My PI has tasked me with coming up with a way to quickly and cheaply genotype thousands of seedlings to determine whether they are the products of hybridization or self pollination. I am considering various ways to get reduced representation of the genome and multiplex hundreds or thousands of individuals into a library for NGS. Ideally, this technique will involve taking a leaf punch and either using the alkaline lysis or boiling method to get "quick and dirty DNA", or just putting the leaf punch directly into the reaction as you can do with some PCR kits. If we do full quality DNA extractions it is a huge cost in terms of time and labor just to find out if we want to keep the plant or throw it away. So my question is, what can one accomplish with really dirty plant DNA? Just PCR, or has anyone managed restriction digestion and ligation with this sort of DNA?
Hi Lindsay,
to the best of my knowledge, you can do RE digestions with your 'dirty DNA preps' since many REs are quite tolerant to sub-optimal reaction conditions. However, I think this is not true for Ligases, since they are sensitive enzymes and require optimal conditions for optimal results.
best of luck
SD
I have done PCR with boiling method (some lysis too) - worked great and saved TONS of time when genotyping hundreds of plants. Even debris in the rxn mix - still worked fine!
Are you also referring to 'dirty' DNA if leaf chips is used as DNA template for direct PCR (no extraction needed)? Because there are kits (KAPA3G & TERRA) like you mentioned that can produce PCR products and can be use directly for RE treatment. I think the 'no-extraction' protocol would save time and resources especially if highthroughput screening is required. By the way I'm using the direct PCR products and used it for CAPS assay. Ligation is I think another thing. Maybe you could have DNA cleaning first.
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