INTRODUCTION TO PRIMER - PROBE

The probe is a nucleic acid molecule (single-stranded DNA or single-stranded RNA) with a specific affinity for specific purposes (DNA sequences or RNA). Probes and target base sequences complement each other, but depending on the conditions, they do not necessarily complement. The hybrid (combination probes) can be detected when an appropriate identification and marking system is used. Genetic probes are often used in many blotting techniques and in situ techniques for the determination of nucleic acid sequences. In medicine, they can help identify microorganisms and diagnose genetic diseases, infectious diseases and many other diseases.

2. Sample design

The specimen design depends on the requirement to use a gene template or an oligonucleotide probe.

2.1. Gene Probes

Genetic probes are generally longer than 500 bases and contain the entire (or majority) of the target gene sequence.

They can be created in two ways:

In the first way, cloned probes are used when a specific clone is available or when the unknown DNA sequence needs to be cloned for mapping and sequencing. Target genes were cut with limited enzymes and recovered from agarose gel. In case of homologous vectors, unnecessary cutting reactions proceed. Polymerase chain reaction (PCR) with the ability to amplify DNA simultaneously fights nucleotides, using plasmid DNA or chromosomal DNA, which is the process of producing effective probes.
The second design is carried out when there is a whole sequence of genes from data sources such as GenBank, EMBL, DDBJ. Based on the sequence found, it is easy to design primers to amplify the entire gene or genome by PCR. This saves a lot of time because of the limited enzyme cut-off, electrophoresis, and DNA elution from the vector. However, if PCR is given to products containing non-specific bands, purification of the bands used as probes should be performed to ensure accuracy in subsequent experiments.
2.2. Oligonucleotide probe (Oligonucleotide Probes)

Oligonucleotide probes target specific sequences within the gene. The typical size of this sample is 18-30 bases. However, today's synthesis devices allow for the efficient design of probes containing at least 100 bases. The oligonucleotide probe attaches precisely to the target sequence. Their length is suitable for cross-reactivity under stringent conditions, helping to identify DNA with very small differences in sequence. The selection process for oligonucleotide probes can be made using the known genetic sequences, as follows:

Probe length: 18 - 50 base. Longer lengths will result in longer hybridization times and lower composite ratios, and shorter size will reduce specificity.
The G - C component should be 40 - 60%. Unspecified hybridisation increased when G-C was outside the range.
Make sure there are no additional areas inside the probe, as they can create a hair-pin structure and prevent hybridization.
Avoid sequences that contain single sequences of nucleotides (such as AAAA).
Once the order meets the above requirements, it is still necessary to analyze the sequence on the computer. The probes should be compared with the source sequence or source genome as well as the additional sequences of the source sequences. If the homology to the (non-target)> 70% or> 8 bases in a row is observed, the sample is not eligible.
However, to determine optimum hybridization conditions, crosslinking with specific and specific non-specific nucleic acids should be carried out in the context of large variations in hybrid conditions.

The same procedure applies the