1. OBJECTIVE
This experiment allows learning how fluorescent in situ hybridization (FISH) and comparative genomics (CGH) techniques are performed on chromosomes. In addition, it also allows to know the array comparative genomic hybridization technique (array CGH).
At the end of this experiment, you should be able to:
identify the distribution pattern of fluorescent in situ hybridization, SKY-type fluorescent in situ hybridization and comparative genomic hybridization in chromosomes;
identify the distribution pattern of array comparative genomic hybridization in DNA molecules;
perform fluorescent in situ hybridization, comparative genomic hybridization, and array comparative genomic hybridization experiments;
identify errors during the experiment.
2. WHERE TO USE THESE CONCEPTS?
Fluorescent in situ hybridization allows localization of repetitive or non-repetitive sequences of interest within the genome of an organism when in the form of dividing chromosomes. A variation of this technique is the so-called spectral karyotype, known as SKY, which allows mapping several probes within the karyotype of the species of interest in a single hybridization experiment. It is used in the detection of interchromosomal and intrachromosomal rearrangements and in distribution patterns of microsatellites and genetic diseases. In addition, comparative genomic hybridization and array comparative genomic hybridization tools are widely used in oncological and clinical cytogenetic laboratories in the detection of tumors, tumor markers and gene alterations.
3. THE EXPERIMENT
The experiments carried out in this practice will be: fluorescent in situ hybridization (FISH); spectral karyotype (SKY); comparative genomic hybridization (CGH); and array comparative genomic hybridization (array CGH). In all of them, detection of the target DNA will be sought by means of fluorescence. For this, you will use, among other materials: graduated pipette; thermal cycler; different colored fluorochromes (eg, CY3 and CY5); chromosome solution; blades; stove; and fluorescence microscope. To perform hybridization, known sequences of DNA or chromosomes are laid out on a slide and denatured. Soon after, the probe of interest is joined to this denatured genetic material and detected in microscopy by the emission of fluorescent signals from the probe in one color, and from the target DNA in another.
4. SECURITY
This practice can offer risks to the professional who performs it. Therefore, throughout the experiment, the following personal protective equipment (PPE) must be used: gloves, lab coat, closed shoes, mask and goggles. Such measures are necessary due to the toxic potential that the reagents (eg, formamide, methanol and acetic acid) present.
5. SCENARIO
The experiment environment should be a complete genetics and cytogenetics laboratory. In the absence of this, the practice can be carried out in an environment with benches, running water, oven, refrigerator, freezer and optical fluorescence microscope.
This experiment allows learning how fluorescent in situ hybridization (FISH) and comparative genomics (CGH) techniques are performed on chromosomes. In addition, it also allows to know the array comparative genomic hybridization technique (array CGH).
At the end of this experiment, you should be able to:
identify the distribution pattern of fluorescent in situ hybridization, SKY-type fluorescent in situ hybridization and comparative genomic hybridization in chromosomes;
identify the distribution pattern of array comparative genomic hybridization in DNA molecules;
perform fluorescent in situ hybridization, comparative genomic hybridization, and array comparative genomic hybridization experiments;
identify errors during the experiment.
2. WHERE TO USE THESE CONCEPTS?
Fluorescent in situ hybridization allows localization of repetitive or non-repetitive sequences of interest within the genome of an organism when in the form of dividing chromosomes. A variation of this technique is the so-called spectral karyotype, known as SKY, which allows mapping several probes within the karyotype of the species of interest in a single hybridization experiment. It is used in the detection of interchromosomal and intrachromosomal rearrangements and in distribution patterns of microsatellites and genetic diseases. In addition, comparative genomic hybridization and array comparative genomic hybridization tools are widely used in oncological and clinical cytogenetic laboratories in the detection of tumors, tumor markers and gene alterations.
3. THE EXPERIMENT
The experiments carried out in this practice will be: fluorescent in situ hybridization (FISH); spectral karyotype (SKY); comparative genomic hybridization (CGH); and array comparative genomic hybridization (array CGH). In all of them, detection of the target DNA will be sought by means of fluorescence. For this, you will use, among other materials: graduated pipette; thermal cycler; different colored fluorochromes (eg, CY3 and CY5); chromosome solution; blades; stove; and fluorescence microscope. To perform hybridization, known sequences of DNA or chromosomes are laid out on a slide and denatured. Soon after, the probe of interest is joined to this denatured genetic material and detected in microscopy by the emission of fluorescent signals from the probe in one color, and from the target DNA in another.
4. SECURITY
This practice can offer risks to the professional who performs it. Therefore, throughout the experiment, the following personal protective equipment (PPE) must be used: gloves, lab coat, closed shoes, mask and goggles. Such measures are necessary due to the toxic potential that the reagents (eg, formamide, methanol and acetic acid) present.
5. SCENARIO
The experiment environment should be a complete genetics and cytogenetics laboratory. In the absence of this, the practice can be carried out in an environment with benches, running water, oven, refrigerator, freezer and optical fluorescence microscope.
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