Working with nucleic acids

Learning Outcome (LO)

LO 11: menjelaskan tahapan isolasi DNA dan

RNA
LO 12: menjelaskan jenis label dan cara
pelabelan asam nukleat
LO 13: menjelaskan prinsip hibridisasi asam
nukleat
LO 14: menjelaskan metode sekuensing DNA
SangerCoulson
LO 15: menjelaskan metode sekuensing
otomatis

1 Isolation of DNA and RNA

Metode dasar diperlukan dalam teknik
manipulasi gen:
penanganan
pengukuran
analisis molekul asam nukleat.
Percobaan manipulasi gen membutuhkan
sumber asam nukleat, baik dalam bentuk
DNA atau RNA
Ada
tiga
persyaratan
dasar
untuk
mendapatkan/ mengisolasi asam nukleat:
membuka sel untuk mengeluarkan asam
nukleat
pemisahan asam nukleat dari komponen

LO 11: menjelaskan tahapan isolasi DNA dan RNA

solation of DNA and RNA

cel
l
cell disruption

enzymatic degradation of cell wall material (if

present)
and detergent lysis of cell membranes
phenol or phenol/chloroform mixtures
deproteinisation

precipitation

isopropanol or ethanol

nucle
ic
acids
If a DNA preparation is required, the enzyme
ribonuclease (RNase) can be used to digest the
RNA in the preparation.
LO 11: menjelaskan tahapan isolasi DNA dan RNA

Fig. 3.1. Preparation of mRNA by

affinity chromatography using
oligo(dT)-cellulose.

If mRNA is needed for

cDNA synthesis, a
further purification can
be performed by using
oligo(dT)-cellulose to
bind the poly(A) tails of
eukaryotic mRNAs (Fig.
3.1).
This gives substantial
enrichment for mRNA
and enables most
contaminating DNA,
rRNA and tRNA to be
removed.

LO 11: menjelaskan tahapan isolasi DNA dan RNA

adiolabelling of nucleic acids

A major problem encountered in many cloning
procedures is that of keeping track of the small
amounts of nucleic acid involved.
This problem is magnified at each stage of the
process, because losses mean that the amount of
material usually diminishes after each step.
One way of tracing the material is to label the
nucleic acid with a radioactive molecule (usually a
deoxynucleoside triphosphate (dNTP), labelled with
3
H or 32P), so that portions of each reaction may be
counted in a scintillation counter to determine the
amount of nucleic acid present.

LO 12: menjelaskan jenis label dan cara pelabelan asam

 A second application of radiolabelling is in the

production of highly radioactive nucleic acid
molecules for use in hybridisation experiments.
Such molecules are known as radioactive probes,
and have a variety of uses (see Sections 3.4 and
8.2).
The difference between labelling for tracing
purposes and labelling for probes is largely one of
specific activity, i.e . the measure of how
radioactive the molecule is.
For tracing purposes, a low specific activity will
suffice but for probes, a high specific activity is
necessary.
In probe preparation the radioactive label is usually
the high-energy -emitter 32P.
Some common methods of labelling nucleic acid
molecules are described below.
LO 12: menjelaskan jenis label dan cara pelabelan asam

3.2.1 End labelling

In this technique the enzyme polynucleotide kinase
is used to transfer the terminal phosphate group of
ATP onto 5-hydroxyl termini of nucleic acid
molecules.
If the ATP donor is radioactively labelled, this
produces a labelled nucleic acid of relatively low
specific activity, as only the termini of each
molecule become radioactive (Fig. 3.2).

Fig. 3.2. End labelling DNA using polynucleotide kinase (PNK).

