Analisis berbasis asam nukleat dan

protein
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

Maxam/Gilbert chemical sequencing

Sanger chain termination sequencing

Pyrosequencing

Array sequencing

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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

Maxam-Gilbert sequencing is performed by chain

breakage at specific nucleotides.
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Maxam-Gilbert Sequencing
3′
A
A
G
G G+A T+C C
C
Longer fragments A
A A
C
G
T
G
Shortest fragments C
G
A
G
5′

Sequencing gels are read from bottom to top (5′ to 3′).

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Chain Termination (Sanger) Sequencing

A modified DNA
replication reaction.

Growing chains are
terminated by
dideoxynucleotides.
 Chain Termination (Sanger)
Sequencing
The 3′-OH group necessary for formation of the
phosphodiester bond is missing in ddNTPs.

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

With addition of enzyme (DNA polymerase), the primer

is extended until a ddNTP is encountered.

The chain will end with the incorporation of the ddNTP.

With the proper dNTP:ddNTP ratio, the chain will
terminate throughout the length of the template.

All terminated chains will end in the ddNTP added to
that reaction.

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 Chain Termination (Sanger)
Sequencing

The collection of fragments is a sequencing ladder.

The resulting terminated chains are resolved by
electrophoresis.

Fragments from each of the four tubes are placed in four
separate gel lanes.

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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′

Sequencing gels are read from bottom to top (5′ to 3′).

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 Cycle Sequencing

Cycle sequencing is chain termination sequencing performed in

a thermal cycler.

Cycle sequencing requires a heat-stable DNA polymerase.

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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

In dye terminator sequencing, the fluorescent dye

molecules are covalently attached to the
dideoxynucleotides, labeling the sequencing ladder at the
3′ 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 The fragments are distinguished by

AC size and “color.”
GT
G

T
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Dye Terminator Sequencing

The DNA ladder is resolved in one gel lane or in a capillary.

GA
G A T C TC

G
T
C
T
G
A

Slab gel Capillary

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Dye Terminator Sequencing

The DNA ladder is read on an electropherogram.

Slab gel Capillary

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.

T/T T/A A/A

5′ AGTCTG 5′ AG(T/A)CTG 5′ AGACTG

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 Alternative Sequencing Methods:
Pyrosequencing

Pyrosequencing is based on the generation of light
signal through release of pyrophosphate (PPi) on
nucleotide addition.

DNAn + dNTP
DNAn+1 + PPI

PPi is used to generate ATP from adenosine
phosphosulfate (APS).

APS + PPI
ATP

ATP and luciferase generate light by conversion of
luciferin to oxyluciferin.
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 Alternative Sequencing Methods:
Pyrosequencing

Each nucleotide is added in turn.

Only one of four will generate a light signal.

The remaining nucleotides are removed enzymatically.

The light signal is recorded on a pyrogram.
DNA sequence: A T C A GG CC T

Nucleotide added : A T C A G CT
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 Alternative Sequencing Methods:
Bisulfite Sequencing

Bisulfite sequencing is used to detect methylation in DNA.

Bisulfite deaminates cytosine, making uracil.

Methylated cytosine is not changed by bisulfite treatment.

The bisulfite-treated template is then sequenced.

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 Alternative Sequencing Methods:
Bisulfite Sequencing

The sequence of treated and untreated templates is compared.

Me Me Me
Methylated sequence: GTC GGC GATCTATC GTGCA …

Me Me Me
Treated sequence: GTC GGC GATUTATC GTGUA …

DNA Sequence:

(Untreated) reference: ...GTCGGCGATCTATCGTGCA…

Treated sequence: ...GTCGGCGATUTATCGTGUA…

This sequence indicates that these Cs are methylated.

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 Summary

Genetic information is stored in the order or sequence of
nucleotides in DNA.

Chain termination sequencing is the standard method for the
determination of nucleotide sequence.

Dideoxy-chain termination sequencing has been facilitated by
the development of cycle sequencing and the use of
fluorescent dye detection.

Alternative methods are used for special applications, such as
pyrosequencing (for resequencing and polymorphism
detection) or bisulfite sequencing (to analyze methylated
DNA).

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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:

- DNA: from cell-based cloning or by PCR. Probe is double stranded.

Labeling by DNA polymerase-based DNA strand synthesis.

- RNA: by transcription from DNA cloned in an expression vector.

Probe is single stranded. Labeling by “run-off ” transcription.

- Oligonucleotide: by chemical synthesis. Probe is single stranded.

Labeling is by end labeling.

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 06_01.jpg

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 How Genetic Sequencing Works in DNA biosensor

Separate ds-DNA (Probe DNA).

DNA is denatured by heat or chemical denaturant
and placed in solution or on a solid substrate,
forming a reference segment

Introduce unknown ss-DNA (Target DNA)

Unknown DNA sample is introduced to the reference
segment. The complement of the reference segment
will hybridize to it.

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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

Signal Optical devices Emission

signal of fluorescent or
chemiluminescent lables Surface optical
properties Nanoparticle based
Transducer colorimetric detection

Recognition layer Mass-sensitive devices Frequency

(Probe DNA) signal of oscillating crystal with DNA
probe
DNA Hybridizaion biosensor
- Immobilization of ss-DNA probe onto the transducer surface
- Tranducing (association of an appropriate hybridization indicator)
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Joseph Wang, Nucleic Acids Research, 2000, 28(16), 3011