What is a bacterium made out of?
- protein 55% - DNA/RNA 24% - lipid - 9% - carb - 6% - other organics 3% - inorganics - 1%
- random mutagenesis and see what genes are involved
- find the gene that is mutated
- complementation - 2 mutants restore a normal phenotype
- have sequences of all genes but not known what each gene does.
- look through genome and guess what genes are involved in whatever you want
- gene directed mutagenesis to generate strains deficient in the gene and check for a phenotype.
How Sanger sequencing works
- ssDNA, DNA pol, all ddNTP's, labeled primer, template DNA
- 4 tubes containing all the components needed to polymerize DNA - adds a small amount of ddNTP to each tube
- creates a pool of DNA sequences of different length all ending with that specific nucleotide
- terminates reaction at every A, T, G, and C nucleotide in each tube.
- run on 4 lanes and visualize
- shortest bands travel furthest
Old DNA sequencing
- used radioactively labeled DNA, a gel was run and the bands visualized after exposing the gel to film.
- gels had to be read manually and the amount of sequence was only 200-400 bps.
Fluorescent DNA sequencing
- all the reactions can run in one lane because each base has its own color
-automatic reading of the sequence from the gel from the fluorescent scanner
- sequence is read from the bottom up
- up to 1000 bp read length
- DNA sequenced using dideoxy chain termination but instead of using gels, the bands are separated using electrophoresis in capillary tubes and the signal (flouro color) is read as the sample leaves the tube by automated sequencing machine.
- performed at higher voltages in smaller volumes
- shortens runs times and increases band resolution, so more bp sequenced in single run (up to 1000)
2nd generation sequencing
- 454 or pyrosequencing - light
- illumina sequencing - fluoro (color)
- ion torrent - pH
- no need to clone but each has short reads
- if dNTP incorporated, pyrophosphate released
- converted to ATP by sulfurylase
- luciferase uses ATP to oxidize luciferin to generate light
- peak if nucleotide incorporated
- dNTP not incorporated - no light or ATP - degraded by apyrase
- can't do too many at once because sensor can't tell difference between more than 6
- genomic DNA denatured into short pieces
- add adapters
- add to bead
- PCR - polymerase - pp - ATP - light
- must sequence a lot of DNA 30-100X
- prep the library
- generate clusters of DNA in each flow cell
- template strand - incorporation of base - fluoro cleavage (color)
- clusters are excited by a laser and color seen.
- continuous cycle of extension, identification, and cleavage.
- flood chip with nucleotides
- if the nucleotide is incorporated, a proton is released
- proton release read by the sensor
- if more than one nucleotide incorporated, the H+ level is raised x-fold
- clone very large pieces of DNA, sequence both ends of the pieces, put the clones in order, then design primers to sequence further into the sequence, repeat until the whole genome is done.
- look for start and stop codons in frame and more than 100 bp apart - translate the gene and search the database for homologous genes - if yes, it is likely a good gene, annotate - if not in database, you have a novel gene
- does it overlap another ORF? is it in an operon? - is there a recognizable promotor or ribosome binding site? - Does it use the same codon bias as the rest of the genome
- for every codon for Ile don't want to make all 3 tRA and tRNA synthase so just make one or two codons. - micrococcus luteus only makes one. E. Coli makes two.
- no match in the database
- matches in the database that also have not had a function defined