Nucleic acid hybridization

Table B: Factors influencing the rate of hybridisation3(continued) 

Table B: Factors influencing the rate of hybridisation3(continued) 

When all the factors for hybridisation are considered, one needs to consider about the time and nucleic acid probes that will affect hybridisation. In terms of time, it might appear that long hybridisation incubations would be expected to give the best results3. However, other factors such as double-stranded probe reassociates in solution and non-specific background forms on the filter may affect the rate of hybridisation reaction3. At the same time, nucleic acid probes need to be greater than 15 bases and less than 1.5 kbp in length3. Table C shows the effect if the probes are shorter than 15 bases or the length is more than 1.5 kbp:

Table C: Effects of hybridisation reaction under different types of probes3

All the above conditions / factors are considered in nucleic acid hybridisation in order for the technique to work well.

        The descriptions above are the factors that affect hybridisation and are an example of filter hybridisation, one of the simplest types of hybridisation in molecular biology (which was done in the experiment). There are other types of hybridisation and are described in table D below:

Table D: Types of hybridisations1,2,3

Table D: Types of hybridisations1,2,3 (continued)

Table D: Types of hybridisations1,2,3 (continued)

Table D: Types of hybridisations1,2,3 (continued)

Almost all the types of hybridisation mentioned in table D follows the three basic steps as for filter hybridisation, with only minor differences between them.

        Some types of hybridisation are evolved to be used nowadays in molecular diagnostic assays. The first type is known as Southern Blotting, which was created by Edward Southern1,2,7. Another type of hybridisation technique is known as Northern Blotting. Both techniques are widely used in many areas. Table E shows the characteristics of both types of blotting and how each type of blot is used and figs 2 and 3 below show both types of blotting:

Table E: Characteristics of both northern and southern blots and their respective uses2

Fig 2: Southern blotting2.

First, we electrophorese DNA

fragments in an agarose gel.

Next, we denature the DNA with

base and transfer the single-

stranded DNA fragments from

the gel to a sheet of nitrocellulose

or similar material. We can do this

in two ways: by diffusion, in

which buffer passes through the

gel, carrying the DNA with it

(left), or by electrophoresis (right).

Next, we hybridise the blot to a

labelled probe and detect the

labelled bands by

autoradiography or

phosphorimaging2.

   

Fig 3: A Northern blot2.

Poly (A)+ RNA was isolated from

The rat tissues indicated on the

right, then equal amounts of

RNA from each tissue were

electrophoresed and Northern

blotted. The RNAs on the blot

were hybridised to a labelled

probe for the rat G3PDH gene,

and the blot was then exposed to

x-ray film. The bands represent

the G3PDH mRNA, and their

intensities are indicative of the

amounts of this mRNA in each tissue.

In overall, nucleic acid hybridisation has helped in many areas as shown in table F below:

Table F: Nucleic acid hybridisation and its uses generally1

        In nucleic acid hybridisation, both radioactive (e.g. 32P and 35S) and non-radioactive labelled probes are being used to target nucleic acid1,2,3. However, the total amount of target nucleic acid in a sample is often very low and hence the signal generated by the hybridisation of specific probes is often undetectable1. In order to overcome such limitation, schemes for target amplification (e.g. using PCR) have been developed1. However, no matter how good the schemes are, to produce useful information on the size, identity and quantity of the amplification products, there are some limitations1. One of the main disadvantages is the risk of contaminating untested samples, since the amplification products have to be transferred from the reaction solution to a gel matrix for electrophoresis analysis8. Moreover, the synthesis of non-specific amplification products can be significant when the targets are rare or absent and the sample contains an abundant and diverse nucleic acid hybridisation1. Hence in order to simplify and detect amplified targets in sealed tubes in real time, other types of probes / primers are introduced and listed in table G (fig 4 shows the schematic representation of some nucleic acid hybridisation probes / primers):

Table G: Types of probes / primers, characteristics and their uses1

Fig 4: Schematic representation of fluorescence signal generation with different nucleic acid hybridisation probes1

Fig 4 (continued)1

With these newly introduced probes, nucleic acid hybridisation can be made to detect amplified targets in real time and improves in the techniques of such process.

        Overall, nucleic acid hybridisation has been a great help in terms of speed and simplicity1. It can be combined with other molecular biology techniques and can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations1.  

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