DNA Dyes Stain

DNA dyes stain deoxyribonucleic acid for laboratory purposes such as detection and quantification. Many DNA dyes also bind to RNA and could be more broadly described as nucleic acid stains. Common dyes included ethidium bromide (EtBr), esp. for agarose gel electrophoresis of DNA, and DAPI for staining the cell nucleus in fluorescent microscopy. DNA dyes stain are the most commonly used method for single- or double-stranded DNA ranging from tens to hundreds or even thousands of base pairs, labeled with a special tracer, such as isotopes, enzymes or chromophores. At the appropriate pH, temperature and ionic strength, the DNA can form a hydrogen bond with complementary non-labeled single-stranded DNA or RNA in the sample to be tested to form a double-stranded complex. The stability of the hybrid depends on the degree of complementarity between the two single-stranded nucleotides. Under stringent conditions (high pH, high temperature, low ionic strength), the two strands of the hybrid that are not perfectly matched will dissociate, while the perfectly matched hybrid will remain double stranded. After the unpaired bound probe is washed away, the hybridization reaction result can be detected by a detection system such as autoradiography or enzyme-linked reaction.

Source

DNA dyes stain are classified into three types according to their source: one derived from the genome itself, called a genomic probe, which can be the entire sequence of a gene, or a sequence of genes; the other is from The corresponding gene is transcribed to obtain mRNA, and the probe obtained by reverse transcription is called a cDNA probe. In addition, a 20 to 50 base DNA fragment complementary to the gene sequence can be artificially synthesized in vitro，which is called oligonucleotide probe

Advantage

DNA dyes stain is polyclonal in a plasmid vector, and the preparation method is simple;
DNA dyes stain are not easily degraded relative to RNA probes, and generally inhibit DNase activity;
DNA dyes stain labeling methods are more mature, can be labeled with isotopes or non-isotopes, there are many ways to choose.

Labeing

DNA dyes stain fall into two categories: isotopically labeled probes and non-isotopically labeled probes. Isotopically labeled probes generally have high specific activity, high sensitivity, but short duration of use, and radioactive hazards. Contaminant disposal is difficult, requiring special equipment and equipment, and is not suitable for use in general laboratories. In recent years, non-isotopic labeling methods have been greatly developed, such as enzymatic labeling methods (such as biotin, digoxin labeling) and chemical labeling methods (such as fluorescent biotin, enzyme labeling). Non-isotopically labeled probes have a longer shelf life and avoid isotopic contamination, but are less sensitive than isotope-labeled probes.

Preparation

The acquisition of genomic DNA dyes stain depends on the development and application of molecular cloning techniques. It is often cumbersome to get a specific DNA probe. Taking bacteria as an example, the genome size of a bacterium is about 5 x 106 bases and contains about 3,000 genes. In order to obtain a bacterial-specific nucleic acid probe, it is usually necessary to establish a bacterial genomic DNA library, that is, after cutting the bacterial DNA into small fragments (for example, incomplete hydrolysis by restriction enzymes), the entire information including the genome is cloned. The clone library is then screened with DNA dyes stain from a variety of other strains, and clones that produce hybridization signals are rejected, and the remaining clones that do not hybridize with any other bacteria may contain the bacterial-specific DNA fragments. cDNA probe is a probe obtained by transcribed an mRNA from a corresponding gene and then obtained by a reverse transcriptase, and does not contain an intron sequence. The small fragment single-stranded oligonucleotide probes can be synthesized by machine in vitro. Oligonucleotide probes are stable and highly specific under very stringent conditions. Hybridization assays with oligonucleotide probes can detect single base mutations and single base mismatches, but oligonucleotide probes are short, have fewer markers, and are less sensitive.

Reference:

Latt, SA.; et al. Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. Journal of Histochemistry and Cytochemistry. 1975, 23 (7): 493–505.

Dyes Application

Probe Application

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