Homework 8

We want to design primers to isolate the gene encoding for tRNA-Glu in E.coli from a metagenomic sample. This gene was first sequenced in 1978 by Brosius et al. as part of a DNA fragment that is available in NCBI with accession number J01695.2.

1. Find the gene location in the fragment.

2. Use Primer-BLAST to design primers. We want to find forward primers inside a 100bp region before the start of the gene. The reverse primer should be somewhere in the 100bp region after the gene end.

   Choose database “Refseq representative genomes” and organism “Bacteria”. The melting temperature of the PCR product should be at least 55°C ideally 57°C, and at most 60°C.

3. Describe the sequences reported as “highly similar” to the PCR template. What is their taxonomy?

4. Choose only the sequences related to Escherichia coli and report the primers found by Primer-BLAST.