Flashcards in Lecture 7 Deck (21):
1
Genomics
Brand of science/technology which deals with study of genomes (including molecular characterization and production of proteins, role in health and ease and effects of manipulation of these systems by agents
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Sequencing
Procedures for finding exact order of nucleotides in DNA strand
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Sequencing of virus genome
Sequenced virus first because of small genome
Sanger found genome of MS2 and phi-X174 phage
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First cellular genome
Done on H. Influenzae
E. Coli popular bacteria but genome was twice as big
Started to understand protein and regulations from sequence
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PLUS and MINUS method:
First method and required comparison of both + and - seq to find actual strand
Only for ssDNA
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Maxim and Gilbert Method
Series of complex reactions
More difficult than inhibitor method
Can used for dsDNA
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Sanger et al method
Used idea of inhibitors that can terminate elongation of DNA at specific points.
Remains the standard today
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Sanger Method
Method based on DNA polymerase dependent syntehsis of complementary DNA strand in presence of dNTPS and ddNTPs
Synthesis is terminated when ddNTP is randomly added
Used gel electrophoresis too read out order
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Modern Automated DNA sequencing
Advances in fluorescence detection allowed combining the four terminators into 1 reaction, they are labeled with different colored fluorescent dyes
Throughput increase with advent of capillary arrays allowing many samples to be analyzed in parallel
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"Shotgun-cloning" Method
DNA sheared in to random fragments and ligated into plasmids
Generate thousands of plasmids each with random fragment
Library of plasmid are put into E. Coli so that each can be purified for sequencing
Each colony has different fragment
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"Shotgun-cloning" Method (2)
Plasmids are isolated
Determine 500-100bp of sequence
Possible using primers complementary to plasmid area flanking either end of insert
Sequence 1000s of plasmid and stitch them together using computers for full genome
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Limitations to whole genome shotgun-cloning method
Cloning Bias: Some genes toxic to E. Coli, results in gaps in assembled sequence
Project maintenance issues: Grown different colonies of E. Coli which is difficult
Slow and Expensive: Most advanced Sanger yields <100Kb per run, Typical genome has 5 million bp
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Pyrosequencing
Real time sequencing
Reaction can be performed in liquid or with DNA attached to slide or bead
No electrophoresis - instead readout of light production measured by camera
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Ingredients of Pyrosequencing
Primer annealed to template
Mix of 4 enzymes and 2 chemicals:
DNA polymerase: extend primer with nucleotides
ATP sulfurylase: convert PPi and APS to ATP
Luciferase: convert ATP and luciferin to light
Apyrase - degrades unused NTP after each addition round
APS- adenosine phosphosulfate
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Genome sequencing in microfabricated high-density picolitre reactors
DNA fragmented and primers are ligated to both ends
DNA attached to tiny beads under condition where one DNA fragment binds to one bead
Beads immersed in oil-water droplets and PCR used to amplify strands.
PCR primers complementary to primers attached to ends
Oil isolated droplets enable PCR on each each bead in isolation
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Genome sequencing in microfabricated high-density picolitre reactors (2)
Beads placed in micro wells
Pyro enzymes is added to little beads
Reaction mixture (dNTPSs and apyrase) flowed over each well
Carry out many parallel pyrosequencing with a High Res camera recording each reaction
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454 Pyrosequencing
Nonradioactive/non-hazardous
Sequence DNA straight from bacteria no cloning for plasmid
No chromatography step
Massively parallel
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Illumina/Solexa Sequencing
Differs from 454 in 2 major ways
Uses Sanger-like reaction using dye labeled reversilbe terminator NTPS
Amplifies DNA fragments on a surface of flow cell rather than a bead
LOOK AT DIAGRAM
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454 vs Ilumina
Both needs sheering DNA into small pieces
Both need adapters which have primers ligated to strand
454 uses small beads and ilumina uses clusters of DNA strands
All start at a single piece of DNA that is amplified
Gives off light. In 454 intensity of one nucleotide is recorded at 1 at a time
Ilumina gives off coloured light but is added at the same time
454 is one by one, ilumina is parallel
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Sequencing: next-next gen
Nano-Knife technology rubs small conductive blades over immobilized ssDNA
Determines sequence based on specific size and conductive changes of each base
May even detect modifications like methylations and protein binding
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