BIO 415 DNA Sequencing Part 1 WEEK 12 14 Dec

BIO 415 GENETIC ENGINEERING
14 DEC 2020
Sanger sequencing
What is DNA sequencing?
• Determining the precise order and identity of
nucleotides in a piece of DNA
• DNA sequence is useful in studying
fundamental biological processes and in
applied fields such as diagnostic or forensic
research
• DNA sequencing methods have been around
since the mid-1970s
DNA sequencing is used:
1. To characterize newly cloned DNAs
2. To confirm the identity of a mutation
3. To check for gene polymorphisms
4. To sequence genomes
Founders of sequencing technology
Frederick Sanger Walter Gilbert
Frederick Sanger, Walter Gilbert and Paul Berg

DNA Sequencing
• Gilbert & Maxam, 1973
• Sanger et al., 1977 (Sanger Method, Dideoxy Sequencing,
Chain Termination Method)
• Automated DNA sequencing machines based on the Sanger
method (Applied Biosystems Corp. 1986)
– These machines used fluorescent dyes to tag each nucleotide,
allowing the reactions to be run in one column By running 24
samples at a time, the $100,000 machines yielded 12,000
"letters" of DNA per day.
• single-nucleotide addition (SNA) method of DNA
sequencing called pyrosequencing (Hyman, 1988) 454
sequencing (after the name of the Roche-owned company
that developed it)
The enzymatic method (dideoxy sequencing) developed by
Sanger and the chemical method of Gilbert.
• Both methods use a similar principle. They generate large

numbers of oligonucleotides that begin at a fixed point
and terminate at a specific type of base.
• The sequencing reaction consists of 4 different reactions
that are performed in parallel. There is one for each base
(A, T, G, and C).
• These reactions produce many fragments of different
sizes and these are separated in a polyacrylamide gel
that can resolve differences as small as 1 nucleotide.
• After the 4 reactions are fractionated in the “sequencing
gel” the order of nucleotides can be read directly from
the gel.
The Sanger method uses controlled synthesis of DNA to generate
a population of fragments that terminate at specific points.
• Dideoxy nucleotide triphosphates

(ddNTPs):
• nucleoside analogs that lack the OH

group at positions 2 and 3 of the sugar.
When incorporated into the growing
DNA strand, the chain can grow no
longer (there is no place for the next
sugar to attach).
TIP: What is a nucleoside? What is a
nucleotide
• Nucleoside is a nucleotide without phosphate
group (sugar + base)
• Nucleotide is sugar + base + phosphate
Sanger Method is based on
the principle of chain
termination since
phosphodiester bond can
not be formed with a
dideoxynucleotide
Phosphodiester bond
requires oxygen bonded
to the 3’ carbon atom:
Typically Sanger method uses radio-labeled primers but today
fluorescent dyes are used (see automated sequencing)
1. Start with a primer that binds to one end of the DNA and is
radioactively tagged. Radioactivity allows you to visualize each band
in a gel.
2. Run 4 DNA synthesis reactions in separate tubes. Each reaction has
dATP, dTTP, dCTP, and dGTP, besides to DNA polymerase and buffer.
3. The first reaction also has a small amount of ddGTP, the second
reaction has some ddATP, the third ddCTP, and the fourth ddTTP.
4. In the first tube, the ddGTP stops one of the growing DNA molecules
as soon as it is incorporated. Some DNAs grow a little longer before
the ddGTP is incorporated, some are a little shorter.
• The bottom line is that each tube contains a large population of
DNAs of random and different sizes and all end in G.
5. The same process occurs in the other 3 reactions with ddTTP, ddCTP,
and ddATP.
6. After each of the four reactions is complete, the
contents are loaded into a well of a sequencing
gel. This is a polyacrylamide gel that is capable of
resolving 2 molecules that differ in length by only
1 base pair.
7. The sequencing gel is dried and covered with an
X-ray film to visualize radioactive bands.
8. To obtain the DNA sequence, you start at the
bottom of the gel with the smallest band. By
counting each successively larger
oligonucleotide you can see whether it ends in
the G, C, T, or A.
Tips for sequencing
• The first and most important factor in
automated DNA sequencing is clean, pure
template.
• A260/280 and A260/230 values greater than
1.8 are typically suitable for analysis (see
section “Absorbance” for more information on
Absorbance ratios). Lower A260/280 values
may indicate protein contamination.
Overview of Sanger Method
Example of the sequencing gel of Sanger Method
(historical version)
AUTOMATED SEQUENCING (modern version of Sanger Method)
To day Sanger method has become automated and instead of radioactive labeling fluorescent dyes are used for DNA
labeling and the sequencing results are obtained as a computer file as shown on the right below
Sequencing file: each

