- 1 Difference between cDNA and Genomic DNA library – Construction of Genomic Libraries and cDNA Libraries
- 1.1 A. Genomic DNA Library
- 1.2 B. cDNA Library
- 1.3 Genomic versus cDNA Libraries
- 1.4 Identification of RNA by Northern Blotting
- 1.5 Identification of A Specific Protein Western Blotting (Western Transfer or Immunoblotting)
- 1.6 Determination of the Sequence of an RNA Molecule
- 1.7 Production of Transgenic Organism
- 1.8 A. Gene Transfer via Vector
- 1.9 B. Microinjection
- 1.10 C. Electroporation
- 1.11 D. Embryonic Stem Cell-Mediated Gene Transfer
- 1.12 Enzyme-linked immunosorbent Assay or ELISA
- 1.13 Radioimmunoassay (RIA)
- 1.14 FISH
Immunology is one of the Biology Topics focused on understanding the immune system and its response to pathogens and diseases.
Difference between cDNA and Genomic DNA library – Construction of Genomic Libraries and cDNA Libraries
A gene library represents a collection of all the genes of a species. In common sense, a library means a collection of many books and in this, a book may be present in multiple numbers. A gene library is a collection of cloned segments of DNA that is big enough to contain at least one copy of every gene from a particular organism. A gene library (gene bank) is a collection of randomly cloned fragments of the genomic DNA of an organism (genomic library) or of a specific set of DNA fragments representing for instance a collection of the mRNAs expressed in a cell at a specific time (cDNA library).
The fragments are in each case inserted into suitable vectors (e.g.„ cosmids or bacteriophage vectors, occasionally also plasmids) and transformed into a suitable host. A genomic library ideally encompasses the entire genome of the species from which it originates, and a cDNA library ideally represents all the different mRNA molecules present in a specific cell at a specific time. There are two types of DNA libraries namely genomic DNA library and cDNA library.
A. Genomic DNA Library
The collection of clones that contains all the genes of a species or all the DNA sequences of a species, is known as a Genomic DNA library. The ideal genome library should contain at least one copy of all the genes of the organism. Cloning of all the DNA sequences from a species at random in the host cell helps in producing a genomic DNA library.
Genomic DNA isolated from an organism is exposed to a restriction endonuclease which cleaves the DNA molecule into many fragments. This process produces fragments of DNA that include the organism’s entire genome.” Each bacterial cell during its reproduction produces copies of a recombinant vector by which many copies of a fragment DNA are also produced. This procedure of producing clones of genes or segments of DNA is known as shot shotgun experiment.
Consider each clone a “book” in this “library” of DNA fragments. One disadvantage of creating this type of library for eukaryotic genes is that non-protein-coding pieces of DNA, called introns, are cloned in addition to protein-coding sequences (exons). Because a majority of DNA in any eukaryotic organism consists of introns, many of the clones in a genomic library will contain non-protein-coding pieces of DNA. Another limitation of genomic libraries is that many organisms, including humans, have such large genomes that searching for a gene of interest would be like searching for a needle in a haystack.
B. cDNA Library
The genes in the cell during their expression produce mRNA. Thus mRNA molecules in a cell represent the sequence of expressed genes in the genome. If the mRNA molecule is treated with reverse transcriptase with dNTPs, then a sequence of DNA may be produced by reverse transcription. The DNA thus produced is called cDNA or complementary DNA. cDNA does not contain any intron and it represents the coding sequence. Therefore, cDNA is composed of exons. cDNAs produced using all different types of mRNA represent the cDNA library.
In a cDNA library, mRNA from the tissue of interest is isolated and used for making the library. As, mRNA cannot be cut directly with restriction enzymes, it must be converted to a double-stranded DNA molecule. An enzyme called reverse transcriptase (RT) is used to catalyze the synthesis of single-stranded DNA from the mRNA.
This enzyme is made by viruses called retroviruses so named because they are exceptions to the usual flow of genetic information. Instead of having a DNA genome that can be used to make RNA, retroviruses have an RNA genome. After infecting host cells, they use RT to convert RNA into DNA, so that they can replicate. Human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), is a retrovirus.
