B.Sc. Med. Lab. Technology
M. Phil. Microbiology
Certificate in Health Professional Education
Lecturer, Department of Medical Lab. Technology
Institute of Paramedical Sciences, Khyber Medical
University, Peshawar, Pakistan
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
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. 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
The target will most often form part of a mixture of unrelated
nucleic acid sequences.
METHODS OF LABELING NUCLEIC ACID &
There are five basic methods for labeling nucleic acids. These
Methods based on RNA polymerase
End labeling methods
Direct labeling methods
P-labelling of duplex DNA by nick translation. Asterisks indicate
radiolabelled phosphate groups.
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
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
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
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 [γ32
-P] ATP to DNA or RNA
containing a 5’-hydroxyl terminus. This is termed as a forward
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
END-LABELLING OF NUCLEIC ACIDS
The major advantages of 5’-end labeling are:
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
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 32
P are easily detectable.
They are used often.
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
(c)Detection of hybrids is much faster than by radioactive probes.