What is random priming?|What is non radioactive Labelling?|What are random primers used for?

          

              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 1<sup>st</sup> 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.