DNA probe

Nucleic acid probe

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DNA DNA probe is the most commonly used nucleic acid Probes with length of tens to hundreds or even thousands Base pair Single chain or Double chain DNA， With special tracer (e.g isotope , enzyme or colored group); At proper pH, temperature and ionic strength DNA probes use molecular denaturation 、 Renaturation as well as complementary base pairing Height of Accuracy , unmarked single stranded DNA or RNA Hydrogen bonding (hybridization) to form double chain complex（ Hybrid ）。 The stability of hybrid depends on two single chains nucleotide The degree of complementarity between them. Under strict conditions (high pH value, high temperature, low ionic strength), the two strands of the incompletely matched hybrid will be dissociated, while the perfectly matched hybrid will keep double strands. The unpaired probe can be used after washing Autoradiography or Enzyme-linked reaction And other detection systems to detect the hybridization reaction results.^[1]

Chinese name: DNA probe
Foreign name: DNA probe
Purpose: Detection of nucleic acid

Principle: complementary base pairing
Source classification: Genome probe, cDNA probe, oligonucleotide probe
Dependent on technology: Molecular cloning technology

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DNA probe According to its source, it can be divided into three types: one from genome The gene itself in is called Genome probe (genomic probe), which can be the whole sequence of a gene or a segment of a gene; The other is obtained by transcription from corresponding genes mRNA , and then pass Reverse transcription The obtained probe is called cDNA probe （ cDNA probe）； In addition, 20-50 can be artificially synthesized in vitro Base The DNA fragment complementary to the gene sequence is called Oligonucleotide probe 。^[1]

preparation

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The acquisition of genomic DNA probes depends on Molecular cloning technology Development and application of. To obtain a specific DNA probe is often cumbersome. Take bacteria for example Genome size About 5 × 10 six Base, containing about 3000 genes. To obtain a specific nucleic acid probe for a bacterium, it is usually necessary to establish a bacterium Genome DNA Library The method is to cut bacterial DNA into small pieces (such as Restriction endonuclease Incomplete hydrolysis) were cloned to obtain Full information And then screened with DNA probes of a variety of other bacteria, and the clones generating hybridization signals were eliminated. The remaining clones that did not hybridize with any other bacteria might contain specific DNA fragments of the bacteria.

The cDNA probe obtains mRNA from the corresponding gene through transcription, and then passes Reverse transcriptase The probe obtained by the action of, without Intron Sequence.

Small single stranded oligonucleotide probes can be synthesized by machines in vitro. Oligonucleotide probes are stable and highly specific. Under very strict conditions, hybridization tests with oligonucleotide probes can detect single base mutations and Mismatch However, the oligonucleotide probe is short, with few markers and low sensitivity.^[1]

advantage

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① DNA probe polyclonal Plasmid vector The preparation method is simple;

② DNA probe relative RNA probe （ RNA Probe) is not easy to degrade, and generally can effectively inhibit DNase activity;

③ The labeling methods of DNA probes are mature, which can be labeled with isotopes or non isotopes, and there are many methods to choose from.^[1]

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DNA probes fall into two categories: isotope Marked probe and non Isotope labeling The probe of. Isotope labeled probes usually have high radioactivity Specific activity , hybridization sensitivity is high, but the service life is short, and radioactivity Hazards, difficult disposal of pollutants, special instruments and equipment required, not suitable for ordinary laboratories. in recent years non-isotope labeling The method has been greatly developed, such as enzyme catalysis Markedness (such as biotin digoxin Labeling) and chemical labeling (such as fluorobiotin Enzyme labeling ）。 Non isotope labeled probe Save time It is longer and avoids isotope pollution, but it is less sensitive than the isotope labeled probe. Next Isotope labeling The labeling method of DNA probe is introduced as an example.

