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Third Generation DNA-Sequencing-LifeTechnologies FRET Sequencing Platform
Third Generation DNA-Sequencing-LifeTechnologies FRET Sequencing Platform
Differences between Sanger, Second Generation, and Third Generation
Second Generation
cost-efficient
simultaneous interrogation of more than 100 genes at a time
finding a novel variants by increasing the number of targets sequenced per run
analysis of samples with low-input DNA
sequencing of whole genomes, especially microbial genomes
Sanger
target small genomic region
sequencing of variable regions
based on detection of labelled chain-termination
sequencing of single genes
known as chain-termination sequencing
cost-efficient sequencing of single samples
analysis of longer fragments (~1,000bp in lentgh)
verification sequencing for site-directed mutagenesis
less error-prone than next generation sequencing
specializes in Sanger sequencing application
DNA isolation
Bioinformatics: mapping
Genotyping- determint genetic variants
Primer walking
cloning
PCR establishement with/without primer design
Third Generation
error rate of a single read goes up to 15-40%
whole-genome sequencing requires 30 times to 70 times coverage
the connections between configs are clear
cost- high
much longer reads
History/background on Life Technologies FRET Sequencing Platform
Dominique Francois Jean Arago, thefamous French astronomer repeated Oerstead's experiment and suggested the possibility of a telegraph together with Jacques Babinet
Faraday pictured lines of force as the mechanism by which electrical and magnetic substances interact with themselves and with each other
James Clearn Maxwell created a compeleted mathematical representation of Faraday's descriptions of electricity and mangnetism
First theoretical attempts explaining FRET were applications of this classical EM theory
Then, he introduced the notion of dispalcement current and furnished theoretical setting to predict electromagnetic radiation
Hertz carried out the critical experiment in 1888, and published the theory to explain the EM fields surrrounding his electric oscilliator in 1889
First experiment were conducted by Hertz by producing high frequency repetitive sparks in an air gap of a primary oscillating circuit
His theoretical description the electromagnetic disturbance in the near field where the electromagnetic energy escapes and is carried away as radiation with tranverse oscillations
Maxwell's theory reaches very unsatisfactory state, Planck solved this by introducing the quantum concept
Bohr's theory of the atom be introduced
Quantum mechanical theoty to explain the transfer of energy between atoms at longer distances compared to the collisional radii was proposed by Kallmann and London20
J. Perrin hypothenized that the transfer of the excitation energy could hop from one molecule to the other through interactons between oscillating dipoles of closely speaced molecules
Oppenheimer with Arnold explained about photosythesis
Forster then derive a quantitative theory of non-radiative energy transfer that still use until this day
He also furnished the clear and explicit to experiment
Advantages of the technique
Used in various biological applications . For example, studies in organelle structure,conjugated antibodies,cytochemical identification and oxidative metabolism
Can measure viscoelasticity and biochemical responses of living cells and real time monitorig of cell-membrane motion in natural environments , owing to its nanometer depth resolution and noningtrusiveness
FRET image with improved lateral resolution can control depth of field
Ability to collect serial optical sections from thick specimens with the improved FRET image with lateral resolution
Used for quantitative comparisons of cellular compartments and time-lapse studies for cell motility ,intracellular mechanics and molecular movements
Investigation on thick living tissue specimen by using a tunable laser ( range 700-1000nm ) ,allowing excitation of photobleaching of marker dyes and increased cell viability
How life Technologies FRET Sequencing Platform Technique is formed
Donor fluorophore absorb the energy due to the excitation of incident light. The donor will transfer the excitation energy to a nerby chromorphore, the acceptor through long range dipole-dipole intermolecular coupling
The energy transfer will manifests itself through decrease or quenching of the donor fluorenscence and a reduction of excited state lifetime also acompanied by the rate changes in acceptor fluorenscence
The important criteria in FRET is the fluorenscence emission spectrum of the donor molecule must overlap the absorption spectrum of the acceptor,The donor and acceptor must be in the close proximity to one another, The transition dipole proentations of the donor and acceptor must be in parallel state towards each other and lastly, the fluorenscen lifetime of the donor molecule must be of sufficient duration to allow the process of FRET to occur
The two involved fluophores must be positioned within a range of 1 to 10 nanometers of each other
The distance dependence of the resonance energy transfer process is the primarily basis in investigation of molecular interaction
In living cell studies that involving donor and acceptor fluorophores, resonance energy transfer will occur only between molecules that are close enough to interact biologically with one another
How data is analyzed
New algorithm that removes both the donor and acceptor SBT problems and corrects the variation in fluorophore expression level for all intensity-based FRET techniques has been developed to calculate FRET effieciency (in percent) and estimate the distance (in angstroms) between donor and acceptor molecules in a double-labeled cell
Both SBT and fluorophore expression level corrections are incorporated in mathematical calculations
From the data collected , the bleedthrough component is evaluated based on the individual donor and acceptor specimens and is then eliminated from the FRET data, pixel by pixel to obtain the true (or precision) FRET signal
Disadvantages of the technique
• It is one of centerpieces of their SMS efforts, but presently it is hard to gauge process
• Because of the time and cost have been reduced, the error rates of third-generation sequencing (TGS) reach up to 4-11% and are relatively higher than second-generation sequencing (SGS)
• The choice of filters for fluorescent wavelength selection is also critical to the success or failure of experimental detection of FRET
• Contaminating direct excitation of the acceptor molecule can be accounted for using appropriate controls, but large amounts will make interpretation of the data more difficult
• Only those molecules that interact with one another will result in FRET. If large amounts of donor and acceptor molecules are present, but do not interact, the amount of FRET taking place would be quite low
