Download - The Undergraduate Genomic Sequencing Project
THE UNDERGRADUATE
GENOMIC SEQUENCING
PROJECT
Zachary Bendiks
Jonathan Eisen UC Davis Genome Center Lab focus: “Our work focuses
on genomic basis for the origin of novelty in microorganisms (how new processes and functions evolve).”
Metagenomics – assessing the microbial makeup of environmental samples
Bioinformatics - applying computer technology to phylogeny reconstruction
http://www.ucdmc.ucdavis.edu/medmicro/jeisen.html
16S rDNA Present in all prokaryotes. The 16S gene codes for the small rRNA
subunit. ~1.5 kb in length rRNA can be phylogenetically significant
crucial for protein synthesisConserved structure and function across all
organismsUniversal in cellular organismsDiffering rates of evolution throughout the gene can
help identify species Carl Woese (1970)
The Undergraduate Genomic Sequencing Project First attempt by Eisen lab to incorporate
undergraduates Attempts to strengthen mechanical lab
skills by taking undergraduates through the process of isolating genomic DNA.
Learn simple genetic analysis techniques and methods used to identify organisms
Culturing Bacteria Place environmental
sample in liquid overnight culture
Grow liquid culture on nutrient plate
Use dilution streaking to isolate individual colonies, and thus, individual species
http://www.scienceprofonline.org/science-image-libr/sci-image-libr-bacterial-growth-media.html
Genomic Preparations Extraction of genomic
DNA from the cell Lysis of the membrane Cellular proteins and
other impurities are dissolved
Genomic DNA is suspended in rehydration solution
Presence of DNA is confirmed on agarose gel
http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/dna_lab/images/dna_precipitate/174993-1-eng-AU/dna_precipitate_large.jpg
16S PCR DNA polymerase, forward
and reverse primers, and buffers are combined with the DNA
Solution heated through cycles for several hours. Taq polymerase -
thermophilic Allows polymerase to
repeatedly amplify the regions that the primers specify
Confirmed on agarose gel
http://upload.wikimedia.org/wikipedia/en/a/a9/Amit_Yadav_16S_PCR_Phytoplasma.JPG
PCR Purification Free-floating primers,
buffers, and other impurities are still in the solution. Need pure DNA for sequencing
Use a series of solutions and a filtering tube to remove contaminants
Pure DNA solution can then be diluted to the proper concentration and sent for sequencing
http://www.favorgen.com/jpg/FAEPK-02.jpg
Genetic Analysis Geneious – creates alignments between
fragments and generates consensus sequence
BLAST – compare sequence to a database of organisms
GOLD – lists the number of in progress or future projects for organisms
http://www.geneious.com/geneious_theme-theme/images/biomatters/screenshots/3.7_sequence_organize_sequence_visualize.png
Looking Forward My sequenced organisms so far
Staphylococcus pasteuriEnterobacter ?Bacillus amyloliquefaciens6 more currently in sequencing facility
Undergraduates will be assigned an organism and write a summary report of it and publish the full genome to a database