100 Ideas

"One a year" - A pre-retrospective: 2008-2108

Problem: sequencing a heterologous mix of 16s PCR products


Possible solution: Use gel electrophoresis to separate the DNAs.

DGGE of small ribosomal subunit coding genes was first described by Gerard Muyzer, while he was Post-doc at Leiden University, and has become a widely used technique in microbial ecology. PCR amplification of DNA extracted from mixed microbial communities with PCR primers specific for 16S rRNA gene fragments of Bacteria and Archaea, and 18S rRNA gene fragments of Eukaryotes results in mixtures of PCR products. Because these amplicons all have the same length, they cannot be separated from each other by agarose gel electrophoresis. However, sequence variations (i.e. differences in GC content and distribution) between different microbial rRNAs result in different denaturation properties of these DNA molecules. Hence, DGGE banding patterns can be used to visualize variations in microbial genetic diversity and provide a rough estimate of the richness and abundance of predominant microbial community members.

Solid-phase PCR (Polony PCR) is also intriguing: Dilute and spread the DNAs across a surface and use solid-phase PCR to amplify individual DNAs into polonies. Pick polonies and have them sequenced.

“We describe a method to clone and amplify DNA by performing the polymerase chain reaction (PCR) in a thin polyacrylamide film poured on a glass microscope slide. The polyacrylamide matrix retards the diffusion of the linear DNA molecules so that the amplification products remain localized near their respective templates. At the end of the reaction, a number of PCR colonies, or ‘polonies’, have formed, each one grown from a single template molecule. As many as 5 million clones can be amplified in parallel on a single slide. If an Acrydite modification is included at the 5[prime] end of one of the primers, the amplified DNA will be covalently attached to the polyacrylamide matrix, allowing further enzymatic manipulations to be performed on all clones simul-taneously. We describe techniques to make replicas of these polony slides, and high throughput sequencing protocols for this technology.”

Lastly, I could just give up on the idea of a heterologous PCR product and instead design primers specific to a particular rDNA or other gene of interest. Instead of exploring the space of all rDNAs present in the sample’s metagenome, I would be testing for the presence of a particular DNA. Much less exciting, if you ask me.

Colophon

I'm Mac Cowell. I'm an amateur biologist. I'm a part of diybio.org. I'm interested in synthetic biology and refactoring and reengineering existing biological tools and techniques to be cheaper, easier, safer, and more accessible.

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  1. Hi Mac, What do you think about taking your PCR product and then digesting it with a few enzymes? After electrophoresis take that image and compare it to a virtual electrophoresis run of 1000 bacterial species, and see which bands your sample has. This would work best with probably under a dozen species at once, beyond that I imagine there will be a ton of bands to figure out . Any obvious holes in this idea?

    Titol


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