Designing probe ?

Designing probe: -

1.     A probe is a nucleic acid molecule (single stranded DNA or RNA) with a strong affinity with a specific target

2.     Probe and target base sequence must be compliment to each other beet depending on the conditions the do not necessarily have to be complementary.

3.     Probe design depends upon whether a gene probe an oligonucleotide probe is desired.

Gene Probe: -

Gene probe are generally longer than 500 bp and comprise all or most of the target gene. They can be generated in two ways.

Clone Gene Probe: -

-        Clone probe are generally used when clone species is available or when DNA sequence is unknown must be done first in order to be map and sequence.

-        It is usual to cut the gene with restriction enzyme and excise it from and agarose gel, although if the vector has no this might not be necessary.

PCR: -

-        PCR is α powerful procedure for making gene probe because it is possible to amplify and label at the same time.

-        Long starches of DNA using chromosomal or plasmid DNA template.

-        Labelled nucleotide molecule in extension step.

-        A considerable amount of time, can be sequence when the gene of interest is PCR amplified, for there is no need for restriction enzyme digestion, Electrophoresis, elusion of DNA fragment from vectors.

-        However, if the PCR amplification gives non-specific bond that will be used as a probe.

Oligonucleotide Probe: -

-        Most common oligo robe contain 18-30bp.

-        But current synthesis allows efficient synthesis of probe containing at least 100bp.

-        Oligo probe can match perfectly to its target sequence sufficiently long to allow the use of hybridization condition that that will present the hybridization to other closely related sequence.

-        Making it possible to identify and detect DNA with slight differences in sequence with in highly conserved gene. The selection of oligo probe sequence can be done manually from a known gene sequence using following gaudiness.

Guidelines : -

-        The probe length should between 18 to 50 bases longer probe with result longer hybridization time and low synthesis yield.

-        The base composition should be 40 to 60% GC nonspecific hybridization may increase for GC ratio outside of this range.

-        Be certain that no complementary regions may present within the probe. This may result the formation of hairpin structure that will inhibit hybridization with target.

-        Avoid sequence containing long stretches of a single base

-        Once a sequence making the above criteria has been identified computerized sequence analysis is highly recommended.

-        The probe sequence should be compared with the sequence region or genome from which it was derived as well as to the reverse complement of the region.

-        It homology to non-target regions are grater than 70% or more bases in a row are found that probe sequence should not be used.

Random prime labeling (Prime extension)

A: Double stranded DNA is denatured and annealed with random oligonucleotide primers. (the oligonucleotide serves as a primer for 5’ 3’ fragment of E. coli) DNA polymerase which synthesis labelled probe in the presence of DIG-UTP.

B: In PCR-DIG labeling DIG-UTP is denatured and annealed with random oligonucleotide primer for the 5’à3’ polymerase. (The know fragment of E. coli polymerase I ) Which synthesis a labelled probe in presence of labelled nucleotide precursor.