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I really need an answer on PCR (16s rRNA), PLS PLS PLS! - (Apr/29/2007 )

For the analysis of soil microbiology, is the protocol in the following order?

  • DNA extraction
  • 16s rRNA PCR (to amplify 16S rRNA sequence)
  • PCR product purification
  • Cloning
  • T-RFLP
  • DNA sequencing

There is 1 big doubt which I really need to have an answer. It is regarding on 16s rRNA, which is after running this then followed by agarose gel electrophoresis, how many bands will there be? Is it 1? If there is only 1 band appear but it comprises of many different 16s rRNA sequences, am I correct to say so?
Why do we have to carry out cloning when the 16s rRNA sequences have already been amplified to many copies? Why can't directly perform T-RFLP directly after PCR product purification?
What's the importance of peaks produced from T-RFLP in identification of microbe when DNA sequencing is performed right after T-RFLP?


Yes, there should be single band after your 16S rDNA PCR which consists of different 16S rDNA sequence from various organisms in the soil. Your have to clone the amplicon so that you can separate each of the different 16S rDNA sequence which come from different organism. Then, you have hundreds of clones in your library. Each of the clone carries one of the 16S rDNA fragment. Different clones may carry the same sequence fragment or a different one.

We may not want to sequence each of the clones (imaging how much it costs sequence hundreds of clones). So, we try to sort out which clones are the same, which are not... using a typing method. In your case, the typing method is T-RFLP. I am not familiar with T-RFLP, but I believe the theory is the same with other typing methods. The typing method is used to differentiate the clones. The clones are grouped based on the banding patterns. Clones with different banding patterns indicating that they are different in sequence and from different organisms. Clones with identical banding patterns suggesting that they are the same, but they may not. Then, you may choose some clones from each group for sequence determination.

Hope that this help.


Really thanks for our answer but I have another doubt. In this case, during the cloning duplicate was made so is that being used to carry put T-RFLP and the other send for DNA sequencing? Becos T-RFLP may not give the confirm identity of a microbe as 1 peak may represent more than 1 organism so DNA sequencing is performed to confirm and tally with T-RFLP results in order to construct a phylogenetic tree? Pls clear my doubt, thanks!