Because RT synthesizes DNA that is an exact copy of mRNA, it is called complementary DNA (cDNA). The mRNA is degraded by treatment with an alkaline solution or enzymatically digested; then DNA polymerase is used to synthesize a second strand to create double-stranded cDNA. Because cDNA sequences do not necessarily have a convenient restriction site at each end, short, double-stranded DNA sequences called linker sequences are often enzymatically added to the ends of the cDNA.
Genomic versus cDNA Libraries
One primary advantage of cDNA libraries over genomic libraries is that they are a collection of actively expressed genes in the cells or tissue from which the mRNA was isolated. Also, introns are not cloned in a cDNA library. By contrast, when cloned genomic DNA, containing introns and exons is inserted into bacteria, the cells cannot splice mRNA transcribed from this DNA and remove introns.
For this reason, cDNA libraries are typically preferred over genomic libraries when attempting to clone and express a gene of interest. Another advantage of cDNA libraries is that they can be created and screened to isolate genes that are primarily expressed only under certain conditions in a tissue. For example, if a gene is expressed only in a tissue stimulated by a hormone, researchers make libraries from hormone-stimulated cells to increase the likelihood of cloning hormone-sensitive genes.
Analysis of Cloned DNA Segments or Genes
Selection of the desired gene from the clone DNAs is not a difficult job. With the help of a probe designed against a specific gene/the gene of interest, the desired gene may be identified by Southern hybridization.
Principle of Southern Blotting:
To identify a specific gene among the DNA fragments, first agarose gel electrophoresis is carried out to separate the DNA fragments. Since the charge per unit length (owing to the phosphate groups) in any given fragment of DNA is the same, all DNA samples should move towards the anode with the same mobility under an applied electrical field. However, separation in agarose gels is achieved because of resistance to their movement caused by the gel matrix. The largest molecules will have the most difficulty passing through the gel pores (very large molecules may even be blocked completely), whereas the smallest molecules will be relatively unhindered.
Consequently, the mobility of DNA molecules during gel electrophoresis will depend on size, with the smallest molecules moving the fastest. DNA gels are invariably run as horizontal, submarine or submerged gels; so, named because such a gel is totally immersed in buffer. Nucleic acids can be visualized on the slab gel after separation by soaking in a solution of ethidium bromide, an intercalating dye that displays enhanced fluorescence when intercalated between stacked nucleic acid bases. The mobility of nucleic acids in agarose gels is influenced by the agarose concentration and the molecular size and molecular conformation of the nucleic acid. Agarose concentrations of 0.3 to 2.0% are most effective for nucleic acid separation.
After the separation of the restriction fragment by gel electrophoresis, the DNA molecules in the agarose gel were transferred to a nitrocellulose or nylon membrane by electroblotting. Blotting (blot transfer) is any procedure that transfers electrophoretically separated DNA fragments, RNA fragments, or proteins from a separation gel (e.g., agarose, polyacrylamide) to a paper or membrane matrix (e.g., nitrocellulose, nylon-based membranes). The transfer process retained the same positions on the membrane as the DNA molecules had in the gel. The DNA molecules that were transferred to a membrane were de-natured, bound to the membrane, and hybridized with a radiolabeled complementary single-stranded DNA probe to search for certain nucleotide sequences among DNA molecules blotted from the gel. This technique of detecting specific DNA molecules or desired genes in the nitrocellulose membrane by DNA-DNA hybridization is called Southern blotting after Edwin Southern, who devised the original DNA blotting strategy. RNA and proteins may also be separated by electrophoresis. But for identification of a specific RNA, the technique used is called northern blotting, and that for protein is called western blotting.
Use of Southern Botting
Southern blotting can determine the copy number of a gene within the genome of an organism. For example, Southern blotting has revealed that rRNA genes are found in multiple copies within a genome, whereas many structural genes are unique. In the study of human genetic diseases, Southern blotting can also detect small gene deletions that cannot be distinguished under the light microscope.
