DNA Sequencing
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Transcript of DNA Sequencing
![Page 1: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/1.jpg)
DNA Sequencing
Sanger Di-deoxy method of Sequencing
Manual versus Automatic Sequencing
![Page 2: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/2.jpg)
Sequencing of DNA• Requires DNA amplification
• Labeling of DNA fragments
• Termination of extension
• Separation of fragments
• Detection of fragments
![Page 3: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/3.jpg)
![Page 4: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/4.jpg)
ddNTP (one) ddNTP (one)
5’-labeled primer5’-labeled primer
ATP, TTP, CTP, GTPATP, TTP, CTP, GTP
Terminated fragmentsTerminated fragments
Followed by autoradiogramFollowed by autoradiogram
The Reaction Mixture
![Page 5: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/5.jpg)
Sanger Method dideoxynucleotides: lack a 3’ OH
But have the 2’ and 3’ have hydrogen
NO DNA EXTENSION
Dideoxy key point!
![Page 6: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/6.jpg)
![Page 7: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/7.jpg)
DNA PCR amplification is performed prior to gel electrophoresisNotice the reaction of this “one” reaction mixture!
![Page 8: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/8.jpg)
![Page 9: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/9.jpg)
![Page 10: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/10.jpg)
Separation followed by Detection
![Page 11: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/11.jpg)
Can you sequence without using radioactivity?
![Page 12: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/12.jpg)
Example of Label the ddNTPs
• ddGTP• ddATP• ddTTP• ddCTP
• Orange• Black• Red• Blue
![Page 13: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/13.jpg)
![Page 14: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/14.jpg)
Automated Sequencing
• Efficient
• Does not require radioactivity
![Page 15: DNA Sequencing](https://reader036.fdocuments.in/reader036/viewer/2022062517/56813223550346895d9886cb/html5/thumbnails/15.jpg)
The End
• Onto DNA Mutations