Disease diagnostic methods

conventional diagnostic methods:

Microscopy: we use microscopes for the detection of casual organisms in various specimens
such as tissue, cells. Body fluids, excreta etc. example we go for stool examination to detect
the presence of ova or cyst.
Culturing methods: we will culture the organism on a media and check its susceptibility to a
drug.
Immunological assays: these are used to detect the presence of antigen in a sample or to
detect the presence of antibodies in response to these antigens in our body

these conventional methods are very tedious and time consuming and cannot be applicable in
certain cases such as detection of antibodies in case of a latent viral infection. Novel
diagnostic methods have now been developed. These come under molecular diagnostic
methods.

PROBES : these are small nucleotide sequences usually 15- 30 basses long and are used to
detect the presence of complementary sequences in nucleic acids samples. Both DNA and
RNA can be used .single stranded DNA are more convenient and preferable. But denatured
double stranded DNA can also be used as probes

preparation of probes
1. Highly purified samples of mRNA can be used as they are naturally single stranded.
2. Single stranded RNA can be prepared by cloning methods. Corresponding DNA sequences
are first inserted into special vectors such as GEM and then recombinant vector is linearized
and transcribed with RNA polymerase to obtain RNA molecules.
3. Double stranded DNA probes can be prepared by isolating them from the desired organism
and then cloning them in E.Coli. Single stranded DNA probes can be prepared by cloning
them in vectors such as Phage M13 vector of E.Coli.
4. We can prepare single stranded c DNA by limiting the copies of mRNA by reverse
transcriptase to one strand.
5. Probes can be synthetically prepared by the use of PCR techniques.

labeling of probes
can be done radioactively by P32 or non radioactively by biotin, digoxygenin or fluorescent
molecules.
Disadvantage of radioactive labeling:
1.have very short life span
2. Problem in handling and disposing of probes
3. To detect radioactive label we require auto radiography which is a very time consuming
technique
advantage : filter can be used again
Northern Blotting.