Designing a primer for an Mtb gene: Eg Rv1524

1.       Get the nucleotide sequence of the gene of interest.

a.       >M. tuberculosis H37Rv | Rv1524

atgaaatttgtcgtggccagctatgggactcgcggcgacatcgagccctgcgcagcggtcggcctggagctgcagcggcg
cggccatgatgtgtgccttgccgtgccgcccaacctgattggtttcgtggaaacggccgggctgtctgctgtcgcatacg
gaagcagggactctcaggagcagctcgacgagcagttcctgcacaacgcgtggaaacttcagaaccccatcaagctgctg
cgtgaagcgatggcgcccgtcaccgagggctgggcggagctgagcgcgatgttgacgccggtggccgccggggccgacct
gctgttgaccggtcagatctaccaggaggtggtcgccaacgtcgccgagcaccacggcattccgttggccgcgctgcatt
tttatccggtgcgagccaatggcgagatcgcctttcccgcgcggctgccggcgccactggtccgctccaccatcacggcc
atcgactggctgtattggcgcatgacgaaaggtgttgaggacgcgcagcggcgtgaactgggcctgccgaaggcgtcaac
tcccgcgccgcggcgaatggccgtacgcgggtcgctggagatccaagcctacgacgcgctttgcttcccggggctggcag
cggaatggggcggccgacgcccgttcgtcggcgcgttgacgatggaatcggcgaccgacgcggacgacgaggtcgcttca
tggatcgctgccgatacaccgccgatttatttcggctttggcagcatgccgatcggatccctggccgaccgggtcgccat
gatcagtgcggcctgcgcggagttgggcgagcgcgcgttgatttgctcgggacccagcgatgcgaccggaatcccgcagt
tcgatcacgtgaaggtggtgcgtgtggtcagccacgcggcggtctttcccacctgccgtgcggtcgtccaccatggcggc
gcgggcaccaccgccgccggtcttcgagccggtatccccaccttgattctgtgggtcacctccgaccagccgatctgggc
tgctcagatcaaacagctgaaagtaggccgggggagacgcttttcaagcgccaccaaagaatcgctgattgccgaccttc
gaacgatacttgcgccggactatgtcacccgagcgcgggagatcgcgtctcggatgaccaaacccgccgccagcgtcacg
gccaccgccgatctgctcgaagatgcagcccgccgtgcgcgctaa

 

2.       For our purposes, we require the full length gene. Thus, the regions you are choosing should cover the entire length of the gene.

Choosing the forward and reverse priming region:

atgaaatttgtcgtggccagctatgggactcgcggcgacatcgagccctgcgcagcggtcggcctggagctgcagcggcg
cggccatgatgtgtgccttgccgtgccgcccaacctgattggtttcgtggaaacggccgggctgtctgctgtcgcatacg
gaagcagggactctcaggagcagctcgacgagcagttcctgcacaacgcgtggaaacttcagaaccccatcaagctgctg
cgtgaagcgatggcgcccgtcaccgagggctgggcggagctgagcgcgatgttgacgccggtggccgccggggccgacct
gctgttgaccggtcagatctaccaggaggtggtcgccaacgtcgccgagcaccacggcattccgttggccgcgctgcatt
tttatccggtgcgagccaatggcgagatcgcctttcccgcgcggctgccggcgccactggtccgctccaccatcacggcc
atcgactggctgtattggcgcatgacgaaaggtgttgaggacgcgcagcggcgtgaactgggcctgccgaaggcgtcaac
tcccgcgccgcggcgaatggccgtacgcgggtcgctggagatccaagcctacgacgcgctttgcttcccggggctggcag
cggaatggggcggccgacgcccgttcgtcggcgcgttgacgatggaatcggcgaccgacgcggacgacgaggtcgcttca
tggatcgctgccgatacaccgccgatttatttcggctttggcagcatgccgatcggatccctggccgaccgggtcgccat
gatcagtgcggcctgcgcggagttgggcgagcgcgcgttgatttgctcgggacccagcgatgcgaccggaatcccgcagt
tcgatcacgtgaaggtggtgcgtgtggtcagccacgcggcggtctttcccacctgccgtgcggtcgtccaccatggcggc
gcgggcaccaccgccgccggtcttcgagccggtatccccaccttgattctgtgggtcacctccgaccagccgatctgggc
tgctcagatcaaacagctgaaagtaggccgggggagacgcttttcaagcgccaccaaagaatcgctgattgccgaccttc
gaacgatacttgcgccggactatgtcacccgagcgcgggagatcgcgtctcggatgaccaaacccgccgccagcgtcacg
gccaccgccgatctgctcgaagatgcagcccgccgtgcgcgctaa

3.       Typically, a primer is between 18-24 bases long. The longer it is, the more specific it will be. The priming region we have chosen in the above sequence are :

Forward: 5’ atgaaatttgtcgtggccagctatg3’

Reverse: 3’gaagatgcagcccgccgtgcgcgctaa5’

4.       The forward primer is good as it is. But remember, the DNA polymerase only an act in one direction and that is 5’-3’. Thus we need a reverse complement of the reverse primer. This is how we make a reverse complement:

Sequence:       3’ gat gca gcc cgc cgt gcg cgc taa5’

Complement: 3’ cta cgt cgg gcg gca cgc gcg att 5’

Reverse complement: 5’tta gcg cgc acg gcg ggc tgc atc 3’

 

(Reverse complement is reading the complement sequence backward, i.e  ATT at the end of the complement becomes the beginning of the sequence “TTA”).

5.       Now we a have a set of primer for our gene. The next thing we need to check is the Tm value. A very crude way of calculating the Tm is by using the following formula:

[(A+T)X2]+[(G+C)X4]=Tm

Thus for our above sequence: the Tm of forward primer will be: [(A+T=14)X2=28]+[(G+C=11)X4=44]= 72

And the Tm of the reverse primer will be: [(A+T=6)X2=12]+[(G+C=17)X4=68]=80

 

An ideal primer will have a Tm of around 60C.

However, while going through your exercise of designing primers, you will often find that working on the bench is far from ideal conditions described in your tutorials or books.

 

This is where you will gain actual experience in working in a lab!!

 

This is one of the genes in Mtb which has a very high GC content (as is apparent from the sequence above). For carrying out PCRs for these high GC genes, often special PCR technique called Step-down or Touch-down PCRs are employed. These specialized techniques will be described to you when you face a tight spot!