Lecture 21: DNA Sequencing

Question 1

What is required for DNA synthesis

Answer 1

1. Template DNA strand
2. Primer DNA
3. DNA polymerase
4. Nucleotides

Question 2

How does sanger sequencing work?

Answer 2

1. Nucleotides incorporated are                
   "di-deoxynucleotides", as they're 
    missing 2 oxygens (C3)
2. Without the hydroxyl group (OH) at 
    the 3' end, another nucleotide cannot 
    be added
3. ddNTPs can be labelled with 
    fluorescent dye
4. In the mixture of nucleotide; only a 
    limited amount of ddNTP is added 
    (e.g. 1%), allowing the strand to 
    elongate and by chance then have 
    the ddNTP incorporated
5. By repeating this technique many 
    times, a pool of DNA fragments is 
    generated, with each fragment being 
    terminated at (for eg) a different A 
    (ATP)
6. These can be ordered by size ("size 
    fractionation) on denaturing PAGE or 
    capillary electrophoresis
7. If this is done with all different 
   ddNTPs (ddATP, ddGTP, ddCTP, 
   ddTTP), all with different coloured 
   dyes, and size fractionized, we have a 
   read of the sequence going from short 
   to long. I.e., bp1, bp2, bp3...

Question 3

What is next generation sequencing (NGS)

Answer 3

1. 454 (2005) - GONE BUST
2. Illumina (2006) - 80-90% use illumina
3. Solid (ABI) (2007) - GONE BUST
4. Pacific Biosciences (PacBio) (2014)
5. Nanopore (2014)

Question 4

How does NGS work?

Answer 4

1. Insert (DNA to be sequenced)
2. don’t need to known any of the
   sequence (like you would with sanger to
   create a primer)
3. Adapter will is ligated to each end
   (adapter includes a 6nt index)
   a. ONE ADAPTER HAS NO INDEX
4. Can allow sequencing of different
   species. E.g., 1 bacteria uses 1 type of
   index, another uses a different one etc
   a. adapter with no index looks the same
   b. genomes is still different ofc