A common use of Southern blotting is to identify gene families. A gene family is a group of two or more genes derived from the same ancestral gene. The members of a gene family have similar but not identical DNA sequences; they are homologous genes. Southern blotting can distinguish the homologous members of a gene family. Researchers may produce transgenic organisms that carry new genes or modified genes. Southern blotting may be used to determine if transgenic organisms carry such genes.
Steps of Southern Blotting
Developed by Edward Southern in 1975, Southern blotting begins by digesting chromosomal DNA into small fragments with restriction enzymes. DNA fragments are separated by agarose gel electrophoresis. Following electrophoresis, the gel is treated with an alkaline solution to denature the DNA molecules into single-stranded DNA; then the fragments are transferred onto a nylon or nitrocellulose membrane using a technique called blotting. Blotting can be achieved by setting up a gel sandwich in which the gel is placed under the nylon membrane, filter paper, paper towels, and weight to allow for the wicking of a salt solution through the gel, which will transfer DNA onto the nylon by capillary action. The single strand of DNA sticks to the blot, positioned in bands exactly as on the gel. Alternatively, pressure or vacuum blotters can be used to transfer DNA onto nylon. The nylon blot is then baked or briefly exposed to UV light to attach the DNA permanently.
Next, the blot is incubated with a radiolabeled single-stranded probe designed against the desired DNA sequence/gene. Following the principle of nucleic acid hybridization the radiolabeled probe will hybridize with its complementary DNA sequence (desired DNA/gene) in the membrane and the presence of a hybridized probe may be detected. The probes that do not bind with the filter paper are wasted away. Over five X-ray films, the DNA fragments hybridized with the probe molecules appear as black stripes (a band).
Identification of RNA by Northern Blotting
One of the most basic ways to characterize a cloned gene is to determine when and where in an organism the gene is expressed. The expression of a particular gene can be followed by assaying for the corresponding mRNA by Northern blotting. It is possible with this technique not only to detect specific mRNA molecules in a particular tissue but also to quantify the relative amounts of the specific mRNA. This method can determine if a specific gene is transcribed in a particular cell type, such as nerve or muscle cells, or at a particular stage of development, such as fetal or adult cells.
In northern blotting, special care is taken for RNA isolation because RNA may easily be degraded by RNase. In the first step, the RNA molecules from the tissue are isolated and denatured by treatment with an agent such as formaldehyde that disrupts the hydrogen bonds between base pairs (to prevent the attainment of secondarily double-stranded condition by the RNA molecules), ensuring that all the RNA molecules have an unfolded, linear conformation.
Then the individual RNAs are separated according to size by agarose gel electrophoresis and transferred by electroblotting to a nitrocellulose/nylon filter to which the extended denatured RNAs adhere. It is usual to separate the mRNA transcripts by gel electrophoresis under denaturing conditions since this improves resolution and allows a more accurate estimation of the sizes of the transcripts.
Subsequently, by using single-stranded radioactive probes (DNA) that is complementary to the gene of interest the location of the desired RNA molecule on the nitrocellulose paper is identified following the principle of nucleic acid hybridization and autoradiography. Over the X-ray film, the RNA fragments hybridized with the probe molecules appear as black stripes (a band). Because the amount of a specific RNA in a sample can be estimated from a Northern blot, the procedure is widely used to compare the amounts of a particular mRNA in cells under different conditions.
Identification of A Specific Protein Western Blotting (Western Transfer or Immunoblotting)
Western blotting is an in vitro technique for the detection of specific proteins that are separated by polyacrylamide gel electrophoresis and then transferred (“blotted”) onto a solid support (e.g., a nitrocellulose or nylon membrane). This membrane (Western blot) can then be probed with specific radioactively labeled, fluorescence-conjugated or enzyme-conjugated antibodies (“immunoprobes”).It is possible with this technique not only to detect specific protein molecules but also to quantify the relative amounts of the specific protein in comparison to a standard control. This method can determine if a specific protein is made in a particular cell type or at a particular stage of development.
