next generation in dna sequencing

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Next Generation in DNA Sequencing An ELECTIVE REPORT Submitted to the University of Rajasthan, Jaipur In partial fulfillment for the Degree of M. Sc. Biotechnology Submitted by Pallav Singh

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History of DNA 1869 Swiss physiological chemist Friedrich Miescher first identified "nuclein“ or "deoxyribonucleic acid or “DNA"). 1894 Kossel and Neumann identified the bases in DNA. 1944DNA as genetic material was identified by Avery, Macleod and McCarty. 1950Chargaff demonstrating a consistency to the base ratios in DNA from different souces. 1952 Rosalind Franklin takes the X- ray diffraction pictures that indicate DNA is a helix structure. She was also called “Dark Lady of DNA”

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1953 Watson and Crick derive the three-dimensional, double-helical model for the structure of DNA .

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DNA Sequencing:- Determination of nucleotide sequence Reason of delay in DNA Sequencing :- chemical properties chain length existence of only four bases in DNA no base-specific DNAases RNA Sequencing:- Escherichia coli alanine tRNA was the first nucleic acid molecule to be sequenced by Holley and coworkers in 1965 Restricted enzymes:- Discovery of type II restriction enzymes by Hamilton Smith and coworkers (1970) DNA Sequencing technologies developed since 1975

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Principle of Sequencing:- Sequencing reaction :- Based on PCR, reaction mixture having ddNTPs. Separation by Gel Electrophoreses. Detection on an automated sequencer.

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Gel electrophoresis scanning and detection system

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D) Assembling of the sequenced parts of a gene snapshot of the detection of the molecules on the sequencer

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Human Genome Project and sequencing Human Genome Project Developed and preferred by Celera - Celera eliminating the BAC step from the HGP's approach. - Hierarchical approach is advantageous for sequencers when assembling the shotgun fragments into contigs as long as full chromosomes

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A) DNA fragmentation B) DNA Cloning and amplification C) Sequence reading Methods of DNA fragmentation :- A) Enzymatic digestion :- restriction endonucleases or restriction enzymes - recognition sequences are typically four, six, eight, ten, or twelve nucleotides long. - artificial plasmids – polylinker = multiple cloning sites For sequencing DNA of any organism we have to undergoes three steps :-

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B) Sonication C) Nebulization Ultra sonic sound produce a gaseous cavitation in the liquid due to alternation of pressure - Cavitation and a thermal or mechanical effect . - Size of the fragments determined by the speed at which the DNA solution passes through the hole - Applying the pressure through nitrogen gas. - Electrophoresis not required.

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D) Hydrodynamic Sharing - Fragmented by hydrodynamic shear stress when they are forced through a point-sink such as a small orifice at high velocity. Length of fragments determined by flow velocity of the solution and diameter of the orifice. - Independent of the initial DNA size/concentration and salt concentration Computer-controlled syringe pump for automation.

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Method of DNA Cloning and amplification :- Plasmid Cloning - Plasmid are defined as self replicating circular duplex DNA molecules exit in cell as extracellular controlled unit. B) M13 Cloning Vector - M13 bacteriophage infect E.coli, replicate through lysogenic (prophage ) and lytic cycle. - Have its own mechanism for infecting cells. - Size of DNA insert = 8 kb (mainly). - Size of DNA insert = 3 kb (1.5 kb most common).

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C) PCR D) RT-PCR - Amplification occur. - Both amplification and quantification occur. - Detection occur at plateau phase. - Detection occur at exponential phase. - Taqman polymerase enzyme - SYBER green (fluorescence dye), Scorpion probe, Taq Man probe (fluorescence labeled probe )

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E) Emulsion-PCR (em-PCR) - 6 bln P1-coupled beads + template, PCR components, and primers. - Amplification in 10-µm microreactors. - Enrichment of template beads through amplification. - Glycerol gradient to separate enriched bead from unenriched bead.

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Sequence reading :- Various methods are as follows- A) Allan Maxam and Walter Gilbert :- Chemical cleavage Full length DNA is end labelled 5 (polynucleotide kinase) or 3 (deoxynucleotidyl transferase)prime end. Cleave it with base specific chemical reagent. Run gel to isolated fragments. Read the sequence. G only - DMS+ Piperidine G +A - DMS(acidic) + Piperidine T+C - Hydrazine + Piperidine C only - Hydrazine + 1.5M NaCl Primer not require i.e. no extension chain require.

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B) Chain termination :- enzymatic procedure Kelnow fragment as polymerase enzyme. In a reaction system ddATP is about 1% of normal dATP. - Read sequence from bottom to top in agar gel.

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C) Automated Dye Sequencing :- - Each ddNTP by different dye. - Single track system.

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E) Pyrosequencing :- - Based on "sequencing by synthesis" principle. - Relying on the detection of pyrophosphate release on nucleotide incorporation.

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F) :- Solexa sequencing - Occurs in microfluidic flow cells, which are coated with two different chemically-ligated primers.

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G) :- SOLiD (Sequence by Oligonucleotide Ligation and Detection.) a) Library Preparation b) Emulsion PCR/Bead Enrichment c) Bead Deposition d) Sequencing by Ligation

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H) :- Single Molecule Real Time Sequencing (also known as SMRT) 1) Zero-mode waveguide 2 ) Single DNA polymerase enzyme is affixed DNA to bottom of a 3)The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase.

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G) :- Nanopore sequencing is a method - The nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it. - The amount of current is very sensitive to the size and shape of the nanopore

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Application of DNA Sequencing:- - Full genome sequencing : - Human evolution. - Amplicon Sequencing : - Target sequence. - Transcriptome sequencing :- SNP s , insertion, deletion. - Metagenomics and etc.

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Thank You