PRINCIPLES OF CROP PRODUCTION
ABT-320
(3 CREDIT HOURS)
LECTURE 14
TECHNIQUES FOR GENETIC ENGINEERING,
ISOLATION OF TOTAL CELLULAR DNA
NUCLEIC ACID HYBRIDIZATION
METHODS FOR LABELING NUCLEIC ACIDS
CHOICE OF LABELS
TECHNIQUES FOR GENETIC ENGINEERING
Recombinant DNA technology deals with isolation and manipulation of
genes. Before nucleic acids can be cut or otherwise manipulated, they
must be either isolated and purified or artificially synthesized. The
isolated genes are also sometimes sequenced for a better understanding
of its structure that needs to be manipulated.
ISOLATION OF TOTAL CELLULAR DNA
• DNA is very easily damaged by shear forces, even rapid stirring of solution
can break even high DNA molecular weight into much shorter fragments.
Consequently, DNA is recovered from cells by gentlest possible method of
rupture, in the presence of EDTA to chalate the Mg+2 ions needed for DNase
activity. Cell walls, if present, are digested enzymatically (e.g. lysozyme
treatment of bacteria) and the cell membrane is solubilised using detergent.
Cell disruption is performed at 4℃ using autoclaved glassware and solutions
to destroy DNase activity.
• After release of nucleic acids from the cells, RNA can be removed by
treatment with ribonuclease (RNase). The other major contaminant, protein,
is removed by shaking the solution gently with water-saturated phenol, or
with phenol/chloroform mixture, either of which will denature protein but
not nucleic acids. Centrifugation of the emulsion formed by this mixture
produces a lower, organic phase, separated from the upper aqueous phase
by an interface denatured proteins. The aqueous solution is recovered and
deproteinised repeatedly, until no matter is seen at interface. Finally, the
deproteinised DNA preparation is mixed with ethanol and allowed to
precipitate out in freezer. After centrifugation, the DNA pellet is dissolved in
a buffer containing EDTA for protection against DNases and this solution can
be stored at 4℃ for at least a month.
ISOLATION OF TOTAL CELLULAR DNA
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DNA solutions can be stored frozen, but repeated freezing and thawing
tends to damage long molecules by shearing; so preparation in frequent
use are normally stored at 4℃.
• This procedure is suitable for total cellular DNA. If the DNA from specific
organelle or viral particle is needed, it is best to isolate the organelle or
virus before extracting its DNA.
NUCLEIC ACID HYBRIDIZATION
If a double-stranded DNA molecule is exposed to high temperature, or to
very alkaline conditions, then the two strands will break apart. The
molecule is said to have become denatured. The temperature at which
denaturation occurs is termed as melting temperature or Tm. If the
denatured DNA is returned to a temperature below its Tm or to neutral
pH when alkali was used to denature it, each strand will, after a time,
find its complementary strand. The two strands will ‘zipper’ back
together to re-form a double stranded DNA molecule. This ability of
complementary sequences to anneal, or hybridize, to one another is
called nucleic acid hybridization. This technique helps in determining the
gene structure and in identifying molecules which contain same
sequences of nucleotides. In a complex mixture of nucleic acid
molecules, nucleic acid hybridization technique helps in separation of
complementary sequence.
The principle of hybridization using an inert support. The target DNA is most
often localized to one part of the support and, in most cases, the aim of the
hybridization experiment is to identify this region. The probe is prelabelled
by some method, most often by the incorporation of radioactivity. After
hybridization, the annealed probe is detected by autoradiography.
NUCLEIC ACID HYBRIDIZATION
• When employed analytically, hybridization is normally performed using
one labeled sequence, termed the probe, and an unlabelled sequence
called the target. Probe is a short synthetic oligo deoxyribonucleotide
which is complementary to target DNA sequence. Target DNA may be
obtained from total cellular DNA preparations. The probe is labeled by
incorporation of either radioactively labeled nucleotides or with some
chemicals. The probe is the known, pure species in the hybridization and
the target is the unknown species to be identified.
• The target will most often form part of a mixture of unrelated nucleic
acid sequences. The probe is usually added in considerable excess over
the target, and its concentration determines the rate of reaction.
Hybridization reaction time is extended to ensure complete reaction. The
target and probe can be hybridized to each other in solution, or
alternatively the target can be immobilized on an inert support such as
nitrocellulose.
METHODS FOR LABELING NUCLEIC ACIDS
For identification of hybridized nucleic acid duplexes, labeling of probe
or target is necessary. As probe is short synthetic oligonucleotide it can
be easily labeled. A 32P isotopic label can be added to the 5’ hydroxyl
group using polynucleotide kinase to a probe. Alternatively, labeled
nucleotides can be incorporated at the 3’ end using terminal transferase.
Because label is added at only one place within the oligonucleotide, such
end-labelled probes contain relatively little radioactivity per unit weight
of DNA. This low specific activity limits their sensitivity as probes. If a
high specific activity probe is required, then some method of uniform
labeling must be used. Here the probe sequence is copied along part or
all of its length using DNA polymerase and labeled nucleoside
triphosphate.
METHODS OF LABELING NUCLEIC ACID &
PROBES
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There are five basic methods for labeling nucleic acids. These are:
Nick translation
Primer extension
Methods based on RNA polymerase
End labeling methods
Direct labeling methods
32P-labelling
of duplex DNA by nick translation. Asterisks indicate
radiolabelled phosphate groups.
