Question

A peptide from a novel bacterial protein was isolated and sequenced. The amino acid sequence is NH2-Leu-Met-Pro-Tyr-Ser-Ala-Ala-Pro-Cys-Leu-Trp-Glu-Ala-Gly-Asp-Arg-COOH. To verify and clone the gene encoding this protein from this uncharacterized bacterium, a student has to purify the bacterial genomic DNA and use it as the template to isolate the DNA fragment that encodes this peptide using PCR. The student plans to design a pair of oligonucleotides as the forward and reverse primers to PCR the DNA fragment that encodes this peptide. How does the student design these forward and reverse oligonucleotides? How does he/she identify the bacterial DNA fragment encoding this peptide with confidence? What is the chance to obtain this DNA fragment (how many DNA fragments he/she has to minimally sequence)?

Answer 1

What we have is the following protein sequence
NH2-Leu-Met-Pro-Tyr-Ser-Ala-Ala-Pro-Cys-Leu-Trp-Glu-Ala-Gly-Asp-Arg-COOH (Protein sequence - LMPYSAAPCLWEAGDR)

So, Leucine is coded by the following codons in bacteria (Leu) – TTA/TTG/CTT/CTC/CTA/CTG

Methionine (Met) – ATG

Proline (Pro) – CCT/CCC/CCA/CCG

Tyrosine (Tyr) – TAT/TAC

Serine (Ser) – TCT/TCC/TCA/TCG

Alanine (Ala) – GCT/GCC/GCA/GCG

Cysteine (Cys) – TGT/TGC

Tryptophan (Trp) – TGG

Glutamic Acid (Glu) – GAA/GAG

Glycine (Gly) – GGT/GGG/GGA/GGC

Aspartic Acid (Asp) – GAT/GAC

(Source - https://www.genscript.com/tools/codon-frequency-table)

To amplify from genomic DNA, forward and reverse primer, both to be designed should be about 15-18 basepairs each.

Step 1

Go to NCBI Blast and do a protein BLAST search. The link is

https://blast.ncbi.nlm.nih.gov/Blast.cgi

From there, choose the best suited protein sequence. Make sure you choose

1. Complete cDNA sequences (do not use ESTs, partial cDNAs, exons or introns).
2. Sequences of bacteria (and not humans, or other kingdoms or archaebacteria, etc)

Generally, the first codon in an amino acid sequence is “Methionine”. So, I would consider the first and only Methionine in the sequence to be the first amino acid of the protein.

So, the first sequence from the list is of bacteria

Step 2

I would take the first sequence, and then go to select “Sequence ID”.

A new page will open, which looks like this

Go to FASTA on top and it will show you the protein sequence. From the protein sequence, you can get the gene sequence and check which all codons have been used more for each amino acid given.

Now the next step is primer design. The key to primer design based on an alignment of amino acid sequences is focusing on the relationship between the amino acid sequence and the nucleotide sequence(s) that may code for it.  This step in molecular cloning by PCR is very important.  Nearly every other step requires adherence to a strict protocol, but luck also matter with regards to with degenerate primer design.

Since the gene is unknown, the primers have high possibility that they would end up being degenerate primers and the whole process would be a hit and trial. A "good" primer pair has the following characteristics:

• Minimal degeneracy to insure the maximum likelihood of a sufficient concentration of the correct primer.
• Maximum specificity at the 3' end to optimize binding of primer to template.
• Minimal hairpin formation within the primer, especially producing a 3' end annealed to a potential template.
• Minimal primer dimer formation with one primer itself or with the other member of the pair, especially leaving an extendable 3' end.
• Approximately equal melting temperatures to simplify establishing the PCR reaction conditions.

So, starting with Methionine as N’terminal Amino acid, the sequence of Forward Primer could be

ATGCCTTATTCTGCT (15 bp)

ATGCCTTATTCTGCC (15 bp)

ATGCCTTATTCTGCA (15bp)

ATGCCTTATTCTGCG (15 bp)

ATGCCTTATTCCGCT (15 bp)

ATGCCTTATTCCGCC (15 bp)

And so on… There could be many possibilities of a degenerate Forward primer. Similarly, for Reverse primer, considering the last codon to be a stop codon (TAA/TAG/TGA)

Hairpin, dimer formation, and the melting temperature (Tm), can be calculated using a Java applet called NetPrimer, available online at

http://www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html.  Hairpins and primer dimers should be avoided if at all possible, and melting temperatures for a forward and reverse pair should be within 5^oC of each other.

It is a good plan to design two forward primers and two reverse primers for each sequence you wish to amplify. At least one combination may work for you! It is impossible to predict the success of any particular primer pair. Following these suggestions should help you in your cloning efforts, but there are no guarantees. Of course, the very best primers are those that give you one well-amplified product of the size and sequence of the thing you’re after!