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Learning Outcome (LO)

menjelaskan analisis gen terklon menggunakan teknik blotting


Clone identification
requires a

Highly specific method which enables Isolation of


specific genes

and can involve for further analysis by

Selecting a clone Screening a clone bank Translation of mRNA in vitro Restriction mapping

which requires two main methods two methods in conjunction with


may incorporate

Genetic Use of Nucleic acid Immunological Hybrid Hybrid Blotting Sub-cloning


selection •Chromogenic substrates Hybridisation screening arrest release Tech-niques
•Insertional inactivation

for direct selection Using a Detects proteins from to enable followed by

Presence Complementation Replica filter cDNA Identification DNA


of of a defined expression of protein sequence
vector mutation and a library product analysis

Labelled
probe

which may be

RNA cDNA Genomic DNA Oligonucleotide


Blotting Techniques

• Blotting – Transfer of DNA, RNA or Proteins, typically from a


electrophoresis gel to a membrane e.g. nitrocellulose. This
membrane can then be subject to further techniques such as
hybridization.
• Hybridization – Process where two complementary single
strands of nucleic acid (DNA or RNA) form a double helix.
Blotting Techniques

• Using specific probes that are labelled specific sequences of DNA can be
identified.
• There are three main hybridization techniques which vary in the sample
blotted and the probes used;
1. Northern Blot-Transfer of an RNA sample separated and identified using
DNA or RNA probes.
2. Southern Blot-Transfer of an DNA sample separated and identified using
DNA or RNA probes.
3. Western Blot- Transfer of an Protein sample separated and identified
typically using an antibody.
Applications

• The main use of this technique is to identity any changes in DNA


sequencing or genes expressed, e.g. comparing genes expressed by a
diseased cell to genes expressed by an healthy cell.

• Other uses include- Testing for hereditary disease, Evolutionary


history of species, Screening e.g.food supply

• Applications to synthetic biology


- identification of various parts in natural organisms,
Blotting techniue
 Dalam banyak percobaan, bagian fragmen DNA terklon
yang mengandung daerah yang diinginkan perlu
ditentukan.
 Penentuan daerah yang diinginkan dapat dilakukan
menggunakan berbagai metode berdasarkan blotting
molekul asam nukleat ke membran dan hibridisasi
dengan probe khusus.
 Metode ini hampir sama dengan metode identifikasi klon
menggunakan hibridisasi koloni atau plak.
Southern blotting
 Teknik blotting dikembangkan
pertama kali oleh Edwin M.
Southern, dan dikenal sebagai
Southern blotting.
 Dalam metode ini fragmen DNA
yang dipotong enzim restriksi
dielektroforesis gel agarosa.
 Fragmen terpisah kemudian
ditransfer ke nitroselulosa atau
membran nilon menggunakan
teknik blotting (seperti vacuum
blotting dan electroblotting).
1) The gel is placed on a filter
paper wick and a
nitrocellulose or nylon filter
placed on top.
2) Further sheets of filter paper
and paper tissues complete
the setup.
3) Transfer buffer is drawn
through the gel by capillary
action, and the nucleic acid
fragments are transferred
out of the gel and onto the
membrane.
Fig. 8.9 Blotting apparatus.
Fig. 8.10 Southern blotting. A hypothetical 20 kb fragment from a genomic clone is under
investigation. A cDNA copy of the mRNA is available for use as a probe. (a) Gel pattern of
fragments produced by digestion with various restriction enzymes; (b) autoradiograph resulting
from the hybridisation. Lanes 1 and 6 contain λ HindIII markers, sizes as indicated. These have
been marked on the autoradiograph for reference. The intact fragment (lane 2) runs as a single
band to which the probe hybridises. Lanes 3, 4, and 5 were digested with EcoRI (E), PstI (P), and
BamHI (B). Fragment sizes are indicated under each lane in (a). The results of the
autoradiography show that the probe hybridises to two bands in the EcoRI and BamHI digests;
therefore, the clone must have internal sites for these enzymes. The PstI digest shows
hybridisation to the 7 kb fragment only. This might, therefore, be a good candidate for
subcloning, as the gene may be located entirely on this fragment.
Northern blotting
 Meskipun Southern blotting adalah teknik yang sangat
sederhana, namun memiliki banyak aplikasi dan telah
menjadi metode yang berguna dalam analisis gen.
 Teknik yang sama juga dapat digunakan untuk RNA yang
dikenal sebagai northern blotting.
 Berguna untuk:
 menentukan pola hibridisasi dalam sampel mRNA
 menentukan daerah fragmen DNA terklon yang akan
menghibridasi mRNA tertentu.
 mengukur tingkat transkrip selama ekspresi gen
tertentu
Northern blotting
Dot blotting/Slot blotting
 Pada teknik blotting ini, sampel asam nukleat tidak dielektroforesis
tetapi diteteskan ke filter
 Teknik ini sangat berguna dalam memperoleh data kuantitatif
dalam studi ekspresi gen

