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Hai, I need a clear protocol for extraction of each and every bacterial DNA that is present in human milk (without using any kit and with using cost effective chemicals). If I need to amplify 16s rRNA region of all bacteria by PCR, then, is that DNA extraction protocol useful? My doubt is 'if I extract all bacterial DNA from breast milk, is that bacterial DNA contains 16s rRNA regions all bacteria'?. Please help me anyone, I need this.

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Hi,

i am not exactly sure if I understand your concern. But if you extract DNA from a sample of human milk, you will have any DNA (or at least most of it) that is contained in that sample. And yes that should include 16s rDNA from the different bacteria contained in the sample.

Cheers

Thomas

Thank you sir for your reply. Sir, my aim is to identify bacterial communities present in human milk and infant feces by using DNA Barcoding. But I was unable to find clear protocols starting from the extraction to sequencing. I want to use culture independent methods and pyrosequencing. So, please help sir in this way.

Dear D.Esther Lebonah,

I am actually not working with bacteria but with plants and herbal products. But anyway, if you want to extract DNA directly from milk or feces I am quite sure there are protocols out there. E.g. http://aem.asm.org/content/73/6/1882.short

After DNA extraction you will have to amplify the 16s rDNA and clone the products. So it won't be completely culture independent anyway :-)

Cheers

Thomas

Respected sir,

               Thank you very much sir for your helping nature. Even though you are  not working with bacteria, you are trying to help me, thank you very much sir again.

 Sir, is it necessary to clone amplified 16s rRNA regions? Because I don't want to clone, but directly I'll go for pyrosequencing that amplified 16s rRNA regions. Is my thought right? or not?

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It seems like you do not need to clone the different subpopulations of 16s rDNA because NGS pyrosequencing can handle those. In my line of work - with plants - we usually need to isolate the respective DNA from one plant at a time, because we are building the DNA Barcoding database (DNA Taxonomy). In your case the result of the sequencing will be analysed by comparison of the retrieved sequences to an existing library of sequences (DNA Barcoding). That we usually don't have in plants.

However the approach seems useful for analyzing commercial products containing DNA from different plants. By trying to help you I also helped myself :-) Thanks!

kind regards

Thomas

Thank you sir for providing the requested information and for your consideration.

Sir, if you have time, please clear me this doubt also.

                      I don't know about using universal bacterial barcoded primer sets but I found one article (http://www.nature.com/nmeth/journal/v5/n3/abs/nmeth.1184.html ) that gives clear information on barcoded primer sets, can I use them directly?, because I just want to identify bacterial communities present in human milk and infant feces by using DNA Barcoding. I think, there is no need to design barcoded primer sets again, so please send me your opinion.

                            Thanking you sir

                                                                            D.E.Lebonah                      

Dear D.E.Lebonah,

I think we first should make a distinction between barcoded primers and DNA Barcoding primers. In the publication you mentioned the topic is to tag primers with barcode sequences to increase the number of samples that can be sequenced in one run and to help detect sequencing errors. This method can theoretically be used with any primer - also non DNA Barcoding primers.

Cheers

Thomas

Respected sir,

    So, what is your opinion on using that set of primers in my work?Because they prepared primer set for all the species of bacteria. Using them directly may amplify particular part of 16s rRNA region. But you wrote that method can theoretically be used with any primer - also non DNA Barcoding primers. Theoretically means, only theoretical ? but not practical? now I'm in confusion. 

And don't know the distinction between barcoded primers and DNA Barcoding primers, I think both are same i.e., tagging primers with barcode sequences, and it shows same results on the web also. So, what is your opinion on this also?

                                Thanking you sir

                                                                                                                                                           D.E.Lebonah                      

Dear D.E.Lebonah,

in my opinion barcoding primers are those which bind to a conserved region in the genome of a wide variety of organisms. They are also called universal barcoding primers. There are some good working primers for the cytochrome c oxidase I (COI) gene in animals and the ribulose-bisphosphate carboxylase (rbcL) gene in plants. In the publication you mentioned they use the broadly conserved bacterial primer 27F and the broad range bacterial primer 338R, any additional parts of the final primers they use for their study (see the supplementary data 1) is not there for the amplification of the target region but for the sequencing methodology and for the reasons mentioned in my previous answer.

I would advice you to invest a little more time in reading, especially on the topics that help you understand what you are trying to do. I am not an expert on bacteria, I do not know what kind of bacteria to expect in your samples and how diverse the priming site of the 16s rDNA might be in those groups.

See for example the following publication:

Coverage evaluation of universal bacterial primers using the metage...

Have a nice day

Thomas Horn

Thank you sir for taking the time. I'll follow all of your suggestions.

                                                                               D.E.Lebonah 

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