Coding sequence may be different but does the same function

If u retrieve the sequence from NCBI u can compare and identify the difference

Coding sequence may be different but does the same function

If u retrieve the sequence from NCBI u can compare and identify the difference

Coding sequence of genes does vary among bacteria. In many cases nucleotide sequences vary but amino acid sequences remain the same. For designing a primer you can download the sequences from NCBI. Do a CLUSTALW analysis and based on the conserved region you can design a primer. You can also design a degenerate primer for better results. good luck.

Housekeeping genes are conserved like gyrase, groel, rpod etc and can be captured by primers directed at the conserved sequence sites.

For gelatinase you can design primers but that would not guarantee that you capture all gelatinase positive, you can have gene fragments that can give false positive results, and for novel gelatinases it will be negative.

I am sure there are published papers where ppl have designed, validated and used it.

So at the end of the day a standard biochemical assay would give better chances of capturing a gelatinase positive organism from your samples.

No, the genes would most probably not be conserved. Even for house-keeping genes the DNA sequences deviate with taxonomic distance. But you may still be able to generate primers which may amplify related gelatinases from a number of different isolates. As Naveen wrote already download all gelatinase sequences from NCBI and do a multiple alignment to look for conserved regions. However, additionally look for the taxonomic information NCBI provides. If for example, all sequences in the database are from proteobacteria, then a conserved region (and the primers based on it) may only allow to amplify other sequences from Proteobacteria, since that region may look completely different in other bacterial groups. If you find more or less conserved regions in more than one of the main taxa your chances amplify the gene from many environmental strains are better, but even then genes may be missed. You may also have to use degenerate primers to obtain an amplification of more distantly related strains at all. So the basic question is: Do you want to make a survey about the occurence and distribution of gelatinase genes in your environmental samples (then you would probably need more than one pimer set, or may not be able to find one at all) or do you look for different gelatinases from as many taxa as possibly for a biotechnological application (which may be feasible)?.

Why do you want to use a DNA-based assay in the first place ? First of all, chances are that you could miss genes whose sequence is too divergent to be picked by your probe, and second, finding a sequence that is amplified by your probe is by no mean proof that it expresses significant amounts of a protein that has significant activity. It could be a silent gene, or a pseudogene whose sequence has drifted and no longer encodes an active enzyme. If you can grow your strains, why don't you test for the real thing, i.e. gelatinase activity ? There are plate tests aplenty for that.

If you are measuring gelatinase activity that means you are looking at proteases. Even the amino acid sequences can be very very diverged amongst proteases and the DNA even more so. Proteases of completely different classes can still degrade gelatin, so you don't even know what enzyme you are looking at. Therefore unless you know the sequence of the corresponding protease from a very closely related organism, it is highly unlikely you will be able to make any progress using PCR.