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Edited by Endah Puspitasari, S.Farm., M.Sc., Apt.
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Topik
Analisis asam nukleat:
Sekuensing DNA
Hibridisasi DNA
Nothern Blot
Southern Blot
Analisis protein:
Elektroforesis protein
Western Blot
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Sekuensing DNA
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Introduction
Reading the blueprint of life
The blueprint of life is contained in the DNA in the
nuclei of eukaryotic cells and simply within prokaryotic
cells.
Human genome project – just obtain the list of
approximately 3x109 bases (As, Cs, Gs and Ts) in the 23
chromosomes.
Extraction of useful information from this list and
genome sequence of other organisms relies on
computer-intensive data handling – Bioinformatics.
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Sequencing
The DNA from the genome is chopped into bits- whole
chromosomes are too large to deal with, so the DNA is
broken into manageably-sized overlapping segments.
The DNA is amplified by cloning into bacteria (PCR, see
later, doesn’t produce enough and requires sequence
information for the primers).
It is then denatured (ie. melted), so that the two
strands split apart.
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Sequencing- continued
Denatured DNA is added to reaction mix with:
a primer (to start complementary pairing),
DNA polymerase
nucleotides including special ones called dideoxynucleotides.
These special nucleotides do not allow further nucleotides to be
added to the chain. So in a mix with dideoxy-A, every time a
dideoxy-A is added (small proportion of As), the reaction ends. This
results in different length fragments. The dideoxynucleotides are
fluorescently tagged.
Fragments can be separated out on a gel by
electrophoresis and their length calculated. Working out
DNA sequence ~ jigsaw puzzle.
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Sequencing Methods
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Maxam-Gilbert Sequencing
DMS FA H H+S
G G C C
G A T C
G G T
G G C C
C
A T
G C
A C
A T
Chain terminates
at ddG
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Chain Termination (Sanger)
Sequencing
A sequencing reaction mix includes labeled primer and
template.
Primer
5′OP- -3′ OH
TCGACGGGC…
Template
Template area to be sequenced
Dideoxynucleotides are added separately to each of the
four tubes.
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Chain Termination (Sanger)
Sequencing
ddATP + ddA
A
four dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddC
C
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP + dAddG
G four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
ddTTP + dAdGdCddT
T
four dNTPs dAdGdCdTdGdCdCdCdG
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Chain Termination (Sanger)
Sequencing
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Chain Termination (Sanger)
Sequencing
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Chain Termination (Sanger)
Sequencing
3′
G
G A T C G
Longer fragments T
A
ddG
A
A
T
C
Shorter fragments A
ddG T
G
5′
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Fluorescent Dyes
Fluorescent dyes are multicyclic molecules that absorb and
emit fluorescent light at specific wavelengths.
Examples are fluorescein and rhodamine derivatives.
For sequencing applications, these molecules can be
covalently attached to nucleotides.
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Fluorescent Dyes
In dye primer sequencing, the primer contains
fluorescent dye–conjugated nucleotides, labeling the
sequencing ladder at the 5′ ends of the chains.
ddA
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Dye Terminator Sequencing
A distinct dye or “color” is used for each of the four
ddNTP.
Since the terminating nucleotides can be distinguished by
color, all four reactions can be performed in a single
tube.
T
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Dye Terminator Sequencing
GA
G A T C TC
G
T
C
T
G
A
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Dye Terminator Sequencing
The DNA ladder is read on an electropherogram.
Electropherogram
5′ AGTCTG
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Automated Sequencing
Dye primer or dye terminator sequencing on capillary
instruments.
Sequence analysis software provides analyzed sequence in text
and electropherogram form.
Peak patterns reflect mutations or sequence changes.
Nucleotide added : A T C A G CT
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Alternative Sequencing Methods:
Bisulfite Sequencing
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Alternative Sequencing Methods:
Bisulfite Sequencing
Me Me Me
Methylated sequence: GTC GGC GATCTATC GTGCA …
Me Me Me
Treated sequence: GTC GGC GATUTATC GTGUA …
DNA Sequence:
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Hibridisasi DNA
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DNA Hybridization
Diagnostic test for mutations
Monitoring gene expression (sequence)
Screening for targets known to play a role in disease
Assessment of medical treatment
Environmental investigations
Biological warfare agent detection
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Basic DNA
5’-ATG-3’
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Preparation of nucleic acid probes:
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06_01.jpg
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How Genetic Sequencing Works in DNA biosensor
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http://www.devicelink.com/ivdt/archive/98/09/009.html
How Hybridization is Identified
Analyte
(Target DNA) Electrochemical devices Current
signal of a redox indicator