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nucleic acid sequencing (chemical degradation method (enzymatic, Dimethyl…
nucleic acid sequencing
chemical degradation method
enzymatic
Dimethyl sulfate add chemical to specific nucleotide and then stop extension of the strand
pependine degrade the added chemical resulting in short strand
run gel electrophoresis
chain terminator sequencing
based on the addition of dideoxynuclotide (ddNTPs) thus no phosphodiester bond between nucleotides
ddNTPs labeled either radioactively or fluorescence dyes
the reaction is done in thermocycle: denaturation , annealing and extension steps
Next generation seq (NGS)
steps
template preparation
clonally amplified template
emulsion PCR
library fragments with adaptors containing universal priming site
beads with complementary primers to capture the DNA strand
amplification and enrichment
solid -phase amplification (Bridge amplification )
forward and reverse primers attached to surface
fragments added and bind to these primers making bridge
amplification
single molecule templates
sequencing and imaging
cyclic reversible termination : one nucleotide is added >imaged >cleaved >washed
seq by ligation : DNA ligase and fluorescent probe added to fragment
single -nucleotide addition (pyrosequening ) : measure the release of phosphate once it converted to light by enzymes (sulphurlae and luciferase )
ion-semiconductor seq : measure the pH change as a results of H ion release in dNTP incorporation
real-time sequencing : Nanopore two protein ; one unzip the helix into two strand , the other protein make pore in membrane and attached to adaptor that keeps bases in place when they passed so that can be identified electronically through the change in the current
genome alignment : aligned created sequences against a reference
application
human disease and health
personlised medicine /predictive medicine
drug resistance
diagnosis of disease
microbiology
typing pathogens epidemiologically : source of outbreaks
identification of microogranisms
studies of microbes drug resistance
evoulionary relationship studies
metagenomics