(a) DNA is dephosphorylated using hosphatase, to generate 5-OH
groups. (b) The terminal phosphate of [32P]ATP (solid circle) is then
transferred to the 5 terminus by PNK. The reaction can also occur as an
exchange reaction with 5-phosphate termini

LO 12: menjelaskan jenis label dan cara pelabelan asam

3.2.2 Nick translation

This method relies on the ability of the enzyme DNA
polymerase I (see Section 4.2.2) to translate (move
along the DNA) a nick created in the phosphodiester
backbone of the DNA double helix.
Nicks may occur naturally, or may be caused by a
low concentration of the nuclease DNase I in the
reaction mixture.
DNA polymerase I catalyses a strand replacement
reaction which incorporates new dNTPs into the DNA
chain.
If one of the dNTPs supplied is radioactive, the result
is a highly labelled DNA molecule (Fig. 3.3).

LO 12: menjelaskan jenis label dan cara pelabelan asam

Fig. 3.3. Labelling DNA by nick translation. (a) A singlestrand nick is introduced into the phosphodiester backbone of a
DNA fragment using DNase I. (b) DNA polymerase I then
synthesises a copy of the template strand, degrading the
nontemplate strand with its 53 exonuclease activity. If [32
P]dNTP
is suppliedjenis
this label
will be
incorporated
into the
newly
LO 12:
menjelaskan
dan
cara pelabelan
asam

2.3 Labelling by primer extension

This term refers to a technique which uses random
oligonucleotides (usually hexadeoxyribonucleotide
molecules sequences of six deoxynucleotides) to
prime synthesis of a DNA strand by DNA
polymerase.
The DNA to be labelled is denatured by heating, and
the oliognucleotide primers annealed to the single
stranded DNAs.
The Klenow fragment of DNA polymerase (see
Section 4.2.2) can then synthesise a copy of the
template, primed from the 3- hydroxyl group of the
oligonucleotide.
If a labelled dNTP is incorporated, DNA of very high
specific activity is produced (Fig. 3.4).

LO 12: menjelaskan jenis label dan cara pelabelan asam

Fig.
3.4.
Labelling
DNA
by
primer
extension
(oligolabelling). (a) DNA is denatured to give single-stranded
molecules. (b) An oligonucleotide primer is then added to give a
short double-stranded region with a free 3-OH group. (c) The
Klenow fragment of DNA polymerase I can then synthesise a
copy of the template strand from the primer, incorporating [32
P]dNTP (filled circles) to produce a labelled molecule with a
LOvery
12: high
menjelaskan
jenis label dan cara pelabelan asam
specific activity.

ucleic acid hybridisation

Fig. 2.10 The principle of nucleic acid

hybridisation.
This feature of DNA molecules is a critical part of
many of the procedures involved in gene
manipulation and is also an essential feature of life
itself. Thus, the simple G C and A T base pairing
has profound implications for living systems and for

LO 13: menjelaskan prinsip hibridisasi asam nukleat

ucleic acid hybridisation

Nucleic acid hybridisation can be used as an
extremely sensitive detection method, capable of
picking out specific DNA sequences from complex
mixtures.
Usually a single pure sequence is labelled with 32P
and used as a probe.
The probe is denatured before use so that the
strands
are
free
to
basepair
with
their
complements.
The DNA to be probed is also denatured, and is
usually fixed to a supporting membrane made from
nitrocellulose or nylon.
Hybridisation is carried out in a sealed plastic bag
or tube at 6568 C for several hours to allow the
duplexes to form.
The excess probe is then washed off and the degree

LO 13: menjelaskan prinsip hibridisasi asam nukleat

DNA sequencing
The ability to determine the sequence of bases in
DNA is a central part of modern molecular biology,
and provides what might be considered as the
ultimate structural information.
Rapid methods for sequence analysis were
developed in the late 1970s,and the technique is
now used in laboratories worldwide.
There are two main methods for sequencing DNA.
In one method, developed by Allan Maxam and
Walter Gilbert, chemicals are used to cleave the
DNA at certain positions, generating a set of
fragments that differ by one nucleotide.
The same result is achieved in a different way in the
second method, developed by Fred Sanger and Alan
Coulson, which involves enzymatic synthesis of DNA
strands that terminate in a modified nucleotide.

