Probe Labeling1

8/22/2019 Probe Labeling1

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Methods to label probes

Nick translation

Random primed labeling(Oligolabeling)

End-labeling

Riboprobes labeling


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(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.

coliDNA polymerase I contributes two enzymeactivities: (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 ofE. coliDNA polymerase I can

synthesize new radiolabeled DNA strands using as a

template separated strands of DNA, and random

hexanucleotide primers.


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(A) Kinase end-labeling of oligonucleotides.

The 5-terminal phosphate of the

oligonucleotide is replaced in an exchange

reaction by the 32P-labeled -phosphate of

[-32

P]ATP. The same procedure can beused 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 begenerated by internal cleavage with a

suitable restriction site to generate two

differently sized fragments which can

easily be size-fractionated.


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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 ofreplication (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).


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Nucleic acids can be labeled by isotopic and nonisotopic methods

Isotopic labeling and detection

Traditionally, labeling of nucleic acids has been conducted by incorporatingnucleotides containing radioisotopes(like 32P, 33P, 35S or3H)

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 labelingreactions have the radioisotope at the -phosphate position, becausethe - 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 adisadvantage under circumstances when fine physical resolution is

required to interpret the resulting image unambiguously.


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33P-labeled nucleotides used in DNA strand synthesis labeling reactions havethe radioisotope at the -phosphate position, because the - and -

phosphates from dNTP precursors are not incorporated into the growingDNA chain.

Kinase-mediated end-labeling, however, uses [-32P]ATP

provide less energetic -particle radiation have been preferred in certainprocedures like DNA sequencing and tissue in situ hybridization

33P have moderate half-lives

35S-labeled nucleotides which are incorporated during the synthesis of DNAor RNA strands, the NTP or dNTP carries a 35S isotope in place of the O- ofthe -phosphate group.

provide less energetic -particle radiation have been preferred in certainprocedures like DNA sequencing and tissue in situ hybridization.

35S have moderate half-lives


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3H-labeled nucleotides carry the radioisotope at

several positions.

3H has a very long half-life.

3

H-labeled nucleotides have been for chromosomein situ hybridization.

but disadvantage as its comparatively low energy -particle emission which necessitates very long

exposure times.


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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 oftenconsists of size-fractionated

DNA or protein samples thatare embedded within a driedgel, fixed to the surface of adried nylon membrane ornitrocellulose filter, or

located within fixedchromatin or tissue samplesmounted on a glass slide.

The photographicemulsions consist of

silver halide crystals insuspension in a cleargelatinous phase.


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. 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 convertedto a visible image once the image is developed, an

amplification process in which entire silver halidecrystals are reduced to give metallic silver.

The fixing process results in removal of anyunexposed silver halide crystals, giving anautoradiographic image which provides a two-dimensional representation of the distribution of

the radiolabel in the original sample.


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Direct autoradiography involves

placing the sample in intimate contact with an X-rayfilm

a plastic sheet with a coating of photographicemulsion; the radioactive emissions from the sampleproduce dark areas on the developed film.

This method is best suited to detection of weak tomedium 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.


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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 asolid inorganic scintillator which are placed behind the filmin the case of samples emitting high energy radiation, such

as 32P.

Those emissions which pass through the photographicemulsion are absorbed by the screen and converted to

light.

By effectively superimposing a photographic emissionupon the direct autoradiographic emission, the image isintensified.


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Nonisotopic labeling and detection

Nonisotopic labeling systems involve the use ofnonradioactive 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


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Direct nonisotopic labeling

(modified nucleotides (often 2 deoxyuridine 5

triphosphate) containing a fluorophore that willbe detected is incorporated).

Fluorophore is a chemical group which can

fluoresce when exposed to light of a certainwavelength.

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


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Fluorescence microscopy and structure ofcommon fluorophores

(A) Structure of fluorophores. The example on topshows fluorescein-dUTP. The fluorescein group islinked to the 5 carbon atom of the uridine by aspacer group so that when the modified nucleotideis incorporated into DNA, the fluorescein group isreadily accessible. Below is the structure ofrhodamine from which a variety of fluorophoreshave been derived.

(B) Fluorescence microscopy.

The excitation filter is a color barrier filterwhich in this example is selected to let throughonly blue light.

The transmitted blue light is of an appropriatewavelength to be reflected by the dichroic (beam-splitting) mirror onto the labeled sample whichthen fluoresces and emits light of a longerwavelength, green light in this case.

The longer wavelength of the emitted greenlight means that it passes straight through thedichroic mirror.

The light subsequently passes through a secondcolor barrier filter which blocks unwantedfluorescent signals, leaving the desired greenfluorescence emission to pass through to theeyepiece of the microscope.


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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 atomspaceris required to separate the nucleotide from the reportergroup).

