Random primer labelling (primer extension)
Ø Gene probes, clone or TCR amplified and
oligonucleotide probes can be random prime labelled with radioactive isotopes
and non-radioactive labelled DIG
Ø Random
prime labelling of DNA fragment (double or single stranded DNA was developed by
Fein Berg and Bolgesteinds an alternative to nicker translation to produce
uniformly labelled probes.
Ø Double
stranded DNA is degenerated and aneled with random oligonucleotide primer.
Ø The
oligonucleotide primer too 5`―› 3` polymerase which synthesis labelled probe in
presence of labelled nucleotide pressure.
PCR DIG labelling: -
DIG DUTP is incorporated during PCR
cycle into the DNA strands amplified from the DNA target.
Photo biotin labelling: -
Photo biotin labelling is
a chemical reaction not an enzymatic one. Biotin and DIG can be ink to
nitrophenyl azido group that is converted by irradiation with UV or strong
visible light to a highly reactive nitrene. That can from stable covalent linkage with DNA
and RNA.
The material for photo
biotin labelling or more stable then the enzyme needed in niche translation or
oligonucleotide labelling and are less expensive and it is a method of choice
when large quantities of prob beet not very sensitive.
End labelling: -
End labelling of probes
for hybridization is mainly use to labelled oligonucleotide probe.
Rache biochemical have
been developed their method for labelling oligonucleotide with dig.
Ø The
3`end labelling of an oligonucleotide 14 t 100 oligonucleotide in length with
one residue of DIG-11-ddUTPper molecule.
Ø The
3` tailing reaction where terminal transferase add a mixture of unlabelled
nucleotide and DIG-11-duTP, producing a tail containing DIG-
Ø The
5`end labelling in a 2-step synthesis with 1st and amino linker
residue on the 5`end of the oligonucleotide, and then after purification
digoxigenin-N-hydroxy succinimide ester are covalently link to the 3`amino
residue.
Target format: -
1.
Solid support: -
A convenient format for the hybridization of DNA to gene probes or
oligonucleotide probes in immobilization of the target nucleic acid (DNA or
RNA) onto a solid support with the probe is free in the solution.
the
solid support can be nitrocellulose or nylon membrane later or magnetic bead or
microtiter plate.
Nitrocellulose
membrane are very commonly used and produce low background signal they can only
used when colorimeter detection will be performed and purpose positively
charged nylon membrane are become and they also ensured an optical signal to
noise ratio
2.
In solution: - Both
the probes and target are in solution because both are free to move, the
chances of reaction are maximum and therefore this format is generally used
faster than other.
3.
In sites: -
In this format the probe solution in added to fixed tissue section are smears
which are then usually examine unlabelled the microscope.
The
probe labelled ( a fluorescent marker) produce visible change in specimen if
the target sequence is present and hybridization is occurred.
However
the sensitivity to be low if the amount of fragment nucleic acid present in
specimen is low. This can be used to gene mapping of chromosome and for the
detection for micro organism in specimen.
Application: -
Ø
The
application of nucleic acid probe has particular been evident in microbial
ecology where probe can be used to detect the unculturable micro-organism and
pathogen in the environment simple provide.
(i.e. Detection of pathogenic microorganism)
Ø
Actinomycin,
Bacteroides, Borrelia, clostridium, campylobacteria, conidia, Hemophilia,
Helicobacter, Lactococcus, Unculturable Uncultivated species like marine
protobacteria and thermophilus.
Ø
Detection
of charge nucleotide sequence charge in certain gene sequence can cause
heritable disease such as cystic fibrous, muscular dystrophies, sickle cell
anemia they can be diagnosed probe detection.
Ø
Several
probes might be used to ensure hybridization to all target sequence.
Ø
Random
sequence repeat are usually 30-50bp length. Their size and distribution are
distinctive for an individual. They can be detect using nuclei acid probe and
PCR.
Ø
Detection
of random repeat sequence.
Ø
Detection
of tumor suppressor gene in human
Ø
Diagnosis
of papilloma virus.
Ø
Molecular
analysis of hepcidin resistance S.enterica.
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