DNA SEQUENCING: MAXAM GILBERT METHOD SANGER SEQUENCING Illumina (Solexa) sequencing Pyrosequencing

1. DNA SEQUNCING
ABOALGASIM GOMA YONIS

2. What is DNA sequencing ?
• Why is DNA sequencing important ?
Molecular medicines
Bioarchaeology
Anthropology
Evolution and Human migration
DNA Forensics
Detect bacteria and other organisms that pollute
air
Agriculture, livestock breeding and
bioprocessing

3. MAXAM GILBERT METHOD
Allan Maxim and Walter Gilbert developed a method
for sequencing single stranded DNA
Purines dimethyl sulphate
Pyrimidines hydrazine
Breaking of glyosidic bonds between ribose sugar and
base
example
The purines (A+G) are depurinated using formic acid,
the guanines (and to some extent the adenines) are
methylated by dimethyl sulfate, and the pyrimidines
(C+T) are hydrolysed using hydrazine.

5. SANGER SEQUENCING
Discovered by Scientist Sanger
Also know as dideoxy method or chain termination
method
2 main things to be done :
1. Denature the DNA
2. Labelling

6. The classical chain-termination method requires a single-
stranded DNA template, a DNA primer, a DNA polymerase,
normal deoxynucleosidetriphosphates (dNTPs), and
modified dideoxynucleosidetriphosphates (ddNTPs), the
latter of which terminate DNA strand elongation. These
chain-terminating nucleotides lack a 3'-OH group required
for the formation of a phosphodiester bond between two
nucleotides, causing DNA polymerase to cease extension
of DNA when a modified ddNTP is incorporated. The
ddNTPs may be radioactively or fluorescently labeled for
detection in automated sequencing machines.

7. Dimethyl
Sulphate
Piperdine
Guanine
Hydrazine
Piperdine
Thymine
+
Cytosine
Guanine
+
Adenine
Dimethylsulphate
Piperdine
Formic acid
Cytosine
Hydrazine
Piperdine
Nacl
DIDEOXYNUCLEOTIDE
In DNA nucleotides are
bond to each other by
phosphodiester bond
In case of no 3’OH no it
cannot form a
phosphodiester bond
CHAIN TERMINATION

12. Next-generation sequencing (NGS), also known as high-
throughput sequencing, is the catch-all term used to
describe a number of different modern sequencing
technologies including:
Next-Generation DNA Sequencing
Illumina (Solexa) sequencing
Pyrosequencing
These recent technologies allow us to sequence DNA and RNA
much more quickly and cheaply than the previously used Sanger
sequencing, and as such have revolutionised the study of
genomics and molecular biology.

13. THANK YOU
Illumina dye sequencing is a technique used to determine the
series of base pairs in DNA, also known as DNA sequencing.
The reversible terminated chemistry concept was invented by
Bruno Canard and Simon Sarfati at the Pasteur Institute in
Paris.It was developed by Shankar Balasubramanian and
David Klenerman of Cambridge University,who subsequently
founded Solexa, a company later acquired by Illumina. This
sequencing method is based on reversible dye-terminators
that enable the identification of single bases as they are
introduced into DNA strands. It can also be used for whole-
genome and region sequencing, transcriptome analysis,
metagenomics, small RNA discovery, methylation profiling,
and genome-wide protein-nucleic acid interaction analysis
Illumina dye (Solexa) sequencing

15. The DNA attaches to the flow cell via
complementary sequences. The strand bends
over and attaches to a second oligo forming a
bridge. A polymerase synthesizes the reverse
strand. The two strands release and straighten.
Each forms a new bridge (bridge amplification).
The result is a cluster of DNA forward and reverse
strands clones.

17. Pyrosequencing is a method of DNA sequencing
(determining the order of nucleotides in DNA) based on
the "sequencing by synthesis" principle. It differs from
Sanger sequencing, in that it relies on the detection of
pyrophosphate release on nucleotide incorporation,
rather than chain termination with dideoxynucleotides.
Pyrosequencing

18. The desired DNA sequence is able to be determined by light emitted
upon incorporation of the next complementary nucleotide by the fact
that only one out of four of the possible A/T/C/G nucleotides are
added available at a time so that only one letter can be incorporated
on the single stranded template (which is the sequence to be
determined). The intensity of the light determines if there are more
than one of these "letters" in a row. The previous nucleotide letter
(one out of four possible dNTP) is degraded before the next
nucleotide letter is added for synthesis: allowing for the possible
revealing of the next nucleotide(s) via the resulting intensity of light
(if the nucleotide added was the next complementary letter in the
sequence). This process is repeated with each of the four letters until
the DNA sequence of the sinand gle stranded template is
determined.

20. THE END