BSCI222 – Lecture 19- Next generation sequencing technologieso Now that we have a reference (the first human genome), we can use cheaper methods for more sequencing.o 2 major companies today. No longer use gel to separate the fragments by size; usehigh-throughput array format, sequencing many molecules at the same time, sequencing as the DNA is being synthesized (not after like the old method does).o Comparison of Illumina and Pacific Biosciences companies. Illumina: take genomic DNA, shear it down to 200-300 base pairs in size (leaves ragged ends, the two are not the same length). Next step: polish theends, by using polymerase to fill in on one short end, and chew away fromone long end. Then ligate on short adapter DNA molecules, to produce a product of fragments with adapters. Next, generate small cluster of DNA molecules that are all identical (ideally, would like to sequence just one, but you get so little fluorescence from just one; by building a bunch of copies in each spot on the slide, don’t have that problem). One of the product strands get attached to a wall of olligose, (then size selected and purified) and then have synthesis (bridge amplification, getting a repeated amplification synthesis of DNA molecules). End result is many identical copies of each DNA fragment, each appearing as a small spot in each reaction. Add one base, wash all the other nucleotides away, probe it with a laser (to light it up and determine the color), and thus have a dot on the image of the slide. Build it up with each step. After detecting the color, add enzymes to remove the fluorescent tag (reversible terminator, not like the old method), and can add nucleotides to the next base, get color, reverse termination, add another base, get color, etc. Takes a long time; foreach step of reading one base, have to go through a whole series of washing steps. To sequence 150 bases this way takes ~1 week. But doing it with millions and millions of spots, so it’s highly parallel, and thus have high throughput. Can get up to 1 Terabyte in one run (1,000GB). Eventually get a few failures in the synthesis of one strand, start getting out of phase of one another, so a limit for the technology is about 150bp/reading. But the throughput is amazing. Pacific Biosciences: Longer reads, but not as many. Easier to put the puzzle pieces together (longer and fewer). Also use sequencing by synthesis. Detect the fluorescent nucleotide while it’s in the polymerase being added to the strand; had to overcome a problem, because if you wantto probe a single polymerase for a single nucleotide being added, and you have to have a really high background signal concentration of nucleotides, had to develop a waveguide: metal layer on a glass substrate – very narrow channel, can only detect the nucleotide in the polymerase because the wavelength can barely fit in there (can only go about 20-30nanometers). Tether a polymerase at the bottom of the channel/well, give it DNA, starts to synthesize, fluorescent nucleotides diffuse in there (fluorophore attached to the phosphate group), and then detect the intensity of fluorescence from that tiny area at the bottom of the well (a single nucleotide is being held in the polymerase and being added to the DNA strand, incredibly short period of time). Always a short pause for thesynthesis to happen and the next nucleotide to appear there (added by the polymerase). Happening thousands of times in parallel.- Chapter 8: Bacterial and Viral Genetic Systems- E. coli: small, rapid reproduction, small genome, many mutants available.o One big circular chromosome (about 4.5million base pairs, about 4300 genes).o Also have smaller circular molecules called plasmids (a few thousand bases), carry additional genes (like antibiotic resistance), can be transferred between bacteria of same or other species. Lateral transfer.- Many reasons for using bacteria and viruses: haploid genome makes it easier to detect mutations (only one copy, don’t have to look for homozygotes), fast reproduction, many progeny, etc.- Place bacteria on loop + sterile solution -> leave overnight at 37 degrees, get billions of copies in the morning. Or can do it on Petri dishes. Next morning, begin to look for phenotypes (usually not visible with naked eye), usually nutritional mutants (mutation in one of the genes for an enzyme that is involved in synthesis of some essential molecule). o Take culture -> plate it out, let grow over night so each individual bacteria makes a colony -> replicate the plate by taking aluminum plug, covering it with velvet (many little hairs), stamp on the cells, which enables the hair to pick up bacterial cells, then stamp onto other plates. o Can do the replication plate in the same medium as the original culture, or another. Leucine: can find colonies that grow fine on the plate with leucine, but do not appear on the Petri dish with a medium missing leucine (leu- mutant). Have to keep careful track of the dishes’ orientations, to see what is missing.o Lederberg and Tatum studied bacteria mutant for many things. Y10 (thr-, leu-, thi-) and Y24 (bio-, phe-, cys-), neither can grow on minimal media. If you could get a recombination between the two, could produce that has all the enzymes it needs in order to grow on minimal media, can synthesize all those six things. That’s what they were looking for, these recombinants. Found that if you mix the two strains in a tube, can get some growth on minimal media, meaning that they have undergone recombination. How did that happen? Only one copy of the genes in each cells (not like myosis where you have diploid); had to have a mechanism by which the DNA from the two strands came together in the same cell and underwent recombination. 3 major mechanisms by which DNA from 2 bacterial cells can undergo recombination: transformation, conjugation, and transduction. (transcription, translation, other trans words, might be on the test!)- Transformation:o Simplesto Bacterial cells can take up DNA from the environment. Techniques in the lab can make them especially good at it (called combinant (sp? Competent?) cells), but most can do it anyway (good food, to them). o If the DNA gets into the cell, can have double recombination event that will swap this new DNA with part of the bacterial chromosome. A single recombination event would only open up the circle (draw)o Series of kind of rare events, relatively rare process, but clearly happens