peak with a color code
represents one of the 4
bases
UNIVERSAL PRIMER LIST
• Universal primers are sequencing (or PCR)

primers that bind to a sequence found in
many plasmid cloning vectors
• These sequences were defined as good PCR
and sequencing sites as they flank the
multiple cloning site where an inserted DNA
sequence would be put.
Examples of
UNIVERSAL PRIMER LIST
SP6 5'-TAC GAT TTA GGT GAC ACT ATA G-3‘
T3 5'-CAA TTA ACC CTC ACT AAA GG-3‘
T7 5'-TAA TAC GAC TCA CTA TAG GG-3‘
EGFP-N5'-CGT CGC CGT CCA GCT CGA CCA-3'
Some animation
• https://www.dnalc.org/view/15479-Sanger-
method-of-DNA-sequencing-3D-animation-
with-narration.html
Some resources
• Sanger method
• https://www.youtube.com/watch?v=vK-HlMaitnE
• Automated sequencing animation (Modern version
of Sanger Method)
http://www.wiley.com/college/pratt/0471393878/in
structor/animations/dna_sequencing/index.html
• Next generation sequencing
• https://www.youtube.com/watch?v=jFCD8Q6qSTM
There are some web portals for primer
design:
• http://bioinfo.ut.ee/primer3-0.4.0/primer3/
Appendix
Protocol
Typical Sanger sequencing reaction
Typical Sanger sequencing reaction
(Big Dye Terminator)
• Primer length should be in the range of 18 to 22 bases.
• Rxn Mix: Big dye rxn
Reagent
Quantity
• Terminator Ready Reaction Mix
• 4.0 ml
• template
• up to 4 ml (30-90 ng*)
• primer (at 3.2 mM)
• 1.0 ml
• ddH2O
• total volume
• 10 ml
• mix well and spin briefly
• Step 2 - Sequencing on the PE 2400 or 9600 thermal cyclers.
Place the tubes in the thermal cycler and begin temperature cycling protocol. Program the
thermocycler as as follows: 25 cycles of [96 C for 10 sec, 50 C for 5-10 sec, 60 C for 4 min], then
ramp to 4 C, purify extension products as below.
Step 3 - Purifying sequencing extension products by isopropanol precipitation.
Briefly spin tubes and transfer by pipeting entire sequencing reactions into 1.5 ml microcentrifuge
tubes. Then . . .
• a. add 40 ml of 75% isopropanol, or 10 ml of deionized water and 30 ml of 100% isopropanol.
• b. mix by vortexing briefly, leave at room temperature for >15 min (and < 24 hrs) to precipitate
products.
• c. spin tubes for a minimum of 20 min at maximum speed in a microcentrifuge.
• d. aspirate the supernatants completely with a separate pipet tip for each sample, being careful not
to disturb the DNA pellet, and discard.
• e. add 125 to 250 ml of 75% isopropanol to the tubes and vortex briefly, centrifuge as before for 5
min at maximum speed, and aspirate the supernatants as in step d.
• f. dry the samples in a vacuum centrifuge for 10 - 15 minutes (to dryness), or place on a thermal
cycler heating block for 1 min at 95 C, and store at -20 C until ready for electrophoresis.
Step 4 - Preparation for electrophoresis.

Redissolve each sample pellet in 3 ml loading buffer [deionized formamide/25 mM EDTA (pH 8.0)
with blue dextran (50 mg/ml), at 5:1 vol/vol] immediately before use. Vortex and spin samples.
Heat samples at 95 C for 2 min, then immediately place on ice until ready to load. Load 1 - 2 ml per
sample.
• (© J. A. Wheeler and M. F. Wojciechowski, Molecular Phylogenetics Lab, UC Berkeley, 1998)

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BIO 415 GENETIC ENGINEERING

Frederick Sanger Walter Gilbert

Frederick Sanger, Walter Gilbert and Paul Berg

• Both methods use a similar principle. They generate large

• Dideoxy nucleotide triphosphates

• nucleoside analogs that lack the OH

Sequencing file: each

• Universal primers are sequencing (or PCR)

Step 4 - Preparation for electrophoresis.

• (© J. A. Wheeler and M. F. Wojciechowski, Molecular Phylogenetics Lab, UC Berkeley, 1998)

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