1. In situ hybridization
We like to locate the physical position of a known DNA sequence on a chromosome. This technique helps in the physical mapping of genes or repeated DNA sequences. In this technique, DNA within the cell is denatured by treating the cells that have been squashed on a coverslip. The squashed cells can then be incubated in a solution of labelled DNA whose position in a chromosome we are interested in knowing. Therefore repeated or unique DNA sequences, isolated from an organism or artificially synthesized, can be utilized as radioactive labelled or biotinylated probe, for the study of the location of these sequences on the chromosomes.
DNA ISH can be used to determine the structure of chromosomes.
- The tissues are treated to fix the transcript so that it could be easily accessed by the probe. (The probe is labelled complementary DNA or most complementary RNA <riboprobe>).
- The probe hybridizes to the target sequence at higher temperatures.
- This transcript can be detected by detecting the labelled hybridized by various techniques such as autoradiography, fluorescence microscopy etc.
TYPES OF PROBES
- RNA probes –should be 250 to 1500 bases in length. Probes approximately 800 bases long exhibit the highest sensitivity and specificity.
- DNA probes- DNA probes can also provide high sensitivity for in situ hybridization but they not hybridize as strongly to target mRNA molecules as RNA PROBES
- Prenatal test during pregnancy – prenatal test for chromosomal abnormalities such as DOWN SYNDROME rely on analyzing the number and appearance of karyotype
- Molecular diagnostic techniques such as microarray, genomic hybridization
- PHARMACOGENOMICS – some of the patient’s slight differences in the DNA can help predict how quickly they will metabolize certain drugs.
- Also used in the diagnosis of certain diseases
2 FLUORESCENCE IN SITU HYBRIDISATION
In in-situ hybridization location of genes on chromosomes are detected by the use of radioactive/non-radioactive isotope followed by the staining solution.
However, in this, a fluorescent molecule can be deposited at the site of in-situ hybridization, if the reporter molecule has an affinity with the fluorescent molecule. The sites thus located will exhibit fluorescence and can be photographed with a fluorescent microscope. The technique is popularly known as FISH.
The gene identification or chromosome begins with the denaturation of the chromosome/ gene under investigation and the already known gene/ chromosome. The known genetic sequence is hybridized by the labelled antibody or fluorescent material which will be then hybridized with the investigatory sequence. The addition of the fluorophore will be aiding in visualization by the aid of epifluorescence microscopy. where fluorophore will bind to the secondary or known sequence and the region of the desired genetic sequence will be detected in a chromosome. In case if the desired sequence is not present in the investigatory sequence then the double-stranded sequence will not be visualized.
ADVANTAGES OVER IN SITU HYBRIDIZATION
- Specificity, sensitivity and speed
- A variety of probe labelling schemes are available for simultaneous detection of two or more sequences in the same nucleus
- Entire genomics, whole chromosomes, chromosome segments or single-copy sequences can be highlighted depending upon the complexity of the probe used
- Hybridization of repetitive sequences can be suppressed by pre-hybridization with unlabelled genomic DNA
- Used for studies involving structural and numerical changes in chromosomes or even for gene mapping.
- Multicolour IN –situ hybridization is a new development.
3. BANDING PATTERNS FOR THE IDENTIFICATION OF CHROMOSOMES AND CYTOGENETIC MAPS
A cytogenetic map is the visual appearance of a chromosome when stained and examined under a microscope. Particularly important are visually distinct regions, called light and dark bands, which give each of the chromosomes a unique appearance. This feature allows a person’s chromosomes to be studied in a clinical test known as a karyotype, which allows scientists to look for chromosomal alterations.
With the possibility of more specific identification and detailed analyses of human chromosomes, a new phase in cytogenetics began. The first method for the visualization of a pattern of bands on human chromosomes was Q-banding. The banding technique is based on the identification of chromosome segments, that predominately consist of either GC or AT-rich regions or constitutive heterochromatin. This technique involves denaturation of DNA followed by slow renaturation, which permits the identification of constitutive heterochromatin because it mainly consists of repetitive DNA.