Methods to label probes Nick translation Random primed labeling(Oligolabeling) End-labeling Riboprobes labeling

(A) Nick translation. Pancreatic DNase I or S1 nuclease introduces single-stranded nicks by cleaving internal phosphodiester bonds (p), generating a 5 phosphate group and a 3 hydroxyl terminus. Addition of the multisubunit enzyme E. coli DNA polymerase I contributes two enzyme activities: (i) a 5 3 exonuclease attacks the exposed 5 termini of a nick and sequentially removes nucleotides in the 5 3 direction; (ii) a DNA polymerase adds new nucleotides to the exposed 3 hydroxyl group, continuing in the 5 3 direction, thereby replacing nucleotides removed by the exonuclease and causing lateral displacement (translation) of the nick.

(B)

Random primed labeling(Oligolabeling). The Klenow subunit of E. coli DNA polymerase I can synthesize new radiolabeled DNA strands using as a template separated strands of DNA, and random hexanucleotide primers.

(A) Kinase end-labeling of oligonucleotides. The 5-terminal phosphate of the oligonucleotide is replaced in an exchange reaction by the 32Plabeled -phosphate of [-32P]ATP. The same procedure can be used to label the two 5 termini of double-stranded DNA.

B) Fill-in end-labeling by Klenow. The DNA of interest is cleaved with a suitable restriction nuclease to generate 5 overhangs. The overhangs act as a primer for Klenow DNA polymerase to incorporate labeled nucleotides complementary to the overhang. Fragments labeled at one end only can be generated by internal cleavage with a suitable restriction site to generate two differently sized fragments which can easily be size-fractionated.

RNA Labeling when Riboprobes are generated by run-off transcription from cloned DNA inserts in specialized plasmid vectors Eg. The plasmid vector pSP64 contains a promoter sequence for phage SP6 RNA polymerase linked to the multiple cloning site (MCS) in addition to an origin of replication (ori) and ampicillin resistance gene (amp). After cloning a suitable DNA fragment in one of the 11 unique restriction sites of the MCS, the purified recombinant DNA is linearized by cutting with a restriction enzyme at a unique restriction site just distal to the insert DNA (Pvu II in this example). Thereafter labeled insert-specific RNA transcripts can be generated using SP6 RNA polymerase and a cocktail of NTPs, at least one of which is labeled (UTP in this case).

Nucleic acids can be labeled by isotopic and nonisotopic methods

Isotopic labeling and detection Traditionally, labeling of nucleic acids has been conducted by incorporating nucleotides containing radioisotopes(like 32P, 33P, 35S or 3H) Characteristics of radioisotopes commonly used for labeling DNA and RNA probes

32P widely used in Southern blot hybridization, dot-blot hybridization, colony and plaque hybridization because it emits high energy -particles which afford a high degree of sensitivity of detection. 32P-labeled nucleotides used in DNA strand synthesis labeling reactions have the radioisotope at the -phosphate position, because the - and -phosphates from dNTP precursors are not incorporated into the growing DNA chain. It has the disadvantage, however, that it is relatively unstable . Additionally, its high energy -particle emission can be a disadvantage under circumstances when fine physical resolution is required to interpret the resulting image unambiguously.

 33P-labeled nucleotides used in DNA strand synthesis labeling reactions have the radioisotope at the -phosphate position, because the - and -phosphates from dNTP precursors are not incorporated into the growing DNA chain. Kinase-mediated end-labeling, however, uses [-32P]ATP provide less energetic -particle radiation have been preferred in certain procedures like DNA sequencing and tissue in situ hybridization 33P have moderate half-lives 35S-labeled nucleotides which are incorporated during the synthesis of DNA or RNA strands, the NTP or dNTP carries a 35S isotope in place of the O- of the -phosphate group. provide less energetic -particle radiation have been preferred in certain procedures like DNA sequencing and tissue in situ hybridization. 35S have moderate half-lives

 3H-labeled nucleotides carry the radioisotope at several positions. 3H has a very long half-life. 3H-labeled nucleotides have been for chromosome in situ hybridization. but disadvantage as its comparatively low energy -particle emission which necessitates very long exposure times.

Specific detection of molecules carrying a radioisotope is most often performed by autoradiography. Autoradiography is a procedure for localizing and recording a radiolabeled compound within a solid sample, which involves the production of an image in a photographic emulsion

the solid sample often consists of size-fractionated DNA or protein samples that are embedded within a dried gel, fixed to the surface of a dried nylon membrane or nitrocellulose filter, or located within fixed chromatin or tissue samples mounted on a glass slide.

