8. SANGER SEQUENCING
It involves following components:
a) 1. Primer
b) 2. DNA template
DNA polymerase
d) 4.. dNTPs(A,T,G,C)
e) 5. ddNTPs
4 Steps:
1. Denaturation
2. Primer attachment and extension of bases
3. Termination
4. Poly acrylamide gel electrophoresis
9. This method uses dideoxynucleotide triphosphates
(ddNTPs) chain terminators :
which have a H on the 3’ carbon of the ribose
sugar instead of the normal OH found in
deoxynucleotide triphosphates (dNTPs).
Therefore in a synthesis reaction, if a
dideoxynucleotide is added instead of the normal
deoxynucleotide, the synthesis stops at that point
because the 3’OH necessary for the addition
of the next nucleotide is absent
•ssDNA addition of dNTPs elongation
• ssDNA addition of ddNTPs elongation
stops
11. The sequence of a single-stranded DNA
molecule is determined by enzymatic
synthesis of complementary
polynucleotide chains.
These chains terminating at specific
nucleotide positions.
Separate by gel electrophoresis
Read DNA sequence
PRINCIPLE OF DNA SEQUENCING
12. In the Sanger method, the DNA strand to be analyzed is used as a
template and DNA polymerase is used, in a PCR reaction, to
generate complimentary strands using primers.
Four different PCR reaction mixtures are prepared, each containing
a certain percentage of dideoxynucleoside triphosphate (ddNTP)
analogs to one of the four nucleotides (ATP, CTP, GTP or TTP).
Synthesis of the new DNA strand continues until one of these
analogs is incorporated, at which time the strand is prematurely
truncated.
Each PCR reaction will end up containing a mixture of different
lengths of DNA strands, all ending with the nucleotide that was
dideoxy labeled for that reaction.
Gel electrophoresis is then used to separate the strands of the four
reactions, in four separate lanes, and determine the sequence of
the original template based on what lengths of strands end with
what nucleotide.
PROCEDURE
13.
14. • A M Maxam
and W Gilbert-
1977
• Chemical
sequencing
Treatment of
DNA with
certain
chemicals
DNA
cuts into
fragments
Monitoring
of
sequences
MAXAM AND GILBERT METHOD
15. CH
The partially cleaved DNA fragment is subjected to five separate
chemical base
Each of which is specific for a particular base.
The resulting fragment terminate at that specific
base followed by high resolution gel electrophoresis and detection of
the labeled fragments by autoradiography
PRINCIPLE
16. The method requires radioactive labelling at one end and
purification of the DNA fragment to be sequenced.
Chemical treatment generates breaks at a small proportion of one
or two of the four nucleotide bases in each of four reactions
(G, A+G, C, C+T).
Thus a series of labelled fragments is generated, from the
radiolabelled end to the first 'cut' site in each molecule.
The fragments in the four reactions are arranged side by side in geL
electrophoresis for size separation.
To visualize the fragments, the gel is exposed to X-ray film for
autoradiography, yielding a series of dark bands each
corresponding to a radiolabelled DNA fragment, from which the
sequence may be inferred.
PROCEDURE
17. Reagents Base Specific modification
Dimethyl Sulphate
(pH)
G Methylation of N7 renders the C8-C9 bond
susceptible to cleavage.
Piperidine formate A+G Weakens the glycosidic bond of adenine
and guanine residues by protonaing
nitrogen atoms in the purine rings resulting
in depurination.
Hydrazine C+T Opens pyrimidine rings, which recyclize in
a five membered form which is valnerable.
Hydrazine + 1.5 M Nacl C Only cytocin reacts with Hydrazine.
DIFFERENT TYPES OF BASE SPECIFIC REACTION USED IN THE CHEMICAL
DEGRADATION METHOD
20. Shotgun sequencing, also known as shotgun cloning, is a method used
for sequencing long DNA strands or the whole genome.
•In shotgun sequencing, DNA is broken up randomly into numerous small
segments and overlapping regions are identified between all the individual
sequences that are generated.
• Multiple overlapping reads for the target DNA are obtained by performing
several rounds of this fragmentation and sequencing.
•Computer programs then use the overlapping ends of different reads to
assemble them into a continuous sequence.
•The shotgun approach was first used successfully with the bacterium
Haemophilus influenzae.
•Craig Venter used this method to map the Human genome project in 2001.
21.
22.
23. PYROSEQUENCING
• Pyrosequencing is the second important type of DNA sequencing
methodology in use today.
• Reaction mixture contains
DNA sample to be sequenced
Primers
Deoxynucleotides
DNA polymerase
Sulfurylase enzyme
Advantages:
Accurate
Parallel processing
Easily automated
Eliminates the need for labeled
primers and nucleotides
No need for gel electrophoresis
DISADVANTAGES
Smaller sequences
Nonlinear light response after
more than 5-6 identical nucleotides
24. Do not require electrophoresis or any other fragment seperation.
Determine which of the 4 bases is incorporate at each step during the
copying of DNA templete.
ddNTPs are not required
.
As the new strand is being made, the order in which the dNTPs are
incorporated is detected.
So the sequence can be read as the reaction proceeds.
In reaction all 4 dNTPs are not added at one time.
Each dNTP is added individually in a sequential manner
25. If particular dNTP are not incorporated then it is rapidly
degraded by nucleotidase or by washed before addition of
next dNTP.
Incorporation leads release of pyrophosphate which is
detected in an enzyme cascade that emits light.
26.
27. •The system uses the Staphylococcus auereus toxin α-hemolysin, a
robust heptameric protein which normally forms holes in membranes.
•DNA and RNA can be electrophoretically driven through a nanopore of
suitable diameter (
