Probe labelingAman UllahB.Sc. Med. Lab. TechnologyM. Phil. MicrobiologyCertificate in Health Professional Education Lecturer, Department of Medical Lab. TechnologyInstitute of Paramedical Sciences, Khyber Medical University, Peshawar, Pakistan

Nucleic acid hybridizationIf a double-stranded DNA molecule is exposed to high temperature, or to very alkaline conditions, then the two strands will break apart. The molecule is said to have become denatured. The temperature at which denaturation occurs is termed as melting temperature or Tm. If the denatured DNA is returned to a temperature below its Tm or to neutral pH when alkali was used to denature it, each strand will, after a time, find its complementary strand. The two strands will ‘zipper’ back together to re-form a double stranded DNA molecule. This ability of complementary sequences to anneal, or hybridize, to one another is called nucleic acid hybridization. This technique helps in determining the gene structure and in identifying molecules which contain same sequences of nucleotides. In a complex mixture of nucleic acid molecules, nucleic acid hybridization technique helps in separation of complementary sequence.

NUCLEIC ACID HYBRIDIZATION When employed analytically, hybridization is

normally performed using one labeled sequence, termed the probe, and an unlabelled sequence called the target. Probe is a short synthetic oligo deoxyribonucleotide which is complementary to target DNA sequence. The probe is labeled by incorporation of either radioactively labeled nucleotides or with some chemicals. The probe is the known, pure species in the hybridization and the target is the unknown species to be identified.

The target will most often form part of a mixture of unrelated nucleic acid sequences.

METHODS OF LABELING NUCLEIC ACID & PROBES

There are five basic methods for labeling nucleic acids. These are:

Nick translation Primer extension Methods based on RNA polymerase End labeling methods Direct labeling methods

32P-labelling of duplex DNA by nick translation. Asterisks indicate radiolabelled phosphate groups.

NICK TRANSLATION This is done by making single-strand cuts (nicks) in the double

stranded DNA molecule by brief exposure to a dilute solution of an endonuclease (usually deoxyribonuclease 1 of E. coli). DNA polymerase 1 is then used in the presence of at least one radioactive precursor to “translate” the nick along the molecule in the 5’ to 3’ direction. The net result is that a nonradioactive strand of DNA is replaced by a radioactive strand. The DNA is then denatured and used as a radioactive probe in hybridization experiments (Southern blots, Northern blots etc).

Nick translation can be used with a variety of labels to generate probes suitable for most hybridization applications. It is also appropriate for the generation of biotinylated probes.

PRIMER EXTENSION METHOD Primers are synthetic oligodeoxyribonucleotides which are

complementary to specific regions of known vector DNA. The 3’ termini of these primers serve as initiation site for template dependent DNA synthesis by enzymes like DNA polymerase 1.

DNA polymerase works by extending a short double-stranded region made by annealing an oligonucleotide primer to the single-stranded template. Thus this method of uniform labeling requires a primer which matches the probe sequence. Radiolabelling of primers can be done with two methods.

If the probe sequence is not known then random oligonucleotide labeling can be used. It is often in the case when natural cellular DNA is used. These primers are made by adding a mixture of all four bases at each step in the chemical synthesis reaction. The DNA is denatured and the two complementary strands are copied in the presence of labelled primers as well as nucleoside triphosphates. The polymerase used is Klenow fragment derived from DNA polymerase-I of E. coli.

PRIMER EXTENSION METHOD Chance homology ensures that these primers anneal to

the separated DNA strands at many points along their length, thus providing a base for polymerase to initiate DNA synthesis. This is only one of several uniform labeling methods.

The second method uses a unique primer to restrict labeling to a particular sequence of interest. In the primer extension method, it is essential to use a polymerase lacking a 5’ 3’ exonuclease activity otherwise degradation of the primer will occur. The Klenow fragment of E. coli DNA polymerase I, which lacks the 5’ 3’ exonuclease activity has been used successfully.

It is an ideal method for situations where high specific activity and low probe concentrations are frequently employed.

The principle of random primed (oligo-) labelling. The DNA to be used as a probe is denatured by heating and mixed with hexanucleotides of random sequence which then act as primers for DNA synthesis.

METHODS BASED ON RNA POLYMERASES

RNA polymerases catalyzes the synthesis of RNA from nucleoside triphosphates using a DNA template. Thus they can incorporate labeled ribonucleotides into RNA during transcription if such labeled nucleotides are provided to it. If a specific site of a vector or DNA is transcribed in such way, RNA probes (or transcripts) of defined length and sequence can be obtained.

END-LABELLING OF NUCLEIC ACIDS

A wide variety of techniques is available for introducing label at either the 3’ or 5’ ends of linear DNA or RNA. Usually only a single label is introduced at the terminus. Nucleic acid can be 5’ end labeled using T4 polynucleotide kinase. Radiolabeled phosphate group is donated by [γ32-P] ATP to DNA or RNA containing a 5’-hydroxyl terminus. This is termed as a forward reaction.

If 5’-phosphate group is present in DNA or RNA, then it is removed with alkaline phosphatase. This reaction is driven by excess ADP which causes the enzyme to transfer the terminal 5’-phosphate from DNA to ADP. This is known as exchange reaction. The DNA is rephosphorylated by transfer of labeled γ-phosphate from [γ32-P] ATP.

The 5’-end labelling reaction using T4 polynucleotide kinase

END-LABELLING OF NUCLEIC ACIDS

The major advantages of 5’-end labeling are:

Both DNA and RNA can be labeled. Location of labeled group is known. Very small fragments can be labeled. Restriction digest fragment can be labeled.

CHOICE OF LABEL There are two technical parameters, resolution and

sensitivity, which determines the success of probe application. High degree of resolution is required to know the relative position of a nucleic acid fragment. High sensitivity is necessary because sequence of interest may be present at low abundance.

Other factors are probe stability, safety and ease of use. Broadly labels can be categorized into radioactive and nonradioactive types.

RADIOACTIVE LABELSThese labels have wider applications as they can be easily detected with autoradiography. Their detection gives two important information, firstly about occurrence of hybridization between probes and target DNA and secondly about their position. Radioactive methods using 32P are easily detectable. They are used often.

NON-RADIOACTIVE LABELS

A number of non radioactive labels for probes are available but biotin is widely used.

BIOTIN LABELLED PROBESBiotinylated probes are prepared through a nick-translation reaction by replacing nucleotides with biotinylated derivatives. After hybridization and washing, detection of hybrids is done by adding avidine and going through a series of cytochemical reactions which finally give a blue color whose intensity is proportional to the amount of biotin in the hybrid. There are several advantages of using biotinylated probes. The major advantages of using biotinylated probes are:

(a)assays employ non-toxic materials, with longer half-life.(b)can be prepared in advance in bulk and stored at -20℃ for

repeated uses.(c)Detection of hybrids is much faster than by radioactive

probes.

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