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Nucleic acid hybridization techniques


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Introduction Nucleic acid hybridization is a fundamental

tool in molecular genetics which takesadvantage of the ability of individual single-stranded nucleic acid molecules to formdouble-stranded molecules (that is, to

hybridize to each other)

Standard nucleic acid hybridization assaysinvolve using a labeled nucleic acid probe to

identify related DNA or RNA molecules (thatis, ones with a significantly high degree ofsequence similarity) within a complex mixtureof unlabeled nucleic acid molecules, thetarget nucleic acid.


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Nucleic acid probes Hybridization probe is a fragment of DNA or

RNA of variable length (usually 100-1000bases long), which is used in DNA or RNAsamples to detect the presence of nucleotide

sequences (the DNA target) that arecomplementary to the sequence in the probe.

The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose

base sequence allows probe-target basepairing due to complementarity between theprobe and target.


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Nucleic acid probes may be made as single-stranded or double-stranded molecules but

the working probe must be in the form ofsingle strands.

Probe typesFour types

Oligonucleotide probes

Single stranded DNA probes

Double stranded DNA probes

RNA probes (cRNA probes or riboprobes)


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Oligonucleotide probes

Produced synthetically by an automatedchemical synthesis. The method utilizesreadily available deoxynucleotides butrequires to know the specific nucleotide

sequence we wish to prepare.When using oligonucleotide probes its notjust a matter of picking any region within thecoding region of the target gene to bind to but

requires careful design taking into account anumber of issues .


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These probes have the advantages of beingresistant to RNases and are small, generally

around 40-50 base-pairs.

This is ideal for in situ hybridization becausetheir small size allows for easy penetrationinto the cells or tissue of interest.

In addition, because they are syntheticallydesigned, it is possible to make a series ofprobes that have the same GC content.

So with oligonucleotides protocols can bestandardized for many different probesirrespective of the target genes beingmeasured.


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Another advantage of the oligonucleotide

probes is that they are single strandedtherefore excluding the possibility ofrenaturation.

Single stranded DNA probes

These have similar advantages to theoligonucleotide probes except they are muchlarger, probably in the 200-500 bp size range.

They can be produced by reverse transcriptionof RNA or by amplified primer extension of aPCR-generated fragment in the presence of asingle antisense primer.


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That is, once you have amplified the

sequence of interest, a subsequent round ofPCR is carried out using the first PCRproduct as template, but only using the anti-sense primers, thus producing single

stranded DNA. This is therefore theirdisadvantage.

They require time to prepare, expensivereagents are used during their preparation

and a good repertoire of molecular skills arerequired for their use.


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Double stranded DNA probesProduced by the inclusion of the sequence of

interest in a bacteria which is replicated, lysed andthe DNA extracted, purified and the sequence ofinterest is excised with restriction enzymes.

On the other hand, if the sequence is known then by

designing appropriate primers followed by PCR,potentially obtaining a very clean sample.

The advantage of the bacterial preparation is that itspossible to obtain large quantities of the probesequence in question. Because the probe is doublestranded,denaturation or melting has to be carriedout prior to hybridization .These probes aregenerally less sensitive because of the tendency ofthe DNA strands to rehybridize to each other and

are not as widely used today.


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RNA probesRNA probes have the advantage that RNA-RNA

hybrids are very thermostable and are resistant todigestion by RNases. This allows the possibility ofpost-hybridization digestion with RNase to removenon-hybridized RNA and therefore reduces thepossibility of background staining.

methods

Complimentary RNA's are prepared by an RNApolymerase-catalyzed transcription of mRNA in the3' to 5' prime direction.

Alternatively, in vitro transcription of linearizedplasmid DNA with RNA polymerase can be used toproduce the RNA probes. Here plasmid vectorscontaining polymerase from bacteriophages T3, T7

or SP6 are used


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These probes however can be very difficult to workwith as they are very sensitive to RNases

(ubiquitous RNA degrading enzymes) and so steriletechniques should be observed or these probes caneasily be destroyed.

RNA probes are still probably the most widely used

probes with in situ hybridization.


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Benefits of oligoNT probes

Oligonucleotide gene probes have multipleadvantages over RNA or cDNA probes whenused for in situ hybridization.

They are

Stability, Availability, Faster and lessexpensive to use, Easier to work with, Morespecific, Better tissue penetration, Betterreproducibility and a wide range of labeling

methods that do not interfere with targetdetection.


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Probe labellingTwo approaches

Radioactive labelling

Non radioactive labelling

Radioactive labelling

Developed in 1970Time consuming and laborious

Recents developments use T4 polyNT kinase orE.coliDNA polymerase I.

Involves 3 methods

Nick translation

Random primed radiolabelling

Probes developed by PCR


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Nick translation

Label dsDNA probes.

Nick is made on dsDNA by Dnase I andtranslated by E.coliDNA polymerase.

5'-3' exocatalyticactivity.

NTs are removedfrom 5'end and newNTs are added at 3'end [dATP,dGTP,dTTP,32 -P-dCTP]

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Random primed radiolabelling

Feinberg & Vogelstein [1983-84].

Restriction fragments purified by gelelectrophoresis from linear or circular DNAare used for preparing probes.

Purified DNA is mixed in a buffer and heatedat 100C for denaturing and transferred to ice

The reacn mix[klenow fragment ofE.coli,DNA Pol I,dATP,dGTP,dTTP,32 -P-dCTP

Primer extension .


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Probes developed by PCRAdvantageous.

Reactn mix [ linear template DNA,amplification buffer, Taq pol, primers,dATP,dGTP,dTTP,32 -P-dCTP

Hybridized DNA is isolated and used asprobe.


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Non radioactive labelling3 ways

Horse radish peroxidase

Digoxigenin

Biotin&Steptavidin

Horse radish peroxidase

HRP is a plant derived enzyme which iscovalently linked to DNA.

chromogenic[chloronaphthol emits purpleproduct] or chemiluminescent[luminol emitsluminesence] substrates detect HRP linkedDNA


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