Determination of the sequence of DNA Molecule: The DNA sequencing method enables researchers to determine the base sequence of DNA found in genes and other chromosomal regions. It is one of the most important tools for exploring genetics at the molecular level. Knowledge of DNA sequences has become indispensable for basic biological research and in numerous applied fields such as medical diagnosis, biotechnology, forensic biology, virology, and biological systematics.
There are two different methods for knowing the sequences in the DNA fragments. One method is developed by Allan Maxam and Walter Gilbert and the other method was developed by F. Sanger. The second method is more popular and widely used. Frederick Sanger et al., 1975 developed this method. Sanger sequenced the genome of a virus and for this, he was awarded the Nobel Prize in 1980. The method is known as the dideoxy chain termination method. By the addition of a dideoxy nucleotide, the growth of the DNA strand may be blocked and the DNA sequence may be determined. A short description of the technique is given below.
Principles and Steps of Sanger’s Dideoxy Method
The DNA which is to be sequenced is called the template DNA. In the Sanger method, the single-stranded DNA to be sequenced is incubated with the Klenow fragment of DNA polymerase I, a suitable primer, and the four deoxynucleoside triphosphates (dNTPs). Either at least one dNTP (usually dATP) or the primer is α32-P-labeled. In addition, a small amount of one of the four ddNTPs is added to the reaction mixture.
DNA polymerase connects adjacent deoxyribonucleo- tides by catalyzing a covalent bond between the 5′ phosphate on one nucleotide and the 3′-OH group on the previous nucleotide. If a dideoxyribonucleotide (ddNTP) is added to a growing DNA strand, no more nucleotides can be added, because there is no 3′ -OH group to form j a phosphodiester bond with an incoming nucleotide. The incorporation of a deoxyribonucleotide into a growing strand is therefore referred to as chain termination.
In the reaction tube, when the dideoxy analog is incorporated in the growing polynucleotide in place of the corresponding normal nucleotide, chain growth is terminated because of the absence of a 3′-OH group. By using only a small amount of the ddNTP, a series of truncated chains is generated, each of which is terminated by the dideoxy analog at one of the positions occupied by the corresponding base. Each of the four ddNTPs is reacted in a separate vessel.
After the reaction is over in four different reaction mixtures, each of the four samples will contain a population of radiolabelled DNA fragments of different lengths (both complete and incomplete) terminating with the same nucleotide. The four reaction mixtures are simultaneously electrophoresed in parallel lanes on a sequencing gel. This is a long, thin (as little as 0.1 mm by up to 100 cm) polyacrylamide slab. It contains -7M urea and is run at -70°C so as to eliminate all hydrogen bonding associations. These conditions ensure that the DNA fragments separate only according to their size. The sequence of the DNA that is complementary to the template DNA can then be directly read off an autoradiogram of the sequencing gel, from bottom to top.
Determination of the Sequence of an RNA Molecule
RNA can be readily sequenced by only a slight modification of the above DNA sequencing procedures. The RNA to be sequenced is transcribed into a complementary strand of DNA (cDNA) through the action of reverse transcriptase. The resulting cDNA may then be sequenced normally. It is important to keep in mind that although genomes remain the same from cell to cell and from tissue to tissue, the RNA produced from the genome can vary enormously.
Production of Transgenic Organism
Transgene is any gene that originates from one and has been transferred to a second organism (cell).
A transgenic animal is an animal whose nuclear genome contains foreign DNA (e.g., genes) that has been transferred by transfection or direct gene transfer. Studies performed on transgenic organisms have contributed to an understanding of gene regulation, tumor development, immunological specificity, molecular genetics of development, and many other biological processes of fundamental interest. Transgenic mice have also played a role in examining the feasibility of the industrial production of human therapeutic drugs by domesticated animals and in the creation of transgenic strains that act as biomedical models for various human genetic diseases. For transgenesis, DNA can be introduced into mice by
- Retroviral vectors that infect the cells of an early-stage embryo prior to implantation into a receptive female.
- Microinjection into the enlarged sperm nucleus (male pronucleus) of a fertilized egg.
- Introduction of genetically engineered embryonic stem cells into an early-staged developing embryo before implantation into a receptive female.
