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Bridgeamplificationpart1 141217163328 Conversion Gate02 PDF

The document describes the process of bridge amplification in Illumina sequencing. It involves: 1) Fragmenting genomic DNA into smaller strands that can be sequenced. 2) Ligating adapters and denaturing the DNA into single strands. 3) Binding the single-stranded DNA to the inside surface of flow cell channels. 4) Initiating solid phase bridge amplification by adding unlabeled nucleotides and polymerase to the flow cell, which causes the single strands to become double stranded and attached to the flow cell surface.

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78 views9 pages

Bridgeamplificationpart1 141217163328 Conversion Gate02 PDF

The document describes the process of bridge amplification in Illumina sequencing. It involves: 1) Fragmenting genomic DNA into smaller strands that can be sequenced. 2) Ligating adapters and denaturing the DNA into single strands. 3) Binding the single-stranded DNA to the inside surface of flow cell channels. 4) Initiating solid phase bridge amplification by adding unlabeled nucleotides and polymerase to the flow cell, which causes the single strands to become double stranded and attached to the flow cell surface.

Uploaded by

John
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BRIDGE AMPLIFICATION

Illumina
Diagram

https://www.youtube.com/watch?v=77r5p8IBwJk
Prepare Genomic DNA
Sample

• Fragment DNA of interest into


smaller strands that are able to be
sequenced
• Sonication
• Nebulization
• Enzyme digestion
• Ligate Adapters
• Denature dsDNA into ssDNA by
heating to 95° C
Attach DNA to Surface

• ssDNA is then bound to inside


surface of flow cell channels
• Dense lawn of primer on the
surface of the flow cell
Flow Cell
Bridge Amplification

• Unlabeled nucleotides and


polymerase enzyme are added to
initiate the solid phase bridge
amplification
Fragments Become Double
Stranded

• In this step it demonstrates the


work done by the sequencing
reagents
• Primers
• Nucleotides
• Polymerase enzymes
• Buffer
Denature the Double
Stranded Molecules

• The original strand is then washed


away, leaving only the strands that
had been synthesized to the oligos
attached to the flow cell
Steps 5-7 Repeats

• Cycle of new strand synthesis and Denaturation to


make multiple copies of the same sequence
(amplification)
• Fragments Become Double Stranded
• Denature the Double Strand Molecules
Work Cited

• http://res.illumina.com/documents/products/techspotlights/techspotlight
_sequencing.pdf
• https://www.youtube.com/watch?v=77r5p8IBwJk
• http://nextgen.mgh.harvard.edu/IlluminaChemistry.html
• http://research.stowers-
institute.org/microscopy/external/PowerpointPresentations/ppt/Methods
_Technology/KSH_Tech&Methods_012808Final.pdf
• http://www.molgen.mpg.de/899148/OWS2013_NGS.pdf

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