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.
• 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;
• Northern Blot-Transfer of an RNA sample separated and identified using DNA or
RNA probes.
• Southern Blot-Transfer of an DNA sample separated and identified using DNA or
RNA probes.
• 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,

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.

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.

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 investigation.
 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 can be used to screen for
both prokaryotic and eukaryotic genes.
 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 study.

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, cytosine alternative.
 If 17 bp would do, the number is dramatically reduced to 128.

 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.
 Then a thymine would be added to the end of each of the different products, and so
on.
 In this way a mixture of all 512 oligonucleotides would be made in one run on the
synthesizer.
 This mixture is referred to as a degenerate oligonucleotide.

 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 each colony is transferred to
a sterile filter (Figure 5.6).

 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 making it single-stranded and
available to hybridize with the probe molecules

 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 UV
light.
 The same procedure can be used to screen a phage library, in which case the lysis step
is not necessary as the phage plaques represent lysed bacteria from which the DNA
has already been released.