The photographic emulsions consist of silver halide crystals in suspension in a clear gelatinous phase.

 . Following passage through the emulsion of a -particle or a -ray emitted by a radionuclide, the Ag+ ions are converted to Ag atoms. The resulting latent image can then be converted to a visible image once the image is developed, an amplification process in which entire silver halide crystals are reduced to give metallic silver. The fixing process results in removal of any unexposed silver halide crystals, giving an autoradiographic image which provides a two-dimensional representation of the distribution of the radiolabel in the original sample.

 Direct autoradiography involves placing the sample in intimate contact with an X-ray film a plastic sheet with a coating of photographic emulsion; the radioactive emissions from the sample produce dark areas on the developed film. This method is best suited to detection of weak to medium strength -emitting radionuclides (e.g. 3H, 35S, etc.). However, it is not suited to high energy -particles (e.g. from 32P): such emissions pass through the film, resulting in the wasting of the majority of the energy.

 Indirect autoradiography is a modification in which the emitted energy is converted to light by a suitable chemical (scintillator or fluor). One popular approach uses intensifying screens, sheets of a solid inorganic scintillator which are placed behind the film in the case of samples emitting high energy radiation, such as 32P. Those emissions which pass through the photographic emulsion are absorbed by the screen and converted to light. By effectively superimposing a photographic emission upon the direct autoradiographic emission, the image is intensified.

 Nonisotopic labeling and detection Nonisotopic labeling systems involve the use of nonradioactive probes. Although developed only comparatively recently, they are becoming increasingly popular and are finding increasing applications in a variety of different areas. Two types of non-radioactive labeling are conducted: Direct nonisotopic labeling Indirect nonisotopic labeling

Direct nonisotopic labeling (modified nucleotides (often 2 deoxyuridine 5 triphosphate) containing a fluorophore that will be detected is incorporated). Fluorophore is a chemical group which can fluoresce when exposed to light of a certain wavelength. Popular fluorophores used in direct labeling include fluorescein, a pale green fluorescent dye, rhodamine, a red fluorescent dye and amino methyl coumarin, a blue fluorescent dye

Fluorescence microscopy and structure of common fluorophores (A) Structure of fluorophores. The example on top shows fluorescein-dUTP. The fluorescein group is linked to the 5 carbon atom of the uridine by a spacer group so that when the modified nucleotide is incorporated into DNA, the fluorescein group is readily accessible. Below is the structure of rhodamine from which a variety of fluorophores have been derived. (B) Fluorescence microscopy. The excitation filter is a color barrier filter which in this example is selected to let through only blue light. The transmitted blue light is of an appropriate wavelength to be reflected by the dichroic (beamsplitting) mirror onto the labeled sample which then fluoresces and emits light of a longer wavelength, green light in this case. The longer wavelength of the emitted green light means that it passes straight through the dichroic mirror.

The light subsequently passes through a second color barrier filter which blocks unwanted fluorescent signals, leaving the desired green fluorescence emission to pass through to the eyepiece of the microscope.

Indirect nonisotopic labeling

(usually featuring the chemical coupling of a modified reporter molecule such as biotin or digoxigenin to a nucleotide precursor. The reporter molecules on modified nucleotides need to protrude sufficiently far from the nucleic acid backbone to facilitate their detection by the affinity molecule and so a long carbon atom spacer is required to separate the nucleotide from the reporter group).

Structure of digoxigenin- and biotin-modified nucleotides

digoxigenin and biotin groups in these examples are linked to the 5 carbon atom of the uridine of dUTP by spacer groups consisting respectively of a total of 11 carbon atoms (digoxigenin-11-UTP) or 16 carbon atoms (biotin-16-dUTP). The digoxigenin and biotin groups are reporter groups

 After incorporation into DNA, the reporter groups can be specifically bound by an affinity molecule, a protein or other ligand (such as streptavidin or a digoxigeninspecific antibody)which has a very high affinity for the reporter group. Conjugated to the latter is a marker molecule like fluorophore, amino methylcoumarin acetic acid (AMCA), fluorescein isothiocyanate (FITC) or other fluorescein derivatives, and tetramethylrhodamine isothiocyanate (TRITC) or other rhodamine derivatives which can be detected in a suitable assay .

