Genetics Question: I did a lab on Identification of Unknown Bacteria by 16S rRNA

Question

Genetics Question:

I did a lab on Identification of Unknown Bacteria by 16S rRNA Gene Sequence and have to write a lab report on it. For the discussion we have to answer a few questions and I am struggling with them. In this lab we attempted to identify an unknown bacterium by isolating the organism’s DNA, amplifying a portion of its 16S rRNA gene using polymerase chain reaction (PCR), having the amplified DNA sequenced, and comparing the sequence to sequences in an online database. We found that all had either 99% or 100% match with only one unknown not having a correct match. I also had an equal match with two or more species of bacteria; therefore, I could not pick a correct bacteria until my teacher provided my class with the correct bacteriums for the unknowns (5 unknowns with 20F primer and 1500R primer each)

Discussion questions I need help with:

Was isolation of DNA necessary prior to PCR amplification of the 16s rRNA gene? If isolation of DNA was necessary for amplification of the 16s rRNA gene in some samples, is it necessary when trying to identify a clinical unknown? Did you have 100% sequence identity with a bacterium in the database? Why might you not have 100% identity even if your exact strain has been sequenced? Did you have an equal match with two or more species of bacteria? Why might this occur and what does this tell you about using 16S rRNA gene sequences for identification of bacterial unknowns?  

Explanation / Answer

DNA extraction is most important and most cumpulsion thing prior to get amplification of 16s rRNA gene.

if you extract DNA of unknown bacterium and then amplify it with the primer then and then only you will have your templet for doing PCR. so that extracted DNA will serve as a templet for your PCR reaction. Now that will give you a apmicon, and you will go for sequencing for that amplicon, so the results of sequencing will reveal the sequence of that amplicon, and you will BLAST that sequence in NCBI, now the most simmilar sequence will be found.

yes it is possible that you get more number of bacteria with 99% or sometime 100% of simmilarity or identity. it is happen when your entered query sequence is too small or less.

look, the all bacteria have this 16s rRNA gene, and it is vary or different in some area and with some nucleotides. number of more bacteria matching will increases with lesser the query sequence. so you should check your sequence length.

second thing is, in sequence, some bases of initials and some of endings are very poor in quality score of sequence, so that regions should be trimed before going to BLAST the sequence. sometime whole sequence has very poor up and down quality score. so we should not rely on that kind of sequence.

if your sequence has good quality score nucleotides, good length and trimed ends, then it can give 100% matching.

this 16s rRNA gene is most variable gene among all bacteria, so it can be used as a indentity.

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