In a transgenic animal, every cell carries new genetic information. In other words, novel genetic information is introduced into the germline, not merely into some somatic cells as in gene therapy. Consequently, the novel genes in a transgenic animal are passed on to its descendants. This novel genetic information generally consists of genes transferred from other organisms and referred to as transgenes. They may be derived from animals of the same species, from distantly related animals, or even from unrelated organisms such as plants, fungi, or bacteria.
Not only in bacteria, transgenesis could be achieved both in plants and animals. In biotechnology, transgenesis has developed a new dimension for human welfare and economic development. In the agricultural sector as well as in poultry and animal husbandry, transgenesis contributed remarkably creating new hope for development towards human civilization.
A. Gene Transfer via Vector
With the help of some vectors, gene(s) from external sources may be introduced in all organisms including bacteria, plants, and animals. In microorganisms and plants usually, plasmids are used as vectors. In higher plants during transgenesis and production of transgenic plant Ti plasmid of Agrobacterium tumefaciens is used as a vector for gene transfer.
Retroviral vectors introduce transgenes in early-stage mouse embryos via retroviral vectors. These vectors use the characteristics of retroviruses to integrate themselves into a site in the genome of the infected cells. The infected viral RNA is reverse-transcribed to DNA and integrates itself into the genome of the cell after the first or second cleavage division so that many embryos consist of at least two cell populations (i.e., cells with and without the retroviral transgene). The infected embryos are then transferred into a pseudo-pregnant female mouse and is delivered by foster mothers. Six weeks after their birth the so-called transgenic founder mice are mated. The transgene is only passed on to offspring if the germ cells of the founder animals contain the retroviral transgene, whereby all offspring of a single founder represent one transgenic line.
Microinjection of DNA is currently the preferred method for producing transgenic mice. This procedure is performed in the following way.
- The number of available fertilized eggs that are to be inoculated by microinjection is increased by stimulating donor females to superovulate.
- Young virgin females (approximately 4-5 weeks of age), are injected with a source of follicle-stimulating hormone (pregnant mare’s serum gonadotrophin).
- Forty-eight hours later, the females are given an artificial surge of luteinizing hormone by administration of human chorionic gonadotrophin and are paired with proven stud males.
- A superovulated mouse produces about 35 eggs instead of the normal 5 to 10. The superovulated females are mated so that eggs become fertilized, and then they are killed.
- The following day, females who have mated (as identified by the presence of a vaginal plug) are sacrificed and fertilized eggs are removed from the swollen ampullae of the Fallopian tubes by dissection.
- Using such a protocol, up to 30 zygotes can be isolated per female, depending on the strain used.
- The zygotes are freed from attached cumulus cells by brief incubation in the presence of hyaluronidase, transferred to an appropriate medium, and stored in a CO2 incubator at 37°C prior to microinjection. The microinjected transgene construct is often in a linear form and free of prokaryotic vector DNA sequences.
The fertilized egg, containing foreign DNA may be transplanted into the uterus of the surrogate mother (in the case of a mammal). In the course of time, a transgenic animal is born.
Electroporation is the use of an electric field pulse to induce microscopic pores within a biological membrane. These pores, called ‘electrophoresed’, allow molecules, ions, and water to pass from one side of the membrane to the other. If a suitable electric field pulse is applied [short (1 msec) electric pulse and potential gradients of 700V/cm], then the electroporated cells can recover, with the electrophoresis resealing spontaneously, and the cells can continue to grow. Pore formation is extremely rapid (approximately 1 ps), while pore resealing is much slower, and is measured in the order of minutes. In this method, a gene may be introduced in the bacterial cell with the help of high-voltage of electric shock. The electric shock increases the permeability of the cell membrane of bacteria and then the bacteria may take up a piece of DNA from the medium through its cell membrane. DNA can enter the bacterial cell before the pores spontaneously reseal.
