DNA Sequencing Methods: Chain termination method Maxam-Gilbert Method F.

F. Sanger nd 2 generation sequence methods Pyrosequencing 454 technology Bridge PCR Illumina Massively Parallel System Emulsion PCR SOLiD system sequencing rd 3 generation sequencing method Single Molecule Sequencing The Maxam-Gilbert Method One of the very first methods invented. Extremely popular for some time Could use purified DNA directly instead of clones Uses chemical cleavage and separation of fragments Procedure Purified dsDNA is denatured into ssDNA ssDNA labeled with 32-P Sample is separated into 4 reaction groups, one for each dNTP G+A o Stock treated with a limited amount of DMS (dimethyl sulfate) + formic acid under alkaline conditions to attach a methyl group to the purine ring: Leads to instability, releases the ring o Piperidine facilitates -elimination, DNA cleaved into 5 and 3 fragments G o Stock treated with a limited amount of DMS (dimethyl sulfate) under alkaline conditions to attach a methyl group to the purine ring followed by Piperidine C+T o Stock treated with Hydrazine, which attacks at 4C & 6C and opens the ring o Pyridine causes -elimination and separation into 5 and 3 fragments. C o Stock treated with Hydrazine in 2M NaCl, which attacks at 4C & 6C and opens the ring followed by Pyridine The products of cleavage are then analyzed by electrophoresis on a polyacrylamide gel Gels are refrigerated with x-ray film, exposed by the radiation from the isotopes Films are read from bottom to top

Base calling involve interpreting the banding pattern relative to four chemical reactions. Eg a band in C & in CT is read as C, where as a band in CT only & not in C is read as T. Similarly for G & GA band Drawbacks Of Maxam-Gilbert Method Not really used anymore Reagents difficult to pack in kits A large amt of radioactive substance(S35 ,P32) used Hydrazine used happen to be neurotoxin Scale-up is difficult Better methods available Sanger method Also called the dideoxynucleotide method. Utilizes dideoxynucleotides, nucleotide bases with no hydroxyl group on 2 or 3 C. Components needed are DNA template which is needed to be sequenced A short DNA Primer complementary to the template DNA DNA polymerase Four deoxynucleotides (dNTPS) Four radio-labeled dideoxynucleotides (ddNTPS) Steps of reactions Reaction mixture: Four reactions tubes are taken and mix all the components in all the tubes, with each ddNTPs in separate tube (i.e ddGTPs, ddATPs, ddCTPs and ddTTPs in respective tubes) Denaturation: To start the sequencing reaction the mixture is heated so the complementary template DNA strand separates Annealing: The temperature is lowered to allow the primer sequence to bind to its complementary sequence in the template DNA Extension: Temperature is then slightly raised so that the enzyme polymerase 3 combines to DNA and creates the new strand of DNA Chain termination: dNTPs are added by the enzyme until a ddNTP is added. Once a ddNTP is incorporated into the strand, the chain is terminated. The strand can be terminated at any position resulting in a collection of DNA strands of many different lengths. This results in four dideoxystrands in their respective tubes Electrophoresis: Next, each reaction mixture is electrophoresed in a separate lane (4 lanes) at high voltage on a polyacrylamide gel. Pattern of bands in each of the four lanes is visualized on X-ray film. Location of bands in each of the four lanes indicates the size of the fragment terminating with a respective radio-labeled ddNTP. DNA sequence is deduced from the pattern of bands in the 4 lanes

Automation: Automated sequencers use 4 different fluorescent dyes as tags attached to the dideoxy nucleotides and run all 4 reactions in the same lane of the gel. Chromatograms processed with software to sort signals, results in dsDNA sequence Two types of machines used for this Capillary sequencer Gel-based sequencer Pyrosequencing It is a technique to sequence DNA by using chemiluminescent enzymatic reactions Principle: First step is the preparation of single stranded DNA molecule as a starting material by denaturation DNA polymerase will start elongation by using dNTPs If the dNTP is incorporated it will release phosphate (DNA)n + dNTP Polymerase (DNA)n+1 + PPi

Pyrophosphate will be converted into ATP from Adenosine phosphosulphate (APS) by sulfurylase APS + PPi Sulfurylase ATP

