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Fluorescent in situ hybridization (fish) assay
- 1. Fluorescent In Situ Hybridization (FISH) Assay
- 2. Content Definition, Principle and Sample Types What is FISH？ The core of FISH technology Probes A quick and simple FISH protocol FISH Procedure Wide range of uses and bright prospects Application 1 2 3 4
- 3. 输入标题文本 What is Fluorescent in situ hybridization (FISH) ? • Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity. • It is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. Definition
- 4. 输入标题文本 FISH works by exploiting the ability of one DNA strand to hybridise specifically to another DNA strand. How does FISH work ? The Structure of DNA The DNA contains two strand-like molecules coiled together into a structure known as a double helix. The principle of complementary base pairing When two complementary sequences find each other they will bind together, or hybridise.
- 6. 输入标题文本 Centromere probes • Alpha and Satellite III probes • Generated from repetitive sequences found in centromeres • Centromere regions are stained brighter • Collection of probes that bind to the whole length of chromosome • Multiple probe labels are used • Hybridize along the length of the chromosome • Specific for telomeres • Specific to the 300 kb locus at the end of specific chromosome • Deletion • Translocation probes • Gene detection & localization probes • Gene amplification probes Telomere Whole chromosome Locus Probes What Kind of Probes Can Be Used？
- 7. 输入标题文本 dsDNA probes Stable, available, easier to obtain Stable, easier to work with, more specific, resistant to RNases, better tissue penetration, without self- hybridize • Higher thermal stability, • Better tissue penetration, • More specific, • Low background noise by RNase •Economical, stable, available, easier to work with, •more specific, resistant to RNases, •better tissue penetration, better reproducibility. RNA probes ssDNA probes Synthetic oligonucleotides probes Probes Characteristics of Different Probes Types
- 8. 输入标题文本 • 32P • 35S • 3H • Biotin • Digoxigenin • Fluorescent dy Probes Labeling
- 9. 输入标题文本 Denaturation of the probe and the target Hybridization of the probe and the target Preparation of the fluorescent probes Protocol Outline
- 10. 输入标题文本 Sample Types • Fixed cell suspension • Formalin fixed paraffin embedded tissues The Sample Preparation and Sample Types Preparation • Sample fixation • Slide tissue
- 11. 输入标题文本 Preparation of the fluorescent probes • Commercial Probes can be provided by many biotech companies • Based on your special needs, custom probes are also synthesized • e.g. 𝒂-satellite DNA is often chosen as the source of centromeric probes.
- 12. 输入标题文本 Denaturation of the probe and the target Dehydration (1) Place the suitable amount of fixed sample on the slide, (2) put into 46°C oven drying for 10 minutes (3) Followed by immersion 50%, 80%, 96% ethanol solution, each for 3 minutes (4) dry in the air Hybridization (1) Add 10 μl of Hybridization buffer to the sample on the glass plate and try to cover the entire sample (2) Add 1 μl probe to Hybridization buffer (3) Put a wet absorbent paper in a 50ml centrifuge tube, put the glass pieces in, then place them in a 46°C incubator for 1.5 hours (4) Preheat the Washing buffer to 48°C for the next step
- 14. 输入标题文本 1Morphology Morphology and population structure of microorganisms 2Patholog y Pathogen profiling, abnormal gene expression 3 Developmental biology Gene expression profiling in embryonic tissues 4 Karyotyping and phylogenetic analysis Unique FISH patterns on individual chromosomes, chromosomal aberrations Applications
- 15. 输入标题文本 Products FISH Services In addition to products, high-quality services are also available for our clients. We provide comprehensive commercial probes and FISH Kits for easy to use. Creative Bioarray
- 16. For more info please contact us: E-mail: info@creative-bioarray.com Go to our website： www. creative-bioarray.com
Notas del editor
- Here are four parts. What is FISH? The probes ,the procedure of FISH and the applications.
- Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.
- The DNA contains two strand-like molecules coiled together into a structure known as a double helix. The bases in each strand are able to bind to each other and hold the DNA together. When two complementary sequences find each other they will bind together, or hybridise. FISH works by exploiting the ability of one DNA strand to hybridise specifically to another DNA strand.
- FISH works by exploiting the ability of one DNA strand to hybridise specifically to another DNA strand.FISH uses small DNA strands called probes that have a fluorescent label attached to them. The probes are complementary to specific parts of a chromosome. When DNA is heated, the patient’s two DNA strands break apart, or denature, and the probes are able to hybridise to their complementary sequence in the patient’s DNA. If a small deletion is present in the region complementary to the probe, the probe will not hybridise. If a duplication is present, more of the probe is able to hybridise.
- Probe is critical to in situ hybridization, and a right probe can help you achieve your goals. Not only the probe types but also the label of probe should you take into account when you choose a probe for in situ hybridization.
- Different probes have different characteristics. dsDNA probes are stable and easier to obtain. ssDNA probes are more specific and better in resistanting to Rnases. For RNA probes, they have higher thermal stability and better tissue penetration.
- There are four steps of FISH:Preparation of the fluorescent probes; Denaturation of the probe and the target; Hybridization of the probe and the target; Detection.
- The samples of FISH are two categories: fixed cell suspension; Formalin fixed paraffin embedded tissues. Sample predation includes sample fixation and slides.
- Probe is critical to in situ hybridization, and a right probe can help you achieve your goals. Not only the probe types but also the label of probe should you take into account when you choose a probe for in situ hybridization.
- When you get the slides，the next steps are dehydration and hybridization.
- And the last step is detection。Fluorescence in situ hybridization, as an important biological experimental technique, it features in situ and without PCR, and can be used for quantitative analysis of specific microorganisms in environmental samples.
- FISH can be used for different areas，such as morphology，pathology，developmental biology，and karyotyping and phylogenetic analysis.
- Creative Bioarray provides comprehensive commercial probes and FISH Kits for easy to use. In addition, high-quality services are also available for our clients.
- We are looking forward to cooperating with you!