16s DNA sequencing protocols often entail the use of genomic DNA extraction. The purified DNA is then used as an input for a PCR reaction that amplifies the 16s region. This is then sequenced.
Is this purification step required? Or can cell lysis be used for amplification of the target region?
I am focused only on a defined community with less than 10 microbes present.
Hello Sammy, I don't understand the question. Using cell lysis for amplification? As in, you burst cells to amplify them? The cell lysate as i understand it contains the intracellular or cytoplasmic contents of the cell. Do you think it would be wise to use the lysate as it is? I thought you were supposed to sequence the 16S rDNA. How would you do that without first extracting the genomic DNA and making sure that what you want to sequence is the correct thing? Please correct me if I am wrong because I don't seem to get you. Or is there something you are not telling us?
All you need for sequencing is a clean pcr amplimer. With recent mutated polymerases such as KOD polymerase you can get good clean amplification from whole blood, stool samples and quite dirty and unpurified tissue samples . It is not much harder than colony pcr so try it and expect it to work. You will get the correct product if the primers are well designed and the sequencing will confirm this
Hi, ^^
My answer is yes, you can. I did it last few months with good result in PCR and sequencing. Briefly, I just used 0.5 ul bacterial culture broth for PCR and got clear band (you have to add a little more taq as 1.5 time higher than normal). Then treated the product with SAP and Exonuclease and sequenced it. I also succeed with mold by the same way. I attached the results as below ^:
(Note: Gel image: band 1 - 2: mold; 3 - 4: antibobacter; 5 - 6: bacteria)
Consider that if you used batch of bacteri (10 microbes as you mentioned) sequencing will mess by many distinct sequences.
You mean full length 16S? It will work using cell lysates but if you have a community of different bacteria for some of them (like Gram+) boiling lysis might be less efficient or even not working.... so if you want to quantify each species in the population it will not work... You have to extract the DNA or at least run beadbeating to be sure that you have the whole community lysed....
I've tried using clarified cell lysates as the template in the past and it worked. It really depends on your sample and how specific your primers are. That said, DNA extraction shouldn't take that long. It is highly recommended to get a higher quality sample as your PCR template. However, if you don't have the means to extract DNA for whatever reasons, then go ahead and try it - you never know! Good luck!
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