Thursday, October 27, 2011

Nucleic acid analysis without Amplification (Nucleic acid probe technology) Part I

A nucleic acid probe is a labeled sequence of single stranded DNA or RNA that can hybridise specifically with its complementary sequence (Smith, 2002).
Nucleic acid hybridization technique
Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification, visualization and localization of microorganisms in many fields of microbiology (Thurnheer et al., 2004 & Amann et al., 2001).
FISH was mainly applied in connection with environmental samples during the first years but it became clear that the method also has advantages in diagnostic microbiology for rapid identification and direct visualization of bacteria (Kempf et al., 2000).
Nucleic acid hybridization is a technique was first described in 1961 by Marmur and Doty. Most molecular diagnostics testing procedures use the basic concept of nucleic acid hybridization.
Nucleic hybridization refer to formation of hydrogen bonds between nucleotides of single stranded DNA and/or RNA molecules that are complementary to each other (Marras et al., 2006). This form a stable double stranded nucleic acid molecule. The resulting double stranded hybrids may be DNA:DNA; DNA:RNA, or RNA:RNA.
This hybridization process called duplex formation. This process is key of component for many testes including blotting methods, PCR, and other molecular based techniques.
The two single stranded nucleic acid molecules used in hybridization techniques are referred to by different terms. One of the strands is known as the target.
The target strand is the DNA or RNA sequence that will be identified by the employed molecular diagnostics method. The target is referred to as the template it can be either immobilized on a solid support mechanism or is suspended in solution.The other strand is called the probe (Jensen TK, et al., 2001) (see figure, 4)
The probe is usually a single stranded DNA or RNA oligonucleotide that is labeled with an attached reporter chemical or a radionucleotide that can be detected either visually, by film, or by an instrument. The probe is produced synthetically to detect a specific target (Mothershed E.A., Whitney A.M., 2006)
Hybridization reaction variables
Several variables affect the outcome of a given hybridization reaction. These variable include;
The stability of a given hybrid can be calculated by determining the melting temperature (™) of a probe. The Tm is the temperature at which 50% of hybrids have formed and 50% of the single stranded nucleic acid molecules are still dissociated. Tm is dependent on the G+C ratio because three hydrogen bonds form between G and C, instead of the two hydrogen bonds that form between adenine (A) and (T);The G\C bond pair is more thermodynamically stable than the A\T bond pair.
-Length of the probe
Another aspect that affects the Tm is the length of the probe ; in general the Tm is lower for a shorter probe.Hybridization reactions tend to occur more rapidly for shorter probes than for longer probes
-Probe Concentration
Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences. However, excessive probe concentrations promote nonspecific binding of the probe to non target sequences
-Salt concentration
The rate of a hybridization reaction will increase as the salt concentration increases, up to a threshold; past 1.2 M Nacl, the rate of the reaction become constant.
Neutral PH is preferable for most hybridization reactions
-Probe selection
Selection of proper probe for nucleic acid hybridization reaction is important as the hybridization method itself. probes may be either DNA or RNA based, and are either radiolabeled or non isotopically labeled.
Radiolabeled probes are rarely used due to have short half lives and un desirable waste.radiolabeled probes have been replaced by nonisotopic labels, including biotin digoxigenin (DIG), and fluorescein, nonisotopic labels have resolution and sensitivity that approache .A probe may also be end labeled or continuously labeled
Hybridization Formats
Hybridization reaction may occur in a solid support mechanism, in situ or in solution
A-Solid support hybridization
Technique often called (blotting) the target nucleic acid is transferred and immobilized to a membrane, composed of either nitrocellulose or nylon. Labeled probe is then hybridized to the immobilized nucleic acid washing steps are used to remove excess probe. Two examples of solid support hybridization techniques are:
1-Southern Blot
The southern blot was first described in 1975 by E.M southern; he described a technique whereby chromosomal DNA has digested with a restriction enzyme then separated by agarose gel electrophoresis, then transferred and immobilized to a nitro cellulose membrane, then labeled probe is hybridized to the specific target DNA sequences. [Figure, 5]The southern blot takes more than one day to perform, it can be used to identified micro organism, to detect mutation, to type strains for epidemiological investigation, and for other purposes (Vanrompay.d., 2000)

2- Northern Blot
A northern blot was first described by Alwine et al 1977; northern blot used to determine the size of particular RNA transcript. It has the same procedure of the southern blot but with difference, the restriction enzyme is not used to digest RNA before separation due to RNA is small enough to be separated by agaros gel electrophoresis. Like southern blotting, northern blotting is not often used in clinical microbiology laboratories (see figure, 6)

3-In Situ Hybridization
In Situ Hybridization (ISH) first described in 1969 by Pardue and Gall, is also not used in clinical microbiology laboratories.This is a method of hybridization wherein DNA or RNA transcript can be detected directly in the tissue with labeled probes.ISH may used to detected low level of viruses in tissue specimen such as human papillomaviruses (HPV) (Vanrompay.d., 2000)
B-In Solution Hybridization
In solution hybridization is the type of hybridization reaction most often used by clinical microbiology laboratories.Hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells, usually detection methods are chemiluminescent based. It used to rapidly identify infectious disease organisms.

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