 In some variants of the apparatus the nucleic acid is applied in a


slot rather than as a dot.
 Not surprisingly, this is called slot blotting!
Western blotting
 Teknik ini melibatkan transfer molekul protein yang telah
dielektroforesis ke membran.
 Teknik elektroforesis yang sering digunakan adalah SDS-
PAGE (SDS-Polyacrylamide Gel Electrophoresis)
 Western blotting dapat mengidentifikasi protein khusus
jika antibodi yang sesuai tersedia.
 Western blotting dapat berguna untuk mengukur jumlah
protein tertentu dalam sel tertentu pada waktu tertentu.
 Dengan membandingkan dengan data lainnya (seperti
jumlah mRNA, dan/atau aktivitas enzim) dapat untuk
membangun sebuah gambaran tentang bagaimana
ekspresi dan kontrol metabolik protein diatur.
Immunological screening for expressed genes
 Skrining imunologis bertujuan mengidentifikasi protein
yang diekspresikan gen terklon.
 Persyaratan teknik: gen terklon harus diekspresi dalam
klon rekombinan.
 Pelacak: antibodi spesifik
Immunological screening for expressed genes
 Antibodi diproduksi hewan dalam menanggapi tantangan
antigen, yang biasanya berupa protein murni.
 Ada dua jenis utama dari preparasi antibodi yang dapat
digunakan.
 antibodi poliklonal, mengenali semua determinan
antigenik antigen.
 antibodi monoklonal, yang mengenali determinan
antigenik tunggal.
 Produksi antibodi monoklonal secara teknik rumit, oleh
karena itu antisera poliklonal berkualitas baik sering
digunakan untuk skrining.
 Filter diambil dari cawan Petri yang
berisi rekombinan (biasanya cDNA / 
konstruksi).
 Protein dan debris sel menempel filter.
 Plak mengekspresikan protein target
(+) tidak bisa dibedakan dari yang lain
(-) pada tahap ini.
 Filter ini diinkubasi dengan antibodi
primer yang spesifik untuk protein
target.
 Kemudian ditambahkan radiolabelled
Fig. 8.7 Immunological screening for protein A berlabel, dan dilakukan
expressed genes. autoradiografi.
 Seperti dalam penyaringan asam
nukleat, plak positif dapat diidentifikasi
dan diambil dari master plate.
 Metode pendeteksian kromogenik/
enzimatik juga dapat digunakan dalam
jenis sistem ini.
C. J. Howe, C.J., 2007, Gene Cloning and Manipulation, Second Edition, Cambridge
University Press, UK
Learning outcomes:
By the end of this lecture you will have an understanding
of:

• the various approaches that can be used to identify the


clone of interest from a library
• DNA–DNA hybridization
DNA library
 If there is enough genome sequence data available, you can
design PCR primers to amplify the region of interest and
simply amplify the target region by PCR.
 If genome sequence data is not available you may need to
make a DNA library.
 With DNA library you have a collection of clones in the form
of either plaques or colonies on a plate.
 The process of determining which of those clones carry the
gene or genes you are interested in is called screening.
 cDNA library may be developed from a starting material that
is rich in the mRNA transcribed from the gene of interest, a
high proportion of clones in the library may encode the gene
of interest.
 Screening methods use two main approaches: those that
rely on expression of the gene and those where you look for
a particular DNA sequence
Screening Methods Based on Gene Expression

