Primer designing - with Primer 3 and UCSC Genome Browser - Bioinformatics Practical 3

PCR or Polymerase Chain Reaction is on of the most basic, most versatile and indispensable techniques in Biotechnology. It is an elegant example of how the principles of Nucleic acid hybridization and DNA replication can be applied to exponentially amplify a specific region of DNA.
A critical factor in the success of PCR amplification is the design of specific primers.
In this short video, I describe the basic concepts in Primer designing and then design specific primers to amplify the GAPDH gene in the human genome. The required sequence is retrieved from the UCSC Genome Browser and the primers are designed using Primer3.
#Primer #PCR #Polymerase_Chain_Reaction #Primer3 #UCSC_Genome_Browser #GAPDH #Primer_designng #lets_grow_together #vipins_e_classroom #tm

[@yemibamikole6590]

Thank you so much for this. So what if I need a primer sequence for a whole set of gene and not the nucleotide. I need the primer for sequencing, let's say interleukin 1. How will I go about it? Thanks prof

[@classroom_vipin]

Hi ... Yemi ... thank you. But i did not understand what you mean when you say " I need a primer sequence for a whole set of gene and not the nucleotide. " - can you please elaborate!

[@yemibamikole6590]

@Dr. Vipin's Classroom thank you so much professor vipin.
So what I wish to do is to perform a a sequencing of interleukin-1 gene of among malaria cases and non malaria controls.
I plan to identify the variations and from there do a genotyping of the variations.
But in doing this first sequencing of interleukin 1 gene, I have my blood sampes, I need to extract my dna and do PCR. In doing the PCR I need primers for interleukin 1 gene.
So my question is how do I amplify the interleukin 1 gene. How do I get the primer to use?

[@classroom_vipin]

@@yemibamikole6590 Hi ... I am looking at HL1A gene in human - it is 10,569 base pairs in size - which means you have to do Long Range PCR - you an design the primers exactly as i suggest for GAPDH ... taking sequence with flanks
You may the sequence your amplicon from cases and control to check for SNPs exclusive to cases and control - if already available in literature, then you can design ARMS or Amplification Refractory Mutation System - meaning that one of the primer pairs will contain the SNP position at its 3' end. This will ensure that amplification happens only for the sample where one of the two varaint nucleotide is present - Reading about the target thoroughly will help

[@mbahchinedu4755]

Good day prof,
Sir can we use the UCSC browser to select genes found in bacteria? And can we design a primer for it using primer 3?

[@classroom_vipin]

Hi Mbah, you can use the UCSC microbial genome browser ... microbes.ucsc.edu/
And one you get your sequence, use primer3 as explained

[@rutuvartak26]

Sir very clear explanation, can you also explain how to design primer for chip qpcr.

[@classroom_vipin]

Ok soon

[@swethach7600]

I am unable to open the UCSC genome browser, Asia..can you help me out in this regard?

[@classroom_vipin]

genome-asia.ucsc.edu/cgi-bin/hgGateway
I just checked, the link is working

[@classroom_vipin]

genome.ucsc.edu/
This is working too

[@ramnathnayak985]

Shouldn't the excluded region be - 501, 3855+500"?

[@classroom_vipin]

No ... what you are instructing primer3 is that from 501 bases until the next 3855 bases is the gene sequence and since you want the amplify the full gene, the primers cannot be inside the gene sequence ... so region to exclude from primer designing starts at 501 and ends 3855 bases beyond the start ...
And you anyways do not have sequence beyond 3855+500 bases

[@ramnathnayak985]

@@classroom_vipin Okay sir, Thank you
And if we have introns and exons, how do we move forward?

[@ramnathnayak985]

Also, how do we make sure that the primer we design using a reference genome binds to the organism that we actually have?

[@classroom_vipin]

@@ramnathnayak985 Depends on how close the organism is to the reference you have taken ... also one odd base mismatch towards the 5' end or in the middle can be tolerated .. so the key would be to identify a conserved region using multiple sequence alignment and then design primers to the region ... plus a bit of mismatch can also be overcome by standardization - a gradient PCR for temperature and MgCl2 can help ... but of course you need to sequence the amplicon to confirm

[@classroom_vipin]

@@ramnathnayak985 if you know the position of exons in the final sequence you are taking ... follow the same procedure ... introns serving as flanks and the exon your target