DNA Sequencing

Method based on the chemical modification of DNA and subsequent cleavage
at a specific base.
Often referred to as “chemical sequencing”.
1) DNA is radio-labeled at one end of the DNA
2) DNA is divided into 4 pools
3) DNA is chemically digested to generate
fragments that are cut at A, T, G, C. 4) Fragments are then loaded and run
over gel via electrophoresis; fragments
separate out by length.
5) The gel is next exposed to X-ray film.
6) Resulting dark bands that appear on the
film represent the nucleotide base “sequence”
as it occurs in the DNA.
7) Bases are then read and the “sequence”
captured

Similar approach to Maxam-Gilbert but no chemcial digest; rather uses dideoxynucleotide triphosphates (ddNTPs) as chain elongation terminators.
1. DNA, DNA Polymerase, dNTPs(A,G,C,T), and fluorescently labled DNA primer are mixed; then divided into 4 pools.
2. ddNTPs are then added to each pool; one ddNTP type per pool (ddATP, ddGTP, ddCTP, ddTTP).
3. Primer extension is carried out multiple rounds per pool; resulting in termination
of extension when a ddNTP is incorporated. Moreover, ddNTP lacks a 3’-OH group required for forming a phosphodiester bond between nucleotide bases = No primer or “Chain” extension.
4. Pools are then subject to gel electrophoresis just as in the Maxam-Gilbert method; only
a fluorescent scanner is used instead.

Similar to “Dye-primer”. Instead, fluorescently labeled dNTPs are used as oppose to labeled primer = more label per sequence = better sensitivity:

1. DNA, DNA Polymerase, non-labeled dNTPs (A, G, C, T), and non-labled DNA primer are mixed; then divided into 4 separate pools (A,G,C,T).
2. Fluorescently labeled dATP and non-labeled ddATP is then added to the A pool. Followed by fluorescently labled dGTP and non-labeled dCTP and non-labeled ddCTP added to the C pool, and fluorescently labeled ddTTP and non-labeled ddTTP added to the T pool.
3. Primer extension is carried out multiple rounds per pool; resulting in termination of extension when a ddNTP is incorporated. Moreover, ddNTP lacks a 3'-OH group required for forming a phosphodiester bond between nucleotide bases = No primer or "Chain" extension.
4. Pools are then subject to gel electrophoresis just as in the Maxam-Gilbert method; only a fluorescent scanner is used instead.

Improved approach to the prior two; does not require running 4 separate lanes per DNA. Method only requires one lane to be run per DNA.

1. DNA, DNA primer, DNA polymerase, unlabeled dNTPS, and labeled ddNTPs are all mixed as single pool; ddNTP’s
are labeled w/ specific dye per type:
ddATP = Green
ddGTP = Yellow ddCTP = Blue ddTTP = Red
2. Primer extension is carried out multiple rounds per pool; resulting in termination of extension when a ddNTP is incorporated. Again, ddNTP lacks a 3’-OH group required for forming a phosphodiester bond between nucleotide bases = No primer or “Chain” extension.
3. Pool is then subject to gel or capillary electrophoresis

1. DNA is obtained w/out knowledge of physical map.
2. DNA randomly sheared into 100-kb fragments.
3. Fragments then cloned into plasmids.
4. Plasmids then transformed into bacteria, grown, plasmid recovered.
5. Sequencing performed on DNA inserts in plasmid.
6. Resulting sequences are assembled into one long contiguous sequence; approach coined “de novo”.

The average number of reads representing a given nucleotide in the reconstructed sequence = the number of reads (N) * the average read length (L)/ the length of the genome (G) thus (N*L)/G

1. Started with long strand of DNA and broke into 3kb strands some strands would be to long or to short, thus loss.
2. Not all of the 3kb pieces were expected into plasmids
3. Not all of the plasmids were excepted into E. Coli
4. Current technology only allows 700-800bp pairs at a time thus the 3kb piece will only have 1400 bp read
5. Sequence repeats

To deal with Gaps in whole-genome sequencing post assembly.

pairwise-end sequencing

Same as Whole-genome shotgun w/ one difference.
Sequencing is performed from both ends of DNA inserts as oppose to just one.

Method conceived to reduce “Gaps” and to reduce assembly error.
Moreover, when DNA fragments from different physical locations contain the same repeats, they could be incorrectly assembled together.