Heliscope single molecule sequencing
Heliscope sequence is sequencing method of single molecules developed by Helicos Biosciences Corporation. It uses a DNA fragment in a poly A tail adapter added is connected to the flow cell surface. In the next step, I have to extend the base sequence annular flow cell cleaning solution fluorescently labeled nucleotide (as in the Sanger method, type of base one by one) and. Reading is performed by Heliscope sequencer. 55 bases in each run, read, is short, recent improvements is allowing you a more accurate reading of the section of a type of nucleotide. The apparatus and method of sequencing this, I was using the genome of bacteriophage M13 in sequence.
And work together as an integrated system, genetic Helicos ™ assay system is made up of several components. DNA molecules for sequencing target hybridizes to the site of a single stream of glass cell. The sample with the probe Helicos loader temperature can be adjusted optimal hybridization, it is loaded into the flow cell. Each of the 25 channels, it can be considered separately in other steps necessary to sample was added to prepare a sample standard cells flow. The sequencing system together ™ synthetic that is inserted in the sample HeliScope When loaded properly when the flow cell, and all reagents needed for sequencing by image. Was the sequence for as long as necessary, the image is processed in real time Helicsope ™ engine analysis sequencing system. It processes the image from the physical location of any analysis engine read from these pictures the construction sequence. After execution, and full-field imaging process is transferred to the cluster data to calculate the reference group or aligned properly.
Single molecule sequencing (SMS), and provides information about both series quantitative accurate, Helicos ™, provides a unique view of genome biology through direct sequencing of cellular nucleic acids in an equitable way. To avoid size variations seen in other techniques and GC content, sample preparation is not required PCR amplification or ligation. Subsequently poly simply, DNA was hybridized to the surface of the flow cell containing oligo dT for sequencing-by-synthesis and molecular parallel shear, the several billion. This process will require much less material compared to other technologies. The measured gene expression is carried out by direct quantitative method based on the first strand cDNA or by the (RNA-seq) so that it is possible to use a new approach that enables direct sequence hybridization and cellular RNA I can.
The successful diversification of applications, chip array using picogram weight DNA copy number variation study of both of FFPE tissue samples and tumor tissue fresh, sequence of DNA was degraded and ancient, RNA, a small study, that is the exact mutation This leads to the identification of a new class of RNA have been made, including the genome sequence for detection and capture an array of RNA from 250 cells only cell mass directly. Most of the next generation technology, since it requires, it is not possible range of sequence-specific amplification and size of the target DNA molecules that do not meet these criteria sequencing reliably. Obtaining is unnecessary, it is possible to use as a template directly the degraded molecules or have changed, the sequence of the single molecule is not subject to these limitations it. ® explaining the principles and methods for using a genetic analysis system Helicos.
Generally, samples for sequencing, poly (A) tail was prepared to be longer than (dT) 50 oligonucleotide on the surface of the flow cell. Residues that are not paired are needed to fill the treatment, to lock Thing avoided. After hybridization, 37 ◦ C, and then in the dGTP and dATP the lowered virtual terminator nucleotides corresponding dTTP, and dCTP, was added together with DNA polymerase temperature. Contains an extra base and due to the chemical structure attached to the nucleotide, the virtual terminator nucleotides prevent more involved. Accordingly, TTP is filled with all of the unpaired DASS present in poly (A) tail. Hybridizing molecules are locked into place when it detects the insertion and non-residue of the first nucleotide virtual polymerase terminator. Color image application will include all molecules incorporation of a nucleotide is possible because there is a need to DNA molecules each have now. Further, since the label can be assigned to any base, it does not give sequence information at this stage. Thus, for the majority of the molecule sequence, starting from the base of the second of the original molecule.
The DNA sample, it is necessary to generate a nucleic acid that is compatible with the purpose of the hybridization of these surfaces normally I was hybridized primers immobilized to the flow cell on for sequencing. Target sequence attached to the surface of the flow cell may have a sequence that can be synthesized in theory, in practice, oligo flow cell standard commercial is (dT) 50. To be compatible oligo surface on the flow cell (dT) primer and 50, it is necessary to generate a 50 NT least the 3 ‘end of the molecule poly tail to be (DA) sequencing. When you lock the step and filling, so will fill, is that rather than on the surface oligo (dT) as long as, T excess, it is desirable at least tail it. Generation poly (DA) tail 3 ‘can be carried out using a polymerase or ligase variety. If the average duration DNA, and sufficient for the mass measurement, it is possible to determine the exact amount of dATP, but added to produce a tail poly (DA) from 90 to 200 nucleotides it is possible to. To generate the tail of this length, is in the sample there, it is the first to determine the 3 ‘end of how to use the terminal transferase in the tail of the range of optimal size, mix of the DNA of the right, the dATP There is a need.