last exon start site (e.g.. you chose 800) when designing primers or I can select any of the exon start sites?
If you are making cDNA using oligo dT, which starts synthesis from poly-A tail of mRNA, it is reassuring to design primers close to poly-A tail. If there is a concern about exonuclease activity, you may design primers targeting exons at the center. No matter which exons I targeted, I had a success rate of over 95% that my primers worked as expected.
hello sir....what about just DNA....can we make probe in case of DNA? another question is that....if the percent identity 100 or 99 percent for other species of organism of the same gene then the primers are not specific i think.....waiting for your valuable comments
Yes Mehran. You will have to purify DNA to remove RNA. You will have to design primers and probes within a single exon and NOT target any exon-exon junction. Viral DNA load is measured by real-time PCR using primers and probes. 'Specificity' depends upon the question you ask. When you take human blood samples, isolate DNA, and try to amplify gene of interest (say FoxP3 gene), you want the product to be specific to your gene of interest, and NOT share any similarity to other genes (say TGF-b) in your HUMAN DNA. There is 99% similarity for several genes between human and mouse, but how does that matter when you are only using human blood sample/human DNA. I am releasing some more videos on this topic. Please subscribe and check it out when released. All the best.
Please watch at 7:31 in the video. Probe is termed as "internal oligo" in Primer 3. Probe is a topic by itself to discuss. Simply remember that probe releases a signal at the end of each amplification cycle. This signal is measured by the instrument in real-time.
Thank you so much for your video! This has really supported me with a research project I'm conducting for my final year of uni
Thank you, sir. This is a very helpful video for designing a primer sequence.
Thank you Dr. Nawal Kishor Singh for your comment. All the best and please let me know if you have any suggestions to make other videos.
Hello Sir
Thank you for the video. One thing I do not understand is that you mention @3:32 that this is an mRNA sequence. Why does it contain thymine nucleotides rather than uracil? Thanks!
I can not thank you enough. This is wonderful.
Thank you sir, you explained this process much better.
Please upload more tutorials on biological data bases. Bioinformatics and also biotechnology. It was really delightful to see this video.. Thank you sir.
Really good video. Could you explain a little bit about how to determine the size of the amplicon when you are designing primers?
You can design the primers for the amplicon size of your choice. For real time quantitative PCR, usually 60-100 bp length is ideal.
thank you so much for this highly educative video
Please check if my other video 'Smart Ways To Design PCR Primers' would save time for you in primer designing.
Thank you so much for this Sir.
Sir reverse primer is from 3 to 5 direction or 5 to 3 direction ?
Nice video . It help me a lot
Thank you so much for this informative video! Great help !
Did you check my other video 'Smart Ways To Design PCR Primers'? That's my first approach before I design my primers.
nice content
hope you keep update :)
Thank you, I will
Hi sir, very nice video, can you make video on how to choose vectors nd selection of restriction sites in cloning.
Thanks sir
Very informative
Are you from Andhra Pradesh India
Yes :) Glad you liked the video. I have videos on Immunology posted from now on. Please do view, if useful to you.
Very nice tutorial. Thanks
Glad it was helpful!
great sir thanks for sharing
Most welcome
very well narrated. thank you.
thank you very much, its been very helpful.
Thanks! Quite useful for the beginners
Thanks.
Thanks for this fantastic video. I've tried this way, but I got a reverse primer with score of 1.90 Self Complementarity, could I still use this primer? How could I get both primer without self complementarity? Thanks in advance.
Yes you can use this primer. Primer 3 settings take care of this so as to avoid primer forming secondary structure. I have used 1.9 score and never had an issue. If at all you have an issues, self complimentarity of 1.9 score is an unlikely reason. All the best.
Thanks a lot!
Great video! I do have a question though. The left and right primers, are they just another term for forward and reverse primers?
Thanks. Yes.
superb
Thank you, this video is very helpful
Thank you for the great explanation. Can I please inquire how I could further determine primers for Nested PCR? Is it the same procedure?
Thank you Avindya. That's correct.
Why the forword of primer diffrent in length from revers?
Great Video!! Just subscribed and looking forward to more videos!!
what is the purpose of probe and where it bind actually in CD2 mRNA transcript ?
Why do you recommend to ignore the cds? I need to make a primer out of a gene with only the cds sequence available, is it still possible?
Yes, you can use CDS. If you align CDS and mRNA sequence, you will see a major portion aligned. Reverse transcription of mRNA generates cDNA, and such cDNA contains the 5' and 3' untranslated regions (UTRs). UTRs are not part of the coding DNA sequence (CDS). With exceptions aside, CDS starts with an AUG codon and stops with one of the stop codons. While using Oligo dTs for cDNA synthesis, you have better chance of designing primers close to polyA tail for PCR if using chosen transcript variant (or complete mRNA).
thank you sir...very helpful..
Glad it was helpful. My other video 'Smart Ways To Design PCR Primers' may be of help too.
what if one of the primers pair, for example the reverse primer, does not match with the sequence of our interest in BLAST search?
If one of the primers does not 'solely' match with the sequence of your interest in BLAST search, that may affect the efficiency of your primer pair. If you see amplification, you may isolate the amplicon and sequence it to confirm your target sequence amplification.
thank you very much
Why should i ignore the complete cds?
Thank you for the fantastic video, I want to know if it will work for dengue viral RNA? and also exons are not mentioned in viral rna data(but mat_peptide is given). Also, how do i design genus and species-specific probes?
