Why is the hybridization probe said to be complementary

Probe - a single-stranded nucleic acid that has been radiolabelled and is used to identify a complimentary nucleic acid sequence that is membrane bound The hyrbridization process involves two different steps. First the nucleic acid must be immobilized on a filter.

This is generally called a "Southern Transfer" procedure. The second step is the actual hybridization of the probe to the filter bound nucleic acid. The following steps describe the Southern transfer procedure. Digest DNA with the restriction enzyme of choice. Load the digestion onto a agarose gel and apply an electrical current.

The distance a specific fragment migrates is inversely proportional to the fragment size. Transfer the DNA to a filter membrane nylon or nitrocellulose by capillary action. Typically a Southern transferst setup contains from bottom to top : buffer sponge filter paper the gel containing the nucleic acid a nylon or nitrocellulose membrane more filter paper paper towels to catch the buffer that passed through all of the above Southern hybridizations with plant DNA is not a trivial matter.

The primary requirement for a successful experiment is that the DNA to be probed is digested to completion. We have already discussed the choice of enzymes in this regard. Even when using compatible enzymes not GC or GXC sensitive monitoring the completeness of the reaction is essential for consistent results.

Once you are satisfied that you completely digested the DNA and are confident that it was successfully transferred to the filter membrane, the next step is perform the actual hybridization. The following steps describe the procedure.

Steps in Southern Hybridization Procedure Prepare a probe by nick translation or random, oligo-primed labelling. Add the probe to a filter nylon or nitrocellulose to which single-stranded nucleic acids are bound. The filter is protected with a prehybridization solution which contains molecules which fill in the spots on the filter where the nucleic acid has not bound.

Hybridize the single-stranded probe to the filter-bound nucleic acid for 24 hr. Today, cytogeneticists are able to use extensive HGP clone resources to precisely identify the sites of chromosomal rearrangements that appear in karyotypes.

At least one clone is available for every megabase segment of chromosomal DNA. The only exception is the Y chromosome , because it is relatively gene-poor. McNeil, N. Novel molecular cytogenetic techniques for identifying complex chromosomal rearrangements: technology and applications in molecular medicine.

Expert Reviews in Molecular Medicine online: September All rights reserved. The detection of chromosome rearrangements with site-specific probes Figure 2b can be a lengthy endeavor, especially if complex rearrangements have occurred or if the rearranged regions are difficult to identify by their banding patterns in a karyotype.

Fortunately, cytogeneticists now have the option of using multifluor FISH, or spectral karyotyping , to quickly scan a set of metaphase chromosomes for potential rearrangements Speicher et al. Multifluor FISH generates a karyotype in which each chromosome appears to be painted with a different color.

Each "paint" is actually a collection of hybridization probes for sequences that span the length of a particular chromosome. In Figure 3a, the probe chromosomes have been physically separated from one another by flow cytometry. Today, investigators would probably use commercially available DNA collections for each chromosome.

In the next step, the DNA samples are labeled with combinations of fluorochromes that produce a unique color for each chromosome. The fluorescent hybridization probes are then combined with and hybridized to metaphase chromosomes. Figure 3b shows images of interphase and metaphase chromosomes as they would appear through a microscope after hybridization.

To human eyes, several of the metaphase chromosomes appear to have the same color, but digital processing of the image would distinguish spectral differences between the chromosomes. A normal human chromosome Figure 3b will have a uniform color along its length, but a rearranged chromosome will have a striped appearance. Although chromosome paints allow rapid assessment of large chromosomal changes in metaphase spreads, the resolution of the method is limited.

If investigators need more detailed information about the actual sequences involved in chromosomal rearrangements, they need to follow up with site-specific probes, as previously described Figure 2. Since the introduction of FISH, cytogeneticists have been able to analyze interphase chromosomes as well as the metaphase chromosomes used in karyotypes Trask, This offers a real practical advantage, in that cells do not need to be cultured for several days or weeks before chromosomes can be prepared for analysis.

In addition, FISH can be used to analyze chromosomes from specimens such as solid tumors, which are of great clinical interest but do not divide frequently. Another useful feature of FISH is that researchers are able to simultaneously monitor multiple sites if the hybridization probes have been labeled with different fluorophores. Figure 4 shows two examples of how interphase FISH can be used to diagnose chromosome abnormalities.

CMT type 1A is a relatively common neurological condition caused by a duplication in a gene on chromosome 17 that encodes one of the proteins in the myelin sheath that surrounds nerve axons. In Figure 4a, the patient's cell has been hybridized with a red-labeled probe corresponding to a sequence within the duplicated region, along with a green probe corresponding to a sequence on chromosome 17 that lies outside of the duplicated region.

