Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional
FISHFISH is a cytogenetic technique developed by biomedical researchers in the early 1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high...
methodology. In Q-FISH, the technique uses labelled (
Cy3Cyanine is a non-systematic name of a synthetic dye family belonging to polymethine group. Cyanines have many uses as fluorescent dyes, particularly in biomedical imaging...
or
FITCFluorescein isothiocyanate is a derivative of fluorescein used in wide-ranging applications including flow cytometry. FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group , replacing a hydrogen atom on the bottom ring of the structure...
) synthetic
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
mimics called peptide nucleic acid (PNA) oligonucleotides to quantify target sequences in
chromosomalA chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
DNA using
fluorescent microscopyA fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
and analysis software. Q-FISH is most commonly used to study
telomereA telomere is a region of repetitive DNA sequences at the end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. Its name is derived from the Greek nouns telos "end" and merοs "part"...
length, which in vertebrates are repetitive hexameric sequences (TTAGGG) located at the distal end of chromosomes. Telomeres are necessary at chromosome ends to prevent DNA-damage responses as well as
genome instabilityUsually, all cells in an individual in a given species show a constant number of chromosomes, which constitute what is known as the karyotype defining this species , although some species present a very high karyotypic variability.Sometimes, in a species with a stable karyotype, random variations...
. To this day, the Q-FISH method continues to be utilized in the field of telomere research.
PNAs and FISH
Due to the fact that PNA backbones contain no charged phosphate groups, binding between PNA and DNA is stronger than that of DNA/DNA or DNA/RNA duplexes. Q-FISH utilizes this unique characteristic of PNAs where at low ionic strengths, PNAs can anneal to complementary single-stranded DNA sequences while single-stranded DNA cannot. By using conditions that only allow labeled (CCCTAA)
3 PNA to hybridize to (TTAGGG)
n target sequences, Q-FISH is able to quantify the hybridization of PNAs to telomeric sequences.
General method/protocol for cultured cells
Prepare metaphase-arrested cells
Several hours prior to harvesting the
culturedTissue culture is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar...
cells,
colcemidUsed in cytogenetics, colcemid, also known as demecolcine, is related to colchicine but it is less toxic. It depolymerises microtubules and limits microtubule formation , thus arresting cells in metaphase and allowing cell harvest and karyotyping to be performed.-Mechanism of Action:Colcemid is a...
is added to the culture medium. Colcemid acts to arrest cells in the
metaphaseMetaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
state by disrupting microtubules in
mitoticMitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
cells. Cells are then
trypsinTrypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when...
ized and resuspended in a
hypotonicTonicity is a measure of the osmotic pressure gradient of two solutions separated by a semipermeable membrane. It is commonly used when describing the response of cells immersed in an external solution...
buffer. This will swell the collected cells and spread the chromosomes.
Fix cells
The hypotonic solution is then removed by centrifugation and resuspended in a methanol/glacial acetic acid fixative.
Prepare slides
Place a few drop of the cell suspension onto a microscope slide and let air dry overnight. The following day, immerse the slide in PBS for several minutes.
Fix slides in formaldehyde
Transfer the slides into a 4%
formaldehydeFormaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...
solution and
fixIn the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...
for several minutes. Wash slides several times with PBS.
Treat slides with pepsin
Slides are then transferred into a
pepsinPepsin is an enzyme whose precursor form is released by the chief cells in the stomach and that degrades food proteins into peptides. It was discovered in 1836 by Theodor Schwann who also coined its name from the Greek word pepsis, meaning digestion...
solution. Pepsin is a
proteaseA protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
and acts to digest proteins into peptides.
Hybridize PNA probe (Cy3 or FITC labelled PNAs)
A small volume of the hybridization mixture is placed onto a coverslip and then placed gently onto the microscope slide which contains the fixed cells.
Heat denature DNA
The slide is then placed into a preheated oven where the chromosomal DNA in the cell is
denaturedDenaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
at 80°C for several minutes. The slide is then left at room temperature for several hours to allow the PNA to hybridize to complimentary DNA.
Wash slides to remove unbound PNAs and counterstain DNA (DAPI or PI)
Slides are then carefully washed in various wash solutions to remove unbound PNA. Microscope mounting medium is then placed onto the cells. This medium generally contains
DAPIDAPI or 4',6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to A-T rich regions in DNA. It is used extensively in fluorescence microscopy...
