CMMB 461 Lecture Notes - Lecture 7: Applied Biosystems, Fluorophore, Electrical Resistance And Conductance
22 Sep 2018
School
Course
Professor
Genome Sequencing — Part 5
APPLIED BIOSYSTEMS SOLID SEQUENCING (SGS)
- Library preparation similar to Roche 454 (beads and emPCR)
- Universal primer hybridize to P1 adapter sequence at end of fragments
- A set of 16 8-mers that are fluorescently labelled is flooded over the fragments
- First two bases of each 8-mer are fixed (dibase probes), and the remaining 6 bases are
degenerate
- Allow probe to bind template and ligate to primer (sequencing by ligation)
- Following several ligation cycles, the template is removed and the process is repeated with a
new primer (extended by one nucleotide)
- Only thing that is different is that you don’t perform a sequencing reaction
- First 2 reactions of SGS are the same: make a library, add adaptors (use them to design
universal primers)
- Use emulsifying PCR to amplify
- Sequencing is not using a polymerase, they are ligating a whole bunch of little fragments
called 8-mers
- No dNTPs or polymerase added
- They try to add many small fragments
- On the bottom is the DNA fragment, which is attached to the adaptor
- Universal primer will bind to the adaptor
- 8-mers are 8 nucleotides long, in which the first two nucleotides are fixed
- Fixed means that they are labelled with a fluorescent dye and those 2 nucleotides are
the same (eg. AANNNNNN, in this, all N are non-specific meaning that they can be any
nucleotide, it is unknown)
- Sequencing by ligation
- Add the first 8-mer, if it binds, remove the fluorophore, then add the next 8-mer, it will
hybridize, then add ligase
- Ligase will help remove the fluorescent molecule
- This action of the ligase will release a colour, that can be used to determine which dNTP
was attached and which ones are located beside each other in the sequence
- Whats the flaw, after one round?
- After one round, you still only know what the fixed nucleotides are, you don’t know what
the other 6 nucleotides are
- After the first round, you only have a partial sequence
- How to solve this?
- Repeat the process
- After one round, we need to figure out what the 6 nucleotides are between the 2 sets
of 2 fixed nucleotides
- To resolve this, in the next round extend the primer sequence by one nucleotide, so
now you’re filling in information in the 3rd nucleotide. In the round after, extend the
Document Summary
Library preparation similar to roche 454 (beads and empcr) Universal primer hybridize to p1 adapter sequence at end of fragments. A set of 16 8-mers that are fluorescently labelled is flooded over the fragments. First two bases of each 8-mer are fixed (dibase probes), and the remaining 6 bases are degenerate. Allow probe to bind template and ligate to primer (sequencing by ligation) Following several ligation cycles, the template is removed and the process is repeated with a new primer (extended by one nucleotide) Only thing that is different is that you don"t perform a sequencing reaction. First 2 reactions of sgs are the same: make a library, add adaptors (use them to design universal primers) Sequencing is not using a polymerase, they are ligating a whole bunch of little fragments called 8-mers. They try to add many small fragments. On the bottom is the dna fragment, which is attached to the adaptor. Universal primer will bind to the adaptor.