 It is necessary to construct an expression library in a vector


designed to ensure that the cloned genes are expressed.
 In this case we could plate out the library, spray each plate
with catechol and pick out the yellow colonies (Figure 5.1).
 A range of bacterial genes can be screened for in this way
including those involved in the utilization of specific sugars
and the genes involved in antibiotic production and
resistance to antibiotics.
Figure 5.1 Screening a DNA library by
detection of a phenotype. It is possible
to detect the activity of the xylE gene of
Pseudomonas aeruginosa using a simple
colorimetric test.
a) A genomic library is made by cloning
fragments of P. aeruginosa DNA into
a vector and transforming into E. coli.
b) The library is plated out and the
colonies are sprayed with catechol.
Clones expressing xylE will turn
yellow and can be isolated and
c) grown up in liquid culture.
Complementation
 Unlike human cells E. coli cells are able to synthesize
organic molecules like amino acids.
 If you want to clone the genes responsible for the synthesis
of, for example, the amino acid lysine.
 You might be able to devise a simple test for the presence of
lysine but, as it is unlikely to be secreted from the bacteria,
any test is likely to involve some complex manipulations.
 One approach to this problem is to use complementation.
 It is relatively easy to make auxotrophic mutants of E. coli,
that are unable to grow on minimal media without
supplementation with specific nutrients, by treating the cells
with UV light or a chemical mutagen.
 If such a mutant is unable to grow in the absence of lysine
(Lys–), it is a reasonably safe assumption that the mutations
fall in one or other of the enzymes involved in the synthesis
of lysine.
 To screen a genomic library, constructed from a wild-type
strain of E. coli, for clones expressing genes involved in the
synthesis of lysine, you need to introduce your library into
the mutant Lys– strain and plate onto minimal medium
without lysine.
 The colonies which grow must contain a clone which is able
to complement the mutation in the Lys strain.
 These colonies can be isolated and cultured for further
 One obvious problem with this approach is that you will only
identify the gene in which the original mutation lies.
 In the case of lysine synthesis, as with most biochemical
pathways, there are a whole series of enzymes which must
act in sequence.
 In bacteria the genes encoding these enzymes are often
arranged next to each other on the chromosome, sometimes
forming a functional unit called an operon.
 If your library was constructed from relatively large
fragments of DNA you may well have already cloned many
of the other genes in the pathway.
 If there are still missing components of the pathway you may
need to screen a whole bank of mutations by
complementation
 Screening by complementation, or marker rescue as it is
sometimes known, has been very useful in the elucidation of
biochemical pathways both in bacteria and also in
eukaryotic organisms such as the yeast Saccharomyces
cerevisiae.
 It relies on being able to make a mutation in a suitable host
strain such as E. coli that can be complemented by the gene
you are trying to clone.
 Auxotrophic mutants of E. coli have been used successfully
in screening for genes involved in metabolism in a wide
range of bacteria, as the genes are usually similar enough to
complement the mutations in E. coli.
Immunological Screening of Expression Libraries
 The approaches to screening DNA libraries by looking for
protein expression which we have discussed so far rely on
techniques that are specific to the protein being studied.
 Ideally, we would like a technique that would be adaptable to
detect a wide range of proteins.
 Antibodies have been used in a wide range of applications,
such as western blotting (Box 5.1), to detect specific
proteins immobilized on a sheet of nitrocellulose (Figure
5.2).
 This technology has been adapted for screening DNA
libraries.
 To use an antibody to screen a library, the library is plated
out and the bacteria lysed to release intracellular proteins.
 Proteins are then transferred to a solid support and probed
with the antibody.
 This approach has a very wide range of applications and
 The main difficulty with antibody-based technology is that it
is necessary to raise a specific antibody for each protein that
you wish to detect.
 This usually means being able to purify the specific protein
in sufficient quantity to be able to inject it into a small
mammal, usually a mouse, rat or rabbit, in order to cause an
immune response.
 Serum is then harvested from the animal and the antibody
may be further purified before use.
 This is a lengthy and costly procedure and can only be
carried out successfully with proteins which can be
produced in reasonably large amounts.
 It is common to use vectors derived from bacteriophage λ
(Section 4.10) in the construction of libraries for antibody
screening.
 This is because when you plate a bacteriophage λ library,
plaques are produced where the bacteria have been lysed
(Figure 4.3).
 This means that proteins are released from the bacterial
cells and are readily available to the antibodies and there is
no need for subsequent lysis of the bacterial colonies.
Preparation of filters
 To use immunological screening we need to immobilize the proteins,
produced by each of the phage in our library, on a solid support, in such
a way that we know which original plaque they were produced by.
 This technique, often called a “plaque lift”, is outlined in Figure 5.3.
 For immunological screening nitrocellulose filters are used, as proteins
will bind to them.
 The filters are then bathed in a solution containing the primary antibody,