(a DNA counterstain) and an antifade solution to preserve the PNA fluorescence and reduce
photobleachingPhotobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing...
.
Image capture and analysis
Before experimental samples are imaged, fluorescent reference beads are imaged in order to ensure the proper set-up of the camera and fluorescent microscope. In addition, these reference beads will be imaged prior to every acquisition session. This will ensure that the differences between samples are not due to errors in the lamp or camera. A metaphase cell is then manually selected and centered for the camera. Two types of images are taken: pictures of the stained chromosomes in their metaphase state and fluorescent images of the telomeres. The two images can then be superimposed to generate a combined image. This image can then be
karyotypeA karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.p28...
d or assigned nomenclature. Furthermore, the intra-chromosomal distribution of telomere length in
p-arms versus q-armsIn the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
can be measured.
Data from different experiments may be used to normalize the fluorescence intensity while plasmids with a known number of telomeric repeats can be used as standards to help relate telomere fluorescence and telomere length. In addition to the fluorescent reference beads, signal strength from sister chromatids should be equal and therefore can be used as another control to gauge the precision of the data. Lastly, it is important the images are not saturated. If the fluorescence intensity reaches saturation, telomere lengths become underestimated. Q-FISH image analysis software is available for free from the Flintbox Network at
http://www.flintbox.com.
Applications and significance
Q-FISH has been used extensively to quantitate information regarding telomere length distribution and associating it with various illnesses. In this context, Q-FISH is particularly relevant because it is able to detect and quantify critically short telomeres. It has been shown that it is the frequency of these critically short telomeres, rather than the average telomere length, that is important in telomere dysfunction.
While Q-FISH supplies accurate information about telomere length, its relevance can be extended by combining Q-FISH with other FISH related techniques, such as
flow-FISHFlow-FISH is a cytogenetic technique to quantify the copy number of specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols...
. In flow-FISH,
flow cytometryFlow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
is utilized to measure fluorescence intensity (and thus telomere length) in a large population of cells rather than just a handful of cells in Q-FISH. Conversely, unlike Q-FISH, flow-FISH is unable to determine telomere length in a particular chromosome within an individual cell. However, it should be noted that although Q-FISH is generally considered low-throughput and not suitable for population studes, groups have developed high-throughput (HT) Q-FISH protocols that use automated machinary to perform Q-FISH on interphase nuclei in 96well plates.
Similarly, other methods like multiplex-FISH and cenM-FISH have been developed which can also be used in conjunction with Q-FISH. Multiplex-FISH uses a variety of probes to visualize the 24 chromosomes in different colours and identify intra- or inter-chromosomal rearrangements. Centromere-specific multi-colour FISH (cenM-FISH) uses the multi-coloured probes from multiplex-FISH as well as
centromereA centromere is a region of DNA typically found near the middle of a chromosome where two identical sister chromatids come closest in contact. It is involved in cell division as the point of mitotic spindle attachment...
specific labeled probes to identify and distinguish centromere regions. The relation between centromere abnormalities or chromosomal rearrangements and telomere length may have high clinical impact, since all appear important in pre- or post-natal diagnostics and tumor developments. These experiments can provide more enlightenment about the role of telomeres and the importance of telomere length.
Another application of Q-FISH is the detection of telomeric fusions, where the ends of chromosomes have been fused together at the telomere, which are sometimes called interstitial (within the chromosome) telomeric sequences (ITSs). Studying telomeric fusions can sometimes show the course of evolution. For example, one human chromosome has an ITS that is hypothesized to be the equivalent of two chromosomes in chimpanzees that fused together. Observing the regulation of telomere length in different species also reveals important information about karyotype evolution and relevance to human illnesses.
In another example, the
non-homologous end joiningNon-homologous end joining is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide...
(NHEJ) protein repairs double-stranded DNA breaks and relies on the Ku70/Ku80
heterodimerIn biochemistry, a dimer is a macromolecular complex formed by two, usually non-covalently bound, macromolecules like proteins or nucleic acids...
to function. Disrupting these proteins causes telomeric shortening, which can be observed by measuring telomere length with FISH. For example, in mice lacking the Ku 80 gene, the telomere lengths are measured by qFISH and are observed to be significantly shorter.
