Nucleic Acid Labeling Methods
The labeling methods for the nucleic acids which become the samples can be divided into methods of labeling before hybridization and methods of labeling after hybridization.
Labeling before Hybridization
Methods of labeling before hybridization can be further divided into: (i) a method of binding fluorescent dye via aminoallyl group, (ii) a direct labeling method using an enzymatic reaction when sample DNA is amplified, and (iii) a chemical modification method.
Method (i) involves incorporation of dUTP which has an aminoallyl group during the reverse transcription reaction or in vitro transcription reaction. The incorporated aminoallyl group is covalently bonded with fluorescent dye. The advantage is the high incorporation efficiency of aminoallyl groups into the product. However, this labeling method necessitates two reaction steps (incorporation and binding of dye) and requires time.
Method (ii) requires only one step for labeling. Thus, it can be completed in a short period of time. However, it is thought to frequently have a low incorporation efficiency of dye.
Method (iii), the chemical modification method, involves covalently bonding fluorescent dye to the G residues in the nucleic acid sequence. As in method (ii), the advantage of method (iii) is the short reaction time for labeling. Its disadvantage is the difficulty of incorporating labels depending on the sequence of target nucleic acids.
Labeling after Hybridization
Methods of labeling after hybridization include: (iv) the A-B-C (Avidin-Biotin-complex) method and (v) detection methods using tag sequences.
Method (iv) is shown in the figure below. Biotinylated nucleic acids are introduced into nucleic acids which will be the sample in advance at the time of synthesis and amplification, and the resulting sample is hybridized on the DNA chip. Then labeling is achieved by the addition of a labeled compound incorporated with avidin, which strongly binds to biotin, and the addition of labeled anti-avidin antibody. The A-B-C method is effective for the one-color fluorescence method for detection, but it is not applicable to the 2-color fluorescence detection.
Method (v) involves the addition of tag sequence at the end when synthesizing the target nucleic acid. Hybridization is performed with only target nucleic acids. Next, the second hybridization is performed using labeled nucleic acid complementary to the tag sequence, and the detection of target nucleic acid is performed. The sensitivity of detection can be improved by changing the degree of labeling of the labeled nucleic acids. Preparation of two types of tag sequences makes the method (v) compatible with the 2-color fluorescence method. Since hybridization is performed twice, the work time increases, and the effects of experimental bias need to be considered.
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