which will bind specifically to the protein it was raised against.
 Excess antibody is washed off and the bound primary antibody is
detected using a secondary antibody conjugate (Figure 5.2).
 Using this technique it is possible to pinpoint which of the original
plaques was produced by a phage expressing the protein of interest.
 It is often difficult to be sure exactly which plaque corresponds to the
signal on the filter, especially if the library was plated at high density.
 In this case a plug of agar is removed from the area of the positive
signal, phage are grown up from it and replated at a lower density and
the screening process is repeated.
 Once a single plaque on an agar plate has been identified, which is
expressing the protein of interest, the phage can be isolated for further
Screening Methods Based on Detecting a DNA
Sequence
 This is perhaps the most commonly used approach to library
screening.
 It relies on the fact that a single-stranded DNA molecule will
hybridize to its complementary sequence (Figure 5.4).
 In this approach the single-stranded DNA molecule is used
as a probe in a hybridization experiment to bind to and thus
identify specific sequences.
 DNA probes used in this way are usually either synthetic
oligonucleotides or fragments of cloned DNA.
 DNA probes can be used to probe DNA released from
bacterial colonies directly onto the nylon membrane (colony
blot, Section 5.8) or, as described in Box 5.2, purified DNA
samples which are either applied directly to the membrane
(dot blot) or size separated on an agarose gel prior to
analysis (Southern blot ).
 In order to use a DNA probe to screen a library you will need
some DNA sequence or cloned DNA from which to derive
the probe.
 In some experimental situations you may already know all or
part of the sequence of the gene you are trying to clone. An
example of this would be when you have a clone from a
cDNA library but you want a genomic clone of the same
gene.
 This might be because you are interested in sequences
outside the coding
Oligonucleotide Probes
 Short synthetic DNA molecules similar to those used as
primers in PCR and sequencing can be used as probes in
hybridization procedures.
 Oligonucleotide probes can be designed from known DNA
sequence but more usually are derived from amino acid
sequence.
 Relatively small samples, containing as little as 5 pmol of
purified protein, can be sequenced by the Edman
degradation.
 This is a sequential procedure in which the N-terminal
amino acid is chemically cleaved from the protein and
identified by liquid chromatography; the procedure is
repeated with subsequent amino acids.
 This is now an automated process; it can reliably identify the
first 20 amino acids of a protein, although some proteins are
blocked making sequencing by this approach very difficult.
Designing an oligonucleotide probe
 Imagine that we want to use an oligonucleotide to
screen a human DNA library for a clone of the
insulin gene.
 Have a look at Figure 5.5 which shows the N-
terminal sequence of the B chain of human insulin.
 We would need 2 × 4 × 2 × 2 × 2 × 2 × 4 × 2 or
1024 different oligonucleotides of 21 bp in length to
be sure of having all the possible combinations.
 By thinking carefully about how long the probe
really needs to be we can reduce the number of
combinations required.
 If a 20 bp oligo would be long enough then there
would only be 512 combinations because you do
not need to take into account the last thymine,
 You can see that when using DNA probes derived from an
amino acid sequence you will need to use a mixture of
oligonucleotides to represent all of the possible DNA
sequences, which could encode the amino acid sequence.
 However, the individual sequences do not have to be
synthesized separately.
 The automated DNA synthesizer can be programmed to use
a mixture of nucleotide monomers at specific positions in the
molecule, resulting in a mixture of oligonucleotides
representing all the possible sequences.
 In the case of the 20 bp oligo derived from the insulin
sequence shown in Figure 5.5 the synthesizer would be
programmed to start with a guanine (G) residue.
 It would then add a thymine (T).
 The oligos produced by this stage will all be identical.
 For the next residue it would use a mixture of all four
residues; these would be incorporated randomly into the
products giving four different classes of product.
Colony and Plaque Hybridization

 Once you have labeled


your DNA probe, you
need to transfer the DNA
from individual library
members onto a solid
support, in such a way
that they can be related
back to the original library
member, before
screening can begin.
 This is done using a
method analogous to the
plaque lift described in
Section 5.4, the library is
plated out and a small
amount of bacteria from
 The filters are then
aken through the
series of treatments
outlined in Figure 5.7
to produce filters with
DNA from the bacterial
colonies bound to
them.
 Firstly, the bacteria
are lysed by treatment
with the detergent
SDS and sodium
hydroxide, to release
the DNA, which will
include chromosomal
DNA as well as library
DNA encoded on the
plasmid.
 The alkaline
conditions will also
denature the DNA
 Next the alkali has to be
neutralized, otherwise it will
interfere with annealing of
the probe.
 DNA is covalently cross-
linked to the filters before
hybridization.
 It is bound to the filter by its
sugar–phosphate backbone
leaving the bases free to
hybridize with the
complementary bases of the
DNA probe.
 Both nylon and
nitrocellulose filters can be
used in this procedure as
DNA can be bound to both
substrates.
 However, nylon filters are
usually preferred as they are
more robust than
nitrocellulose and the DNA
can be fixed to them either
by heating or exposure to