Structure of digoxigenin- andbiotin-modified nucleotides

digoxigenin and biotin groups

in these examples are linkedto the 5 carbon atom of theuridine of dUTP by spacergroups consisting respectivelyof a total of 11 carbon atoms

(digoxigenin-11-UTP) or 16carbon atoms (biotin-16-dUTP). The digoxigenin andbiotin groups are reportergroups


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After incorporation into DNA, the reporter groupscan be specifically bound by an affinity molecule, a

protein or other ligand (such as streptavidin or adigoxigenin-specific antibody)which has a very high

affinity for the reporter group.

Conjugated to the latter is a marker molecule likefluorophore, amino methylcoumarin acetic acid

(AMCA), fluorescein isothiocyanate (FITC) or otherfluorescein derivatives, and tetramethylrhodamineisothiocyanate (TRITC) or other rhodaminederivatives which can be detected in a suitable assay .


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General principles of indirectnonisotopic labeling

The protein recognizing thereporter group is often a specificantibody, as in the digoxigenin

system, or any other ligand that hasa very high affinity for a specificgroup, such as streptavidin in thecase of using biotin as the reporter.

The marker can be detected invarious ways.

If it carries a specific fluorescentdye, it can be detected in afluorimetric assay.

Alternatively, it can be an enzyme

such as alkaline phosphatase whichcan be coupled to an enzyme assay,yielding a product that can bemeasured colorimetrically.


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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.
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Nucleic acid hybridization is a method for identifying closely relatednucleic acid molecules within two populations, a complex targetpopulation and a comparatively homogeneous probe population

Nucleic acid hybridization involves mixing single strands of two sources ofnucleic acids, a probe which typically consists of a homogeneouspopulation ofidentified molecules (e.g. cloned DNA or chemicallysynthesized oligonucleotides) and a target which typically consists of acomplex, 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 alkalinetreatment.

After mixing single strands of probe with single strands of target, strandswith complementary base sequences can be allowed to reassociate.

Complementary probe strands can reanneal to form homoduplexes, ascan complementary target DNA strands. However, it is the annealing of aprobe DNA strand and a complementary target DNA strand to form alabeled probe-target heteroduplex that defines the usefulness of anucleic acid hybridization assay.

The rationale of the hybridization assay is to use the identified probe toquery the target DNA by identifying fragments in the complex target

which may be related in sequence to the probe.


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nucleic acid hybridization assay requires the formation of heteroduplexes between labeledsingle-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 manytypes of nucleic acid molecule in the target.

Mixing of denatured probe and denatured target will result in reannealed probe-probehomoduplexes (bottom right) and target-target homoduplexes (bottom left) but also inheteroduplexes formed between probe DNA and any target DNA molecules that are significantlyrelated in sequence (bottom centre).

If a method is available for removing the probe DNA that is not bound to target DNA, theheteroduplexes can easily be identified by methods that can detect the label.
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Melting temperature and hybridization stringency

Denaturation of double-stranded probe DNA is generally achieved byheating a solution of the labeled DNA to a temperature which disruptsthe hydrogen bonds that hold the two complementary DNA strands

together. The energy required to separate two perfectly complementaryDNA strands is dependent on a number of factors:

strand length - long homoduplexes contain a large number of hydrogenbonds and require more energy to separate them; because the labelingprocedure typically results in short DNA probes, this effect is negligibleabove an original length (i.e. prior to labeling) of 500 bp

base composition - because GC base pairs have one more hydrogenbond than AT base pairs, strands with a high % GC composition are moredifficult 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 asformamide and urea destabilize the duplex by chemically disrupting thehydrogen bonds.

A useful measure of the stability of a nucleic acid duplex is the


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A useful measure of the stability of a nucleic acid duplex is themelting temperature (Tm). This is the temperature correspondingto the midpoint in the observed transition from double-strandedto single-stranded form.

Conveniently, this transition can be followed by measuring theoptical density of the DNA.

The bases of the nucleic acids absorb 260 nm ultraviolet (UV) lightstrongly.

However, the adsorption by double-stranded DNA is considerably

less than that of the free nucleotides. This difference, the so-calledhypochromic effect, is due to interactions between the electronsystems of adjacent bases, arising from the way in which adjacentbases 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 orOD260) towards the value characteristic of the free bases.

The temperature at which there is a midpoint in the opticaldensity shift is then taken as the Tm


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Denaturation of DNA results in an increase in

optical densityODSS and ODDS indicate the optical density of single-

stranded and double-stranded DNA respectively.

Oft h b idi ti diti h t


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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 stablethermodynamically when the region of duplex

formation contains perfect base matching.

Mismatches between the two strands of aheteroduplex 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

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Probe Labeling1