SangerCoulson (dideoxy or enzymatic)

sequencing

Although the end result is similar to that attained by

the chemical method, the SangerCoulson procedure
is totally different from that of Maxam and Gilbert.
In this case a copy of the DNA to be sequenced is
made by the Klenow fragment of DNA polymerase
(see Section 4.2.2).
The template for this reaction is single-stranded DNA,
and a primer must be used to provide the 3 terminus
for DNA polymerase to begin synthesising the copy
(Fig. 3.8).

LO 14: menjelaskan metode sekuensing DNA SangerCoulson

Fig. 3.8. DNA sequencing

using the dideoxy chain
termination
(Sanger
Coulson) method. (a) A
primer is annealed to a
single-stranded
template
and (b) the Klenow fragment
of DNA polymerase I used to
synthesise a copy of the
DNA. A radiolabelled dNTP
(often
[-35S]dNTP,
filled
circles) is incorporated into
the
DNA.
(c)
Chain
termination occurs when a
dideoxy
nucleoside
triphosphate
(ddNTP)
is
incorporated. (d) A series of
four
reactions,
each
containing one ddNTP in
addition
the four dNTPs
LO 14: menjelaskan metode sekuensing
DNAto
SangerCoulson

 The production of nested fragments is achieved by

the incorporation of a modified dNTP in each reaction.
These dNTPs lack a hydroxyl group at the 3 position of
deoxyribose, which is necessary for chain elongation
to proceed.
Such modified dNTPs are known as dideoxynucleoside
triphosphates (ddNTPs).
The four ddNTPs (A,G,T and C forms) are included in a
series of four reactions, each of which contains the
four normal dNTPs.
The concentration of the dideoxy form is such that it
will be incorporated into the growing DNA chain
infrequently.
Each reaction therefore produces a series of
fragments terminating at a specific nucleotide, and
the four reactions together provide a set of nested
fragments.
LO 14: menjelaskan metode sekuensing DNA SangerCoulson

LO 14: menjelaskan metode sekuensing DNA SangerCoulson

5 Automated DNA sequencing

In 1986, Leroy Hood and Lloyd Smith automated
Sangers method.
In this new sequencing technology, radioactive markers
are replaced with fluorescent ones.
Each ddNTP terminator is tagged with a different color of
fluorophore: red, green, blue, or yellow.
Thus, instead of having to run four separate sequencing
reactions, the reactions can be combined into one tube.
The first automated sequencer made use of a
polyacrylamide gel to resolve the samples, a laser to
excite the dye molecules as they reached a detector
near the end of the gel, and a computer to read the
results as a DNA sequence.
In this system each automated sequencer was able to
produce 4800 bases of sequence per day.
The current automated systems replace the old-style gel
with arrays of tiny capilliaries, each of which acts as a
LO 15:
menjelaskan metode sekuensing otomatis
lane.

 A pump loads special capillaries with a polymer that

serves as the separation matrix.
DNA samples in a 96-well plate are loaded into the
capillary array by a short burst of electrophoresis,
called electrokinetic injection.
The capillary array is immersed in running buffer
and the DNA fragments then migrate through the
capillary matrix by size, smallest to largest.
As the DNA fragments reach the detection window,
a laser beam excites the dye molecules causing
them to fluoresce.
Emitted light from 96 capillaries is collected at
once, spectrally separated into the four colors and
focused onto a CCD camera.
Computer software interprets the pattern of peaks
to produce a graph of fluorescence intensity versus
time (electropherogram), which is then converted to
themenjelaskan
DNA sequence
(Fig.sekuensing
8.16).
LO 15:
metode
otomatis

Figure 8.16
Automated
DNA sequencing
LO 15: menjelaskan
metode
sekuensing
otomatis

LO 15: menjelaskan metode sekuensing otomatis

NEXT
TATAP MUKA 4
Kuis 3 Enzim
LO
LO
LO
LO

16: menjelaskan enzim restriksi

17: menjelaskan nuklease
18: menjelaskan polimerase
19: menjelaskan enzim yang digunakan
untuk memodifikasi ujung DNA
LO 20: menjelaskan DNA ligase