D. Embryonic Stem Cell-Mediated Gene Transfer
To produce transgenic animals this method utilizes pluripotent embryonic stem cells which are procured and the desired genes are introduced into these cells. After this, the stem cells containing the desired DNA are inserted into a blastula and the blastula is implanted into a pseudo-pregnant mother. Some of the baby animals will have the transgene stably integrated into their chromosomes. In others, the process fails and the transgene is lost. Those that receive the transgene and maintain it stably are called founder animals. These eventually lead to the development of transgenic animals.
Mutation may be introduced at some specific site of the DNA and the composition of a genetic codon is altered. This is known as site-directed mutagenesis. Such a technique is useful for the production of some necessary enzymes. Besides this direct replacement of some amino acids in the polypeptide chain may result in alteration in protein structure. Site-directed mutagenesis helps to know the functions of the different parts of a gene.
DNA Microarray or DNA Chip is a technique based on the concept of nucleic acid hybridization in which a known DNA fragment is used as a probe to find complementary sequences. Monitoring the expression of thousands of genes simultaneously is possible with DNA microarray analysis, i.e.„ DNA microarrays can be used to evaluate the expression of many genes at one time. By coupling microarray analysis with the results from genome sequencing projects, researchers can analyze the global patterns of gene expression of an organism during specific physiological responses or developmental processes like cell growth, cell division, cellular differentiation, or signal transduction.
A DNA microarray consists of an organized array of thousands of individual, closely packed gene-specific sequences attached to the surface of a glass microscope slide. It is a small silica, glass, or plastic slide that is dotted with many different sequences of DNA, each corresponding to a short sequence within a known gene. For example, one spot in a microarray may correspond to a sequence within the β-globin gene, whereas another could correspond to a gene that encodes actin, which is a cytoskeletal protein. A single slide may contain tens of thousands of different spots in an area the size of a postage stamp. The relative location of each spot is known.
After the microarray has been constructed, mRNAs are isolated from a particular tissue or developmental stage of the organism. The isolated mRNAs are converted to single-stranded cDNAs using reverse transcriptase and are then labeled with a fluorescent molecule. When the labeled cDNAs are applied to the DNA chip, they anneal to the spots that contain the complementary sequences and emit fluorescence, which can be detected by an automated scanner. This step is analogous to a Southern hybridization. The unhybridized probe can be washed away, and the chip or slide can be examined by a laser for the level of fluorescence in each spot. The brighter the fluorescent signal, the greater the amount of cDNA that is hybridized to the spot. This should correlate to a higher level of transcription of the gene concerned in the tissue from which the mRNAs were isolated.
DNA microarrays are currently being employed to study changes in gene expression that occur during a wide variety of biological events, such as cell division and the transformation of a normal cell into a malignant cell. The microarray technology reveals whether the expression of every gene tested increases, decreases, or remains the same in the test condition (diseased tissue) relative to the control condition (normal tissue). In some cases, microarrays can even help identify which genes encode the proteins that participate in a complicated metabolic pathway. Microarrays can also be used as identification tools. For example, gene expression patterns can aid in the categorization of tumor types. Such identification is used to determine the best course of clinical treatment for a patient.
ELISA is an immunological method for the detection of specific proteins using two antibody preparations. ELISA is a technique for detecting the existence of an antigen-antibody or hormone in a fluid with the help of an antigen-antibody reaction. ELISA is also used in the detection of AIDS, it is a very effective technique to detect the presence of a protein in the body fluid without using any radio-active substance.
At the first step antibody is produced against a protein and then it is conjugated with some inert solid substance (such as polystyrene). If a sample of body fluid contains the protein, then it is brought in contact with the primary antibody which binds to the protein in question (antigen) and is in turn recognized by a secondary antibody, which is linked to an indicator enzyme (e.g., horseradish peroxidase or alkaline phosphatase, which converts X-Phos to a blue dye) whose activity can be easily quantified. The activity measured is directly proportional to the amount of primary antibody and, consequently, of the antigen.
It is a technique for the quantitative determination of minute amounts of antigens in which the specific binding of radioactively labeled antigens or antibodies is measured. Usually, 125I is used as a label. RIAs fall into several broad categories. In the so-called competition RIA an unlabeled target protein competes with a labeled antigen for binding sites on the antibody. The ratio of bound to unbound radioactivity then is a quantitative measure of the interaction.