NICK TRANSLATION
• This is done by making single-strand cuts (nicks) in the double stranded DNA
molecule by brief exposure to a dilute solution of an endonuclease (usually
deoxyribonuclease 1 of E. coli). DNA polymerase 1 is then used in the presence
of at least one radioactive precursor to “translate” the nick along the molecule
in the 5’ to 3’ direction. The net result is that a nonradioactive strand of DNA is
replaced by a radioactive strand. The DNA is then denatured and used as a
radioactive probe in hybridization experiments (Southern blots, Northern blots
etc).
• Nick translation can be used with a variety of labels to generate probes
suitable for most hybridization applications. It is also appropriate for the
generation of biotinylated probes.
PRIMER EXTENSION METHOD
• Primers are synthetic oligodeoxyribonucleotides which are
complementary to specific regions of known vector DNA. The 3’ termini
of these primers serve as initiation site for template dependent DNA
synthesis by enzymes like DNA polymerase 1.
• DNA polymerase works by extending a short double-stranded region
made by annealing an oligonucleotide primer to the single-stranded
template. Thus this method of uniform labeling requires a primer which
matches the probe sequence. Radiolabelling of primers can be done with
two methods.
• If the probe sequence is not known then random oligonucleotide
labeling can be used. It is often in the case when natural cellular DNA is
used. These primers are made by adding a mixture of all four bases at
each step in the chemical synthesis reaction. The DNA is denatured and
the two complementary strands are copied in the presence of labelled
primers as well as nucleoside triphosphates. The polymerase used is
Klenow fragment derived from DNA polymerase-I of E. coli.
PRIMER EXTENSION METHOD
• Chance homology ensures that these primers anneal to the separated
DNA strands at many points along their length, thus providing a base for
polymerase to initiate DNA synthesis. This is only one of several uniform
labeling methods.
• The second method uses a unique primer to restrict labeling to a
particular sequence of interest. In the primer extension method, it is
essential to use a polymerase lacking a 5’
3’ exonuclease activity
otherwise degradation of the primer will occur. The Klenow fragment of
E. coli DNA polymerase I, which lacks the 5’ 3’ exonuclease activity has
been used successfully.
• It is an ideal method for situations where high specific activity and low
probe concentrations are frequently employed.
The principle of random primed (oligo-) labelling. The DNA to be used as a
probe is denatured by heating and mixed with hexanucleotides of random
sequence which then act as primers for DNA synthesis.
METHODS BASED ON RNA POLYMERASES
• RNA polymerases catalyzes the synthesis of RNA from nucleoside
triphosphates using a DNA template. Thus they can incorporate labeled
ribonucleotides into RNA during transcription if such labeled nucleotides
are provided to it. If a specific site of a vector or DNA is transcribed in
such way, RNA probes (or transcripts) of defined length and sequence
can be obtained.
• Initially RNA probes were obtained from RNA polymerase from E. coli.
This enzyme when used in vitro exhibited very little template and
promoter specificity and therefore produces transcripts which have been
initiated and terminated at random. Today, RNA polymerases from a
number of phages are used, which possess a high degree of specificity.
Thus transcription can be limited to sequences cloned downstream of an
appropriate promoter.
END-LABELLING OF NUCLEIC ACIDS
• A wide variety of techniques is available for introducing label at either
the 3’ or 5’ ends of linear DNA or RNA. Usually only a single label is
introduced at the terminus. Nucleic acid can be 5’ end labeled using T4
polynucleotide kinase. Radiolabeled phosphate group is donated by [γ32P] ATP to DNA or RNA containing a 5’-hydroxyl terminus. This is termed
as a forward reaction.
• If 5’-phosphate group is present in DNA or RNA, then it is removed with
alkaline phosphatase. This reaction is driven by excess ADP which causes
the enzyme to transfer the terminal 5’-phosphate from DNA to ADP. This
is known as exchange reaction. The DNA is rephosphorylated by transfer
of labeled γ-phosphate from [γ32-P] ATP.
The 5’-end labelling reaction using T4 polynucleotide kinase
END-LABELLING OF NUCLEIC ACIDS
The major advantages of 5’-end labeling are:
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Both DNA and RNA can be labeled.
Location of labeled group is known.
Very small fragments can be labeled.
Restriction digest fragment can be labeled.
CHOICE OF LABEL
• There are two technical parameters, resolution and sensitivity, which
determines the success of probe application. High degree of resolution is
required to know the relative position of a nucleic acid fragment. High
sensitivity is necessary because sequence of interest may be present at
low abundance.
• Other factors are probe stability, safety and ease of use. Broadly labels
can be categorized into radioactive and nonradioactive types.
RADIOACTIVE LABELS
These labels have wider applications as they can be easily detected with
autoradiography. Their detection gives two important information, firstly
about occurrence of hybridization between probes and target DNA and
secondly about their position. Radioactive methods using 32P are easily
detectable. They are used often.
NON-RADIOACTIVE LABELS
A number of non radioactive labels for probes are available but biotin is
widely used.
BIOTIN LABELLED PROBES
Biotinylated probes are prepared through a nick-translation reaction by
replacing nucleotides with biotinylated derivatives. After hybridization and
washing, detection of hybrids is done by adding avidine and going through
a series of cytochemical reactions which finally give a blue color whose
intensity is proportional to the amount of biotin in the hybrid. There are
several advantages of using biotinylated probes. The major advantages of
using biotinylated probes are:
(a) assays employ non-toxic materials, with longer half-life.
(b) can be prepared in advance in bulk and stored at -20℃ for repeated uses.
(c) Detection of hybrids is much faster than by radioactive probes.
THE END