Viral RNA is not my expertise. One needs exon information to avoid genomic DNA contamination in cDNA analysis. You may ignore exon information for your purpose. Please note that Taq DNA polymerase will not amplify RNA in PCR. To design genus or species specific probes you need to align all the sequences of the genus and species you are concerned with and identify your unique sequence in your species of interest and design probe. I hope I am clear.
How can I find where the primers anneal using xenbank?
I have shown at '7:30' how to check the region where primer anneals. I don't know about Xenbank.
I want to design primers for qpcr.my gene have 5 mRNA variants.2 of them are predicted variants.do i have to take them in to account when i am designing primers for all variant? Moreover, i want to design primers for exon-exon junctions but when i aligne the sequences , the junctions are different in every variants, what should i do?
Option 1: Design primer pairs for all the five mRNA variants and see which works for your study model; andOption 2: Identify the conserved region by aligning all the variants, ignoring the exon-exon junction, and design primer pair.Remember, if you are facing this problem, there will be many facing the same problem working with your gene of interest. Your success here will be appreciated by the community and your article will be well cited. All the best. By the way, did you subscribe to my channel? More videos are coming:)
Thank you. Yes i did
Nice. Thank You!
Hi im looking for primers and probes to work on the sequence with accession number AY151287.1. But i cant find anything on where the exons are in this sequence on ncbi. How do i go about making sure that its spanning an exon exon junction?
Some genes are single exon genes (SEGs), which means they have only one exon. Complete coding sequence (CDS) of your gene of interest AY151287.1 (Cavia porcellus interferon gamma (Ifng)) is 500 nucleotides long (start at nucleotide 69 and end at nucleotide 569) and the protein has 166 amino acids, which means 498 nucleotides are coding for this 166 amino acid protein. This confirms your CDS has no introns, unless I am missing some critical information about your gene of interest. Design your primer and probe sequences and check the efficiency of your pair. All the best.
Sir, why must you choose an overlap junction to design one of the primers?
Please subscribe if you haven't yet. You only want to amplify your cDNA. You choose overlapping region, so that you do not amplify genomic DNA.
Excellent thank you
Glad it was helpful!
I download the primer3 software but it can't open. how I could install it ?
Rani Andaleeb Thanks for viewing the video. Please do subscribe too. The Primer 3 is an open access online program. Please go to the link I mentioned and use it. You don’t have to download anything. Best.
how to know the primer final concentration? in uM?
I don't think I understood your question. If your working concentration of primers in 20 uM and you add 0.25 ul primers in a 25 ul reaction mix, your final concentration of primers is 0.2 uM.
How can i design primers for all variant of mRNA of a specific gene?
You need to align all of your mRNA variants and look for conserved sequences. For example, if you align three variants of CD25 (IL-2RA) mRNA (NM_000417.2, NM_001308242.1, and NM_001308243.1), you will notice that 1-586 nucleotides are perfectly aligned or same in all the three variants. So, you should design primer sequences between 220 (start codon site for this mRNA) and 586, that will cover all three variants. I hope this helps.
very useful.. but would i have to choose the last exon start site (e.g.. you chose 800) when designing primers or I can select any of the exon start sites?
If you are making cDNA using oligo dT, which starts synthesis from poly-A tail of mRNA, it is reassuring to design primers close to poly-A tail. If there is a concern about exonuclease activity, you may design primers targeting exons at the center. No matter which exons I targeted, I had a success rate of over 95% that my primers worked as expected.
sir how to design primer and probe for a snp
SNP is a whole new ball game. Use this link please: pcrsuite.cse.ucsc.edu/SNP_Primers.html
Thank you!
hello sir....what about just DNA....can we make probe in case of DNA?
another question is that....if the percent identity 100 or 99 percent for other species of organism of the same gene then the primers are not specific i think.....waiting for your valuable comments
Yes Mehran. You will have to purify DNA to remove RNA. You will have to design primers and probes within a single exon and NOT target any exon-exon junction. Viral DNA load is measured by real-time PCR using primers and probes. 'Specificity' depends upon the question you ask. When you take human blood samples, isolate DNA, and try to amplify gene of interest (say FoxP3 gene), you want the product to be specific to your gene of interest, and NOT share any similarity to other genes (say TGF-b) in your HUMAN DNA. There is 99% similarity for several genes between human and mouse, but how does that matter when you are only using human blood sample/human DNA. I am releasing some more videos on this topic. Please subscribe and check it out when released. All the best.
thank you so much!
thank you so much.
Great
love you man！！！！great！！
thanks so much..
I just made a new video for other smart ways of designing primers. I am editing it. Will soon upload. Please subscribe and visit again.
It's online now titled 'Smart Ways To Design PCR Primers'. I am sure it will save time designing your primers.
Thank you
Welcome!
This come under bioinformatics
How to devide primers by pulls?
thank you
what is the purpose of probe and where it bind actually in CD2 mRNA transcript ?
Please watch at 7:31 in the video. Probe is termed as "internal oligo" in Primer 3. Probe is a topic by itself to discuss. Simply remember that probe releases a signal at the end of each amplification cycle. This signal is measured by the instrument in real-time.
ok thank you so much 😀
what is the purpose of probe and where it bind actually in CD mRNA transcript ?
what is the purpose of probe and where it bind actually in CD mRNA transcript ?