Sequencing
Sequencing:
• finding the order of nucleotides on a piece of DNA.
• Nucleotide order determines Amino acid order, thus,
protein structure and function (proteomics).
• An alteration in a DNA sequence can lead to an altered or
non functional protein, and hence lead to a harmful effect
on organisms.
DNA sequencing aim to:
• determine the precise sequence of nucleotides in a sample
of DNA.
DNA sequencing types:
➢ First generation
➢ Second generation
(Next Generation Sequencing/NGS)
➢ Third generation
Two methods for sequencing:
1. Maxam and Gilbert method (chemical)
2. Sanger method (enzymatic)
Maxam-Gilbert Technique (chemical degradation)
• Two steps:
1. Base-specific modification:
by chemicals that selectively attack purines and
pyrimidines and break bond between ribose sugar and
base (step 1)
2. Sugar-phosphate Backbone cleavages:
involving piperidine (strong base) which catalyze
phosphodiester bond cleavage (step 2)
Steps:
1. DNA (single or double-stranded DNA) to be sequenced is
cut by a restriction enzyme and purified.
2. DNA fragment is 5’end labelled by radioactive P by the
following steps:
➢ Treatment with alkaline phosphatase to remove the
5’phosphate.
➢ Treatment with polynucleotide kinase, which attaches
P labelled to the 5’end.
3. radioactive P will be added to the 5’ end of the 2 strands
(forward and reverse strands), so another restriction
enzyme is used to cut one of the labelled ends.
4. Then the double strand DNA is denaturated to create a
single stranded DNA fragment carrying a radioactive label
on the 5’ end.
5. These single stranded one-end labelled fragments are
divided into 4 tubes and subjected to four separate
cleavage reactions, each of which would create a labelled
cleavage products ending in known nucleotides.
Step 1 (base modification reactions):
o Tube 1: dimethyl sulfate (DMS) is added to selectively
cleave at G bases.
o Tube 2: dimethyl sulfate and dilute Formic acid is added to
attack and cleave both purines (A+ G).
o Tube 3: Hydrazine is added to cleave at both pyrimidines
(C+ T).
o Tube 4: Hydrazine in 1.5M NaCl (high salt) is added to
selectively cleave C.
Step 2 (sugar–phosphate backbone cleavage):
• Piperidine is added to the 4 tubes to cleave the backbone.
6. The 4 reaction tubes are loaded into 4 separate wells on
high percentage polyacrylamide gels and the fragments
resolved by electrophoresis.
• Four lanes are produced on the sequencing gel.
• Each lane contains fragments ending in a known
nucleotide:
➢ lane 1 fragments end with G
➢ lane 2 fragments end with A or G
➢ lane 3 fragments end with C or T
➢ lane 4 fragments end with C
7. piece of X-ray film placed over the gel, dark autoradiographic
bands will appear on the film corresponding to the location
of the labelled fragments.
• Since in electrophoresis smaller fragments will run faster
than larger fragments, the bands will represent the 5’→3’
DNA sequence when read from bottom to top.
8. Reading the sequence involve interpreting the banding
pattern relative to the four chemical reactions:
➢ band that appear in both C only and C + T lanes would be
called C.
➢ band that appear in C + T lane but not in C only lane it would
be called T.
➢ The same process would obtain for G only and G + A reaction
lanes.
Disadvantages:
• Requires lots of purified DNA
• Requires restriction digest
• Hazards of radioactive and chemical materials
• Relatively short readings
• Automation not available (sequencers)
Sanger method (enzymatic or chain termination method)
• Sanger sequencing uses a special type of PCR called (cycle
sequencing reaction) that differ from conventional PCR in:
1. Use 1 primer that is end-labeled with fluorescent dyes.
2. Only 1 strand is copied in every cycle (not the reverse strand)
3. Since the copied strand is in the same direction of the primer
it cannot act as a template for the next cycle so all
amplifications are done on the original template so we need
sufficient copies of the original strands.
4. dideoxynucleotides (ddNTPs) are used to terminate the
extension reaction. These have no 3’-OH group on the
deoxyribose which is needed by the polymerase to extend
the growing chain.
Methods of Sanger:
1. original method (dye-primer sequencing)
2. automated sequencing (dye-terminator sequencing)
Steps of the original method (dye-primer sequencing)
1. Many copies of the DNA to be sequenced are made by PCR.
2. DNA is purified by isolating it from other contaminating
DNAs or molecules.
3. dsDNA is denaturated and one of the ssDNA strands is
chosen to be sequenced.
4. mixture of ssDNA + Tag polymerase + normal dNTPs (to
allow some chain elongation) + labelled primer is made
and separated into 4 tubes.
5. different chain terminators (ddNTP) are added to the
mixture in specific ratios to ensure only a limited amount of
chain termination.
6. The tubes are placed in the PCR machines.
7. ddNTPs are randomly incorporated into the growing chain
and act as chain terminators.
➢ Partial copies of DNA fragments made with DNA polymerase
➢ Different fragments of DNA with different lengths will be
produced in each tube.
➢ All the fragments in a tube will be ending with the same
ddNTP
8. The four samples are analyzed by PAGE to separate the
fragments based on their size.
9. Reading the DNA sequence from 5’ to 3’ by reading the
order of the bands from bottom to top.
• However the sequence fragments on the gel are the
complement of the actual template. So the sequence of the
template will just be the complementary sequence to the
sequence obtained.
Steps of automated sequencing (dye-terminator sequencing)
• Here the whole process is done in instruments called
DNA sequencers
• Has the same steps as the original method but differ in that:
➢ ddNTPs are fluorescently labelled instead of the primer. This
allows the cycle sequencing reaction to be done on a single
tube instead of 4.
➢ The resulting fragments are separated by capillary gel
electrophoresis.
➢ As the fragments are separated, the florescent dyes are exited
by a laser and florescence is recorded as peaks. So the
reading of the sequence is computerized.
Advantage of Sanger:
• no need for DNA purification
• no restriction digests
Disadvantages:
• good only for 500-750bp reactions
(human genome is about 3 billion bp)
• Expensive
• Takes a while
• For sequencing of long sequence use:
next generation sequencing (NGS)
Which allow for:
1. Whole genome sequencing
2. Whole Exome sequencing
3. RNA sequencing
