Designing primers for family of genes
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5.0 years ago
agata88 ▴ 810

Hi all,

What tool would you recommend for primer designing of family of genes?

I am interested in whole gene amplification, and if it's possible I would like to generate degenerative primers.

Thanks in advance,

Best,

Agata

primers • 2.7k views
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5.0 years ago
natasha.sernova ★ 3.8k

Dear Agata, what is your target organism? One tool is better for bacteria, another one is better for humans.

http://bitesizebio.com/18992/a-primer-for-designing-degenerate-primers/ -

these people describe step be step what to do, for mouse and humans.

Do you need primers for PCR or NGS? Somewhere I saw a list of NGS-primers, I can try to find it.

Here you can choose what exactly you need, there are a lot of choices:

http://omictools.com/degenerate-primers-category

This paper describes the tool for viruses and bacteria.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3600133/

Below I cite the paper above: “Several primer design programs are available (OLIGO [6], OSP [7, 8], Primer Master [9], PRIDE [10], Primer3 [11]). Regardless of each computational working strategy, all of these use a set of common criteria (e.g., G/C content, melting temperature, etc.) to evaluate the quality of primer candidates in a specific target region selected by the user”.

“Alternative programs are aimed at more specific purposes, such as selection of primers that bind to conserved genomic regions based on multiple sequence alignments [12, 13], primer design for selective amplification of protein-coding regions [14], oligonucleotide design for site-directed mutagenesis [15], and primer design for hybridization [16]. Usually, the design of truly specific primers requires the information of the complete nucleotide sequence. This is the starting point for most of the programs described in the literature. However, the need of designing specific primers is not always accompanied by the complete knowledge of the target genome sequence”.

And this is a problem. “However, the need of designing specific primers is not always accompanied by the complete knowledge of the target genome sequence”.

Their conculusion is optimistic:

"In general, the results suggest that FAS-DPD could be used to design generalized degenerate primers for detection of known or unknown members of gene families or organism families, including different types of pathogens".

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5.0 years ago
boczniak767 ▴ 780

I think you can exploit the fact that family of genes share functional domains, and design primers for these sequences using tools as primer3 or primer-blast. Multiple alignment of sequences of the domains could also help.

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5.0 years ago
Erik Wright ▴ 390

You could try the DesignSignatures web tool. It will design degenerate primers that will amplify as many of the input groups (in your case gene families) as possible. It will also tell you which groups won't amplify with high efficiency, and which groups will result in similar PCR products. The paper describing it is here. Hope that helps!

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5.0 years ago
natasha.sernova ★ 3.8k

Look at this papers:

Degenerate Primer Design via Clustering

Clustering

Family-Specific Degenerate Primer Design: A Tool to Design Consensus Degenerated Oligonucleotides

bacteria_viruses

Different tools:

omics_tools

A Primer for Designing Degenerate Primers

human_mouse

Different tools (they seem to be not free) . Go to Tools in the menu:

non-free_tools

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Thank you, I know about those papers etc, and I would like to know which one do you recommend?

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