Q-FISH is commonly used in
cancerCancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
research to measure differences in telomere lengths between cancerous and non-cancerous cells. Telomere shortening causes genomic instability and occurs naturally with advanced age, both factors that correlate with possible causes of cancer.
Advantages of Q-FISH
The greatest advantage of Q-FISH over other FISH techniques is the quantitative ability of the technique. Compared to traditional FISH which uses DNA probes, quantitative information is difficult to acquire because the hybridization probes compete with the renaturation of complimentary genomic DNA strands. Therefore, by using PNAs and hybridizing them under very stringent conditions, it allows one to overcome this issue. Similarly, because one is able to denature the chromosomal DNA in the presence of the PNA probe,it simplifies the FISH procedure. In addition, the method provides greater resolution, allowing user to examine the telomere length of each individual chromosome (
p or q armIn the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
) in a particular cell. Also, unlike southern blots which need over 10
5 cells for a blot, less than 30 cells are needed in Q-FISH.
Drawbacks of Q-FISH
Despite its advantages, Q-FISH is quite labor intensive and is generally not suitable for high throughput analysis. The technique depends on well-prepared metaphase cells and it is vital that the equipment and samples are adjusted/normalized correctly in order for the quantification to be accurate. Also, while only a small quantity of cells are needed, it is difficult to get a sufficient number in metaphase at once. In addition, poor
chromosome morphologyA karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. The term is also used for the complete set of chromosomes in a species, or an individual organism.p28...
may result from overexposure to high temperatures during preparation. Similarly, if one is using different cell types, many of the steps in Q-FISH (such as the length of colcemid treatment) will require optimization.
A common problem in fluorescence microscopy is
photobleachingPhotobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing...
, where the
fluorophoreA fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
loses its activity as a result of exposure to light. This can lead to the inaccurate measurement of fluorescence intensity. Photobleaching, light source stability, and system variability are all sources of error but can be minimized if the user is able to reduce the acquisition time between samples and include the proper controls.
Classical technique
Prior to the development of Q-FISH and PNAs, the classical technique for measuring telomere length was the use of
Southern blotA Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
s. In this method, genomic DNA is digested using
restriction enzymeA Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s and separated by
gel electrophoresisGel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
. The DNA is then transferred onto a membrane and hybridized using radioactive or fluorescent telomeric DNA probes. However, this method is only able to evaluate average telomere length in a cell population and the presence of interstitial telomeric sequences in the genome can yield inaccurate measurements.
Flow-FISH
Similar to Q-FISH,
Flow-FISHFlow-FISH is a cytogenetic technique to quantify the copy number of specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols...
is an adaptation of Q-FISH that combines the use of PNAs with flow cytometry. In this method, Flow-FISH uses
interphaseInterphase is the phase of the cell cycle in which the cell spends the majority of its time and performs the majority of its purposes including preparation for cell division. In preparation for cell division, it increases its size and makes a copy of its DNA...
cells rather than
metaphaseMetaphase, from the ancient Greek μετά and φάσις , is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells...
chromosomes and hybridizes the PNA probes in suspension. Following hybridization, thousands of cells can be analyzed on a flow cytometer in a relatively short time. However, Flow-FISH only provides an average telomeric length for each cell whereas Q-FISH is able to analyze the telomere length of an individual chromosome.
PNA-FISH
Although the quantitative ability of Q-FISH is most commonly used in telomere research, other fields that only require qualitative data have adopted the use of PNAs with FISH for both research and diagnostic purposes. PNA-FISH assays have been developed for identifying and diagnosing infections diseases in a rapid manner within the clinic. Combined with traditional
gram stainingGram staining is a method of differentiating bacterial species into two large groups ....
of positive blood cultures, PNAs can be used to target species-specific rRNA (ribosomal RNA) to identify different strains of bacteria or yeast. Since the test can be performed relatively quickly, the test is being considered for use in hospitals where hospital-acquired infections can occur.
CO-FISH (chromosome orientation-FISH)
Another adaptation that utilizes PNAs and FISH is known as CO-FISH (Chromosome Orientation-FISH) which allows for the labelling of chromosomes with PNAs in a strand specific manner. This method involves the selective removal of newly replicated strands of DNA (through the use of BrdU incorporation), resulting in only single stranded target DNA. By using different colored unidirectional PNA probes, it becomes possible to uiquely label sister chromatids,.