The double-antibody RIA works with a single antibody bound onto a solid support. This antibody is then exposed to an unlabeled antigen. After washing, the target antibody is quantified by a second radioactively labeled antibody. For an immobilized antibody, RIA a single antibody is bound to a solid support and exposed to a labeled antigen, whose amount can be calculated from the amount of bound radioactivity. Finally, the so-called immobilized antigen RIA is based on the exposure of an unlabeled antigen (bound onto a solid support) to a radiolabeled antibody. The amount of bound radioactivity reflects the amount of specific antigen present in the experimental sample.
Radioimmunoassay employs isotopically labeled molecules and permits measurements of extremely small amounts of antigen, antibody, or antigen-antibody complexes. This technique is widely used for detecting the presence of hormones and antigens.
The technique of in situ hybridization is widely used to cytogenetically map the locations of genes or other DNA sequences within large eukaryotic chromosomes. The term in situ (from the Latin for “in place”) indicates that the procedure is conducted on chromosomes that are being held in place – adhered to a surface. To map a gene via in situ hybridization, researchers use a single-stranded DNA probe designed against the gene of interest to detect the location of the desired gene within a set of chromosomes.
The most common method of in situ hybridization uses fluorescently labeled DNA probes and is referred to as fluorescence in situ hybridization (FISH). FISH is a method to identify specific sequences of intact chromosomes by hybridization with complementary nucleic acid probes that are covalently linked to fluorochromes. Usually, the biological materials are squashed on microscope slides, the DNA denatured, and then hybridized to the fluorochrome-labelled probe.
As the probe is fluorescent when the chromosomes are examined under the fluorescence microscope the specific site of the chromosome which harbours the gene of interest and to which the fluorescently tagged probe has bound will exhibit fluorescence. To detect the exact location of a gene on the chromosome as well as its mapping, FISH is very helpful. It also tells us whether a deletion, duplication or translocation of the target gene has occurred or not.
For performing FISH, the cells are prepared using a technique that keeps the chromosomes intact. The cells are treated with agents that cause them to swell, and their contents are fixed to the slide. Classical in situ hybridization involved spreading mitotic chromosomes on a glass slide, denaturing the DNA in the chromosomes by exposure to alkali (0.07 N NaOH) for a few minutes, and rinsing with buffer to remove the alkaline solution. The chromosomes from the cell are spread on a slide, and the DNA is made single-stranded by treating the chromosomes with a hot salt solution that causes the DNA strands to separate and remain apart.
During the ensuing hybridization step, the denatured chromosomes are incubated with a solution of a single-stranded DNA probe which binds selectively to complementary strands of immobilized DNA located in the chromosomes. The probe binds to a site in the chromosomes where the gene is located because the probe and chromosomal gene line up and hydrogen bond with each other. To detect where the probe has bound to a chromosome, the probe is subsequently tagged with a fluorescent molecule. A fluorescent molecule is one that absorbs light at a particular wavelength and then emits light at a longer wavelength. Following the incubation period, the soluble, unhybridized probe DNA is washed away.
To detect the light emitted by a fluorescently labeled probe, a fluorescence microscope is used. Such a microscope contains filters that allow the passage of light only within a defined wavelength range. The sample is illuminated at the wavelength of light that is absorbed by the fluorescent molecule. The fluorescent molecule then emits light at a longer wavelength. The fluorescence microscope has a filter that allows the transmission of the emitted light. Because only the emitted light is viewed, the background of the sample is dark, and the fluorescence is seen as a brightly glowing color on a dark background.
For most FISH experiments, chromosomes are generally counterstained by a fluorescent dye that is specific for DNA. A commonly used dye is DAPI (4, 6-diamidino-2-phenyl-indol) which is excited by UV light. This provides all of the DNA with a blue background. The results of a FISH experiment are then compared with a sample of chromosomes that have been stained with Giemsa to produce banding, so the location of a probe can be mapped relative to the chromosome banding pattern.