From the two green signals, it is possible to locate two copies of chromosome 17 within the nucleus. One chromosome has the normal configuration , while the second, der 17 , contains the duplicated region, which is evident from two nearby red signals. The figure also serves to illustrate another important feature of interphase FISH.

Because interphase chromatin is about 10, times less compacted than mitotic chromatin, it is possible to resolve the duplicated regions on der 17 as discrete points.

This small duplication would have been difficult to resolve in mitotic chromosomes. Figure 4b shows a FISH analysis that was used to detect the presence of a chromosomal translocation in a patient suffering from chronic myelogenous leukemia Tkachuk et al. In most cases of this disease, a segment of chromosome 9 that contains the ABL proto-oncogene fuses with the breakpoint cluster region BCR on chromosome 22 during a reciprocal translocation.

In this image, the normal copies of chromosomes 9 and 22 are detected as red and green spots, respectively. On the other hand, the Philadelphia chromosome is visible as a complex fused spot, which appears to have a central yellow region with red and green subregions on either side.

In fluorescence microscopy, yellow is indicative of very close proximity of red and green probes, such that they appear to overlap. The intricate substructure of the fused spot is detectable in interphase chromosomes, but it would not be resolved in a similar FISH analysis of metaphase chromosomes.

Thus, two-color interphase FISH provides a sensitive method for analyzing chromosome fusion events without the need for a prior cell culture. Another research application of interphase FISH makes use of chromosome-specific paints to obtain information about the organization of chromosomes within the nucleus. Figure 2a upper left shows an interphase nucleus that has been stained with chromosome-specific paints. One can see from the figure that the chromosomes occupy distinct territories within the nucleus.

By creatively combining chromosome-specific probes with gene-specific probes and antibodies, investigators can use FISH to provide exciting new insights about nuclear architecture. Exciting new applications of FISH that extend its range continue to be developed. For example, cytogeneticists now use comparative genomic hybridization to detect quantitative differences, like copy number variations, in the chromosomes of their patients.

With tools such as these, cytogenetics has been able to move from studying whole chromosomes on the macroscopic scale , to studying the DNA of which these chromosomes consist. BAC Resource Consortium. Integration of cytogenetic landmarks into the draft sequence of the human genome.

Nature , — link to article. Gall, J. Proceedings of the National Academy of Sciences 63 , — Lupski, J. Cell 66 , — doi Parra, I. High resolution visual mapping of stretched DNA by fluorescent hybridization.

Nature Genetics 5 , 17 — 21 doi Rudkin, G. Nature , — doi Schrock, E. Multicolor spectral karyotyping of human chromosomes. Science , — doi Speicher, M. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nature Genetics 12 , — doi The new cytogenetics: Blurring the boundaries with molecular biology.

Nature Reviews Genetics 6 , — doi Tkachuk, D. Trask, B. Human cytogenetics: 46 chromosomes, 46 years and counting. Nature Reviews Genetics 3 , — doi Watson, J. Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid.

Chromosome Mapping: Idiograms. Human Chromosome Translocations and Cancer. Karyotyping for Chromosomal Abnormalities. Prenatal Screen Detects Fetal Abnormalities. Synteny: Inferring Ancestral Genomes. Telomeres of Human Chromosomes.

Chromosomal Abnormalities: Aneuploidies. Chromosome Abnormalities and Cancer Cytogenetics. Copy Number Variation and Human Disease. Genetic Recombination. Human Chromosome Number.

Trisomy 21 Causes Down Syndrome. X Chromosome: X Inactivation. Chromosome Theory and the Castle and Morgan Debate. Developing the Chromosome Theory. Meiosis, Genetic Recombination, and Sexual Reproduction. Mitosis and Cell Division. Genetic Mechanisms of Sex Determination. Sex Chromosomes and Sex Determination. Sex Chromosomes in Mammals: X Inactivation. Sex Determination in Honeybees. Citation: O'Connor, C. Nature Education 1 1 Cytogeneticists can now go "FISH-ing" for chromosomal abnormalities, which are deletions and duplications that can cause disease.

How exactly does FISH work? Aa Aa Aa. Fluorescent Probes Are Introduced. Two labeling strategies are commonly used: indirect labeling left panel and direct labeling right panel.

For indirect labeling, probes are labeled with modified nucleotides that contain a hapten, whereas direct labeling uses nucleotides that have been directly modified to contain a fluorophore.