Question

Identification of unknown Bacteria by sequencing rDNA

Having a little trouble understanding the PCR process. This is a lab we did and some homework questions regarding the sequencing rDNA to find unknown bacteria. I hope by answering these I can have better understanding of the process. The more descriptive the better.

Thank you!

1.

A. Name the gene that we will be sequencing in order to identify the unknown. Explain why this region is considered to be the target for identification instead of sequencing the entire genome? What is the size (in bp) of target DNA in E. coli?

16s ribisomal DNA....

B. What do we mean by ‘conserved regions in rDNA’? Name the technique used to amplify desired target DNA and state the purpose of this amplification step. Where, in the target DNA, do primers bind during PCR (conserved vs. variable)? How many variable regions are present in rDNA? (Copy and paste the schematic diagram to show the regions)

Answer 1

B. Conserved regions in rDNA means that the 16 S rDNA sequence of distantly related organisms are similar or identical. It means that this gene has undergone least change over the course of time.

Technique : PCR.

We do the amplification step to get plural amount of sequence we want to work with cause if we have only 1 sequence, and if the experiment fails, we won't have any more sequence to plan another experiment and so we need plural sequences in hand.

Primers bind to known sequences just upstream and downstream the sequence we want to amplify. We need to know the sequence just upstream or downstream to design the primers.