Question

determine the sequence below then answer following questions. IF YOU DONT KNOW IT OR ITS NOT GIVEN SKIP IT!

sequence:

GGGCGGGGTCTATACATGCAAGTCGAGCGAACGGATTAAGAGCTTGCTCTTAAGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGCACCGCATGGTGCAAGATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTATGAGTTGAATAAGCTCATGCCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG

question:

_______ 4.0 pts. B. Name the gene that we will be sequencing in order to identify the unknown. C. Explain why this region is considered to be the target for identification instead of sequencing the entire genome? D. What is the size (in bp) of target DNA in E. coli

__________ 4.0 pts. 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)

_________ 1.0 pt. Will you be able to identify your unknown organism(s) if you used DNA sequence for conserved regions (instead of variable regions) from your PCR product? (Yes or No)

__________3.0 pts. Enter your sequence data here (for A & B). Which variable regions are included in your PCR product? What is the advantage of sequencing these regions?

________ 3.0 pt. Write the acronym for RDP. In addition to using DNA sequence data for identification, list two other uses.

1.

2.

__________8.0 pt. Name the genus and species of the organism that best matches the sequence of your PCR product(s)

Organism A _____________________ ____________________

(Genus 2.0 pts) (species 2.0 pts)

Organism B _____________________ ____________________

(Genus 2.0 pts) (species 2.0 pts)

Answer 1

B. The name of the gene that will be sequenced to find out unknown strain of E.coli will be 16S Ribosomal RNA gene. Apartial sequence of which will be amplified to send for sequencing.

C. Instead of targeting the entire genome of the bacteria this 16S rRNA gene is targeted because they are ubiquitous in nature where ribosomes can not translate mRNA if there is no 16S rRNA component. Hence they are very essential in prokaryotes and also higly conserved. So we can construct universal primers for these highly conserved region and can amplify 16S rRNA gene from divergent bacteria.

D. The size of 16S rRNA gene is around 1400 bp and present in multiple copies hence very easy to amplify to get prominent band.

Conserved regions : These are the similar sequence that remained unchanged which would help us to make phylogenetic relationships among different species.

The technique used for the amplification of this region is  PCR. The primers do bind to conserved region not to variable region. However the sequence amplified which also contain variable region help to distinguish between 2 species.