DNA-RNA hybridization
DNA-RNA hybridization occur when a mixture of DNA and RNA is heated to denaturation
temperatures to form single strands and then cooled RNA can hybridize (form a double helix)
with DNA that has a complementary nucleotide sequence.
Useful for determining relationships between DNAs and RNAs.
Process of creating a hybrid strand of DNA/RNA
The two strands of a DNA molecule are denatured by heating to about 100°C = 212°F (a to
b). At this temperature, the complementary base pairs that hold the double helix strands
together are disrupted and the helix rapidly dissociates into two single strands.
The DNA denaturation is reversible by keeping the two single stands of DNA for a
prolonged period at 65°C = 149°F (b to a). This process is called DNA renaturation or
hybridization.
Similar hybridization reactions can occur between any single stranded nucleic acid chain:
DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript is introduced during the
renaturation process, the RNA competes with the coding DNA strand and forms doublestranded DNA/RNA hybrid molecule (c to d).
These hybridization reactions can be used to detect and characterize nucleotide sequences
using a particular nucleotide sequence as a probe.
In situ hybridization
In situ Hybridization(ISH) is a powerful method to localize nucleic acid sequences in vivo i.e.
in tissues, cells, organelles, nuclei or chromosomes by using appropriate probes. With ISH,
nucleic acids are localized in their original or proper place.
Basic technique:
1. The preparation of biological material that has to be investigated.
2. Probes are labeled.
3. Both probes target nucleic acid are denatured.
4. Single stranded probe gets hybridized to the region where it found sequences
complementary to it.
5. Hybridization is detected Hybridization is visualized.
Different types localized by ISH:
Different types localized by ISH DNA sequences :1. RNA sequences
2. Viral sequences
3. Repetitive seq.
4. Unique seq.
Probe:
• Key feature of DNA probes is that they are STOICHIOMETRIC - thus number of
molecules of probe bound is equivalent to number of molecules of DNA
DNA or RNA seq. which is labeled and used to probe target nucleic acid.
Labelling Of Probes…:
Chemical labelling and Enzymatic labelling: Acetylaminofluorine, Mercury, Biotin,
Digoxigenin
Labels
Radioactive labels and Non-radioactive labelsare used.
1. Radioactive labels are the isotopes which emit β- particles and are detected by
autoradiography . E.g. 35 S , 32 P , 3 H
2. Non-radioactive labelling procedures are of two types:- Direct ISH and Indirect ISH
Non-radioactive ISH procedures :
Direct ISH label is incorporated directly into nucleic acid probe so that hybridization site
could be visualized immediately after hybridization.
Indirect ISH label second molecule in the probe cannot be detected immediately after
hybridization . A reporter molecule is conjugated required to detect the label in probe .
Non-radioactive Labels:
Biotin, Digoxigenin, Acetylaminofluorene, Mercury
Reporter Molecules in non-radioactive ISH:
1. Biotin is detected by avidin or streptavidin
2. Digoxigenin is detected by anti-digoxigenin antibodies.
3. AAF is detected by anti-acetylaminofluorene antibodies.
4. Mercury is detected by ligands having an immunogenic group which can bind to same
antibody.
Signal Generating System:
1. Fluorochromes Enzymes Metals are used and they get excited by light of one wavelength
and emit light of another wavelength which is observed as fluorescence of different colors.
2. Enzymes work by catalyzing the precipitation of a visible product at hybridization site.
3. Colloidal gold which is conjugated to antibodies. Can be visualized with both light and
electron microscope
Enzymes:
Horseradish peroxidise, Diamino benzidine(DAB) ,Red Alkaline phosphatase 5-Bromo 4chloro 3-indolyl phosphate (BCIP) Blue
Fluorochromes :
Fluorescene isothiocyanate (FITC)
Blue Green Tetramethyl rhodamine isothiocyanate (TRITC)
Green Red Texas red or sulphorhodamine
Green Deep red Amino methyl coumarine acetic acid (AMCA) UV Blue
Multiple labeling :
In multiple labeling more than one probe can be employed simultaneously on target nucleic
acid.
This is very important: To determine the relationship of different sequences with respect to each other.
 To identify different chromosomes simultaneously.
 To identify different genomes simultaneously.
Types: 1. Sequential multiple labeling
2. Simultaneous multiple labeling- Indirect method and Direct method.