General principles of indirect nonisotopic labeling The protein recognizing the reporter group is often a specific antibody, as in the digoxigenin system, or any other ligand that has a very high affinity for a specific group, such as streptavidin in the case of using biotin as the reporter. The marker can be detected in various ways. If it carries a specific fluorescent dye, it can be detected in a fluorimetric assay. Alternatively, it can be an enzyme such as alkaline phosphatase which can be coupled to an enzyme assay, yielding a product that can be measured colorimetrically.

 Fluorescence labeling of nucleic acids was developed in the 1980s and has proved to be extremely valuable in many different applications including chromosome in situ hybridization, tissue in situ hybridization and automated DNA sequencing.

 Nucleic acid hybridization is a method for identifying closely related nucleic acid molecules within two populations, a complex target population and a comparatively homogeneous probe population Nucleic acid hybridization involves mixing single strands of two sources of nucleic acids, a probe which typically consists of a homogeneous population of identified molecules (e.g. cloned DNA or chemically synthesized oligonucleotides) and a target which typically consists of a complex, heterogeneous population of nucleic acid molecules. If either the probe or the target is initially double-stranded, the individual strands must be separated, generally by heating or by alkaline treatment. After mixing single strands of probe with single strands of target, strands with complementary base sequences can be allowed to reassociate. Complementary probe strands can reanneal to form homoduplexes, as can complementary target DNA strands. However, it is the annealing of a probe DNA strand and a complementary target DNA strand to form a labeled probe-target heteroduplex that defines the usefulness of a nucleic acid hybridization assay. The rationale of the hybridization assay is to use the identified probe to query the target DNA by identifying fragments in the complex target which may be related in sequence to the probe.

 nucleic acid hybridization assay requires the formation of heteroduplexes between labeled single-stranded nucleic acid probes and complementary sequences within a target nucleic acid The probe is envisaged to be strongly related in sequence to a central segment of one of the many types of nucleic acid molecule in the target. Mixing of denatured probe and denatured target will result in reannealed probe-probe homoduplexes (bottom right) and target-target homoduplexes (bottom left) but also in heteroduplexes formed between probe DNA and any target DNA molecules that are significantly related in sequence (bottom centre). If a method is available for removing the probe DNA that is not bound to target DNA, the heteroduplexes can easily be identified by methods that can detect the label.

Melting temperature and hybridization stringency Denaturation of double-stranded probe DNA is generally achieved by heating a solution of the labeled DNA to a temperature which disrupts the hydrogen bonds that hold the two complementary DNA strands together. The energy required to separate two perfectly complementary DNA strands is dependent on a number of factors: strand length - long homoduplexes contain a large number of hydrogen bonds and require more energy to separate them; because the labeling procedure typically results in short DNA probes, this effect is negligible above an original length (i.e. prior to labeling) of 500 bp base composition - because GC base pairs have one more hydrogen bond than AT base pairs, strands with a high % GC composition are more difficult to separate than those with a low % GC composition chemical environment - the presence of monovalent cations (e.g. Na+ ions) stabilizes the duplex, whereas chemical denaturants such as formamide and urea destabilize the duplex by chemically disrupting the hydrogen bonds.

 A useful measure of the stability of a nucleic acid duplex is the melting temperature (Tm). This is the temperature corresponding to the midpoint in the observed transition from double-stranded to single-stranded form. Conveniently, this transition can be followed by measuring the optical density of the DNA. The bases of the nucleic acids absorb 260 nm ultraviolet (UV) light strongly. However, the adsorption by double-stranded DNA is considerably less than that of the free nucleotides. This difference, the so-called hypochromic effect, is due to interactions between the electron systems of adjacent bases, arising from the way in which adjacent bases are stacked in parallel in a double helix. If duplex DNA is gradually heated, therefore, there will be an increase in the light absorbed at 260 nm (the optical density260 or OD260) towards the value characteristic of the free bases. The temperature at which there is a midpoint in the optical density shift is then taken as the Tm

Denaturation of DNA results in an increase in optical density ODSS and ODDS indicate the optical density of singlestranded and double-stranded DNA respectively.

 Often, hybridization conditions are chosen so as to promote heteroduplex formation and the hybridization temperature is often as much as 25C below the Tm Probe-target heteroduplexes are most stable thermodynamically when the region of duplex formation contains perfect base matching. Mismatches between the two strands of a heteroduplex reduce the Tm: for normal DNA probes, each 1% of mismatching reduces the Tm by approximately 1C. Although probe-target heteroduplexes are usually not as stable as reannealed probe homoduplexes, a considerable degree of mismatching can be tolerated if the overall region of base complementarity is long