1. Notch translation method (nick translation ）

Fig. 1 Notch translation method

Notch translation As the most commonly used probe labeling method, the main components of the reaction system are DNase I（ DNase I ）、 Escherichia coli DNA polymerase I (DNA polymerase I), three Triphosphate Deoxyribonucleotide An isotope labeled nucleotide (e.g. dATP dTTP 、dCTP，”P— dGTP ）The principle is shown in Figure 1. First, apply appropriate concentration of DNase I to probe DNA Double chain Randomly cut a number of gaps on the molecule (not cut DNA or degrade it), and then use the DNA Polymerase I 5 '→ 3' of Exonuclease Activity: cut off the nucleotide at the 5 'end; At the same time, the 5 '→ 3' polymerase activity of the enzyme is used to thirty-two P labeled complementary nucleotides fill the gap DNA Polymerase The alternative action of these two activities of I makes the gap move to the direction of 3 ', and the nucleotides on the DNA chain are constantly replaced by 32P labeled nucleotides. Because the reaction system contains isotope labeled Mononucleotide , make Neosynthesis The band of is isotope labeled, so the gap translation is actually that isotope labeled nucleotides replace the same nucleotides without isotopes in the original DNA chain.

two Random primer method （random primer）

Schematic diagram of random primer method^[2]

The denatured probe solution was added with small random DNA fragments of 6 nucleotides as primer , when the latter and Single strand DNA After complementary combination of multiple sites principle of complementarity By continuously adding isotope labeled single nucleotide to its 3 '- OH end, DNA probes with high specific activity can also be obtained.

3． End labeling （end—labelling）

Schematic diagram of end marking principle^[2]

The end labeling method is not to label DNA in its full length, but only to introduce markers at its 5 'end or 3' end for partial labeling. This labeling method can obtain a full length DNA probe because it carries Marker molecule Less, so the marker activity is not high.^[1]

Hybridization method

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For any DNA probe determination, hybridization reaction includes four basic elements: probe Target substance (nucleic acid to be tested in the sample), test method (according to the Markers or Reporter gene And hybrid mode. molecular hybridization of nucleic acid Can be divided into Liquid hybridization and Solid-phase hybridization 。

1. Liquid hybridization

Liquid hybridization is to make the DNA probe and nucleic acid React in solution. In the solution, both the nucleic acid to be measured and the probe move freely, increasing the chance of combining them. Therefore, the liquid phase hybridization is 5-10 times faster than the solid phase hybridization. However, liquid hybridization is not easy to separate Hybrid And dissociation Nucleic acid probe Conventional application is not easy.

2. Solid phase hybridization

Solid phase hybridization is to first combine the nucleic acid sample to be tested with the solid carrier, and then analyze the hybridization results with the detection signal dissolved in the solution.

The basic procedure of solid phase hybridization is: ① prepare the sample to be tested; ② Preparation and Marking probe ；③ Solid carrier treatment; ④ Prehybridization , hybridization, rinsing; ⑤ hybridization signal detection ；⑥ Result judgment and analysis.

The commonly used solid phase hybridization methods are Spot hybridization 、 Sandwich hybridization Law and In situ hybridization Etc. Several commonly used nucleic acid molecular solid phase hybridization methods are briefly introduced below.

① Dot blot hybridization: the most commonly used hybridization mode. Dot the sample DNA directly on the Nitrocellulose membrane or Nylon membrane The test was carried out after hybridization under strict conditions. Dot hybridization is simple and rapid, and does not need Restriction endonuclease Digest DNA, and do not need gel electrophoresis And transfer, and multiple samples can be detected on one film. This method susceptibility High, but non strict control of hybridization conditions may lead to non Specificity hybridization.

② Sandwich Hybridization method （sandwich hybridization ）: Two adjacent but non overlapping genes DNA sequence To make a probe, first attach the first unmarked probe (probe A, capture probe) to a solid phase support (such as a membrane, hole or tube), capture the complementary target sequence in the sample to be tested, and then add the second labeled probe (probe B, detection probe) Complementary sequence The hybridization signal can be detected after combination. The sandwich hybridization method has the advantage of good specificity and low requirements for the purity of nucleic acid samples.

③ In situ hybridization (in situ hybridization): a method of hybridization and detection of nucleic acids in tissues or cells with nucleic acid probes. First fix the tissue or cell to be tested on the solid carrier, and then use appropriate Detergent and protease , acid and other substances to treat the sample, so that the labeled nucleic acid probe can enter the cell and form a hybrid with the nucleic acid to be tested, so that the nucleic acid sequence in the tissue or cell can be directly detected. In situ hybridization for cells and even Subcellular The level of nucleic acid detection provides a direct method.^[1]

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