Luciferase will use ATP to oxidise luciferin and generate a flash of chemiluminescence Luciferase Oxyluciferin + Light

Luciferin + ATP

The light will be generated as a peak for each one type of nucleotides incorporated Each peak represents a nucleotide so by this whole sequence can be determined

454 Technology: It is based on pyrosequecing DNA is sheared into 300-800 bp fragments, and the ends are polished by removing any unpaired bases at the ends Adapters are added to each end to hold primer. The DNA is made single stranded at this point

One adapter contains biotin, which binds to a streptavidin-coated bead. The ratio of beads to DNA molecules is controlled so that most beads get only a single DNA attached to them Oil is added to the beads and an emulsion is created. PCR is then performed, with each aqueous droplet forming its own micro-reactor. Each bead ends up coated with about a million identical copies of the original DNA. After the emulsion PCR has been performed, the oil is removed, and the beads are put into a picotiter plate. Each well is just big enough to hold a single bead Reagents are then added to the beads places in microwells : o DNA polymerase o Adenosine Phosphosulfate (APS) o ATP Sulfurylase o Luciferin o Luciferase The plate is then repeatedly washed with the each of the four dNTPs in a repeating cycle The plate is coupled to a fiber optic chip. A CCD camera records the light flashes from each well Illumina Massively Parallel System Principle o The idea is to put 2 different adapters on each end of the DNA, then bind it to a slide coated with primers complementary sequences for each adapter. o This allows bridge PCR, producing a small spot of amplified DNA on the slide o The slide contains millions of individual DNA clusters. o The spots are visualized during the sequencing run, using the fluorescence of the nucleotide being added Steps involved o Prepare genomic DNA sample Randomly fragment genomic DNA and ligate adapters to both end of the fragments o Attach DNA to surface Bind ss fragments randomly to the inside surface of the flow cell channels (just like a sheet) o Bridge amplification Add unlabeled nucleotides and enzymes to initiate the solid phase bridge amplification o Fragment become double stranded The enzyme incorporate the nucleotide to form double stranded DNA on a solid phase substrate o Denature the double stranded molecules Denaturation leaves single stranded substrate anchored to the solid phase substrate (basically it is surface attached)

o Complete amplification After multiple cycles amplification completes o Determine first base Add four labelled reversible terminators, primers and DNA polymerase to the flow cell o Image first base After laser excitation, capture the image of emitted fluorescence from each cluster of flow cell o Determine second base Remove the terminal label and add all four reversible terminator bases o Image second base Laser excitation, record the second base o Repeat the cycle until 33-36 bases read. o Align data, compare to the reference, identify sequence

Single molecule sequencing o Single molecule real time sequencing (also known as SMRT) is a parallelized single molecule DNA sequencing by synthesis technology. o Relatively new, Next-Next-Gen technique that does not require the use of cloning, amplification, or ligation o Sequence-by-Synthesis approach o May significantly reduce the cost of sequencing o Applications in: Pharmaceutical R&D Oncology Research Clinical Diagnosis/Personalized Medicine o It requires Zero Mode Waveguide (ZMW) Is a nano-photonic, cylindrical metallic visualization chamber, about 70nm wide This is present in SMRT 8 cell pacs (a tray of 8 packs, a total of 12 SMRT 8 cells pacs can be loaded) Providing a detection volume equivalent to 1 base. Creates an illuminated observation that is small enough to observe only a single nucleotide of DNA being incorporated o Steps A DNA polymerase molecule is affixed to the bottom of a Zero Mode Waveguide with a single molecule of DNA Phospholinked nucleotides tagged with different colored fluorophores are introduced in high concentrations DNA polymerase incorporates a nucleotide (this takes place within the detection volume for a very short period of time) The engaged fluorophore emits light with color corresponding to base identity When DNA polymerase cleaves the bond holding the fluorophore, the dye diffuses out and the signal returns to baseline o Advantages over older methods: Long read length Short cycling time Low cost Reliably good data Rate of reaction within the detection volume is very fast, results in low background Low detection volume (20 zeptoliters, or 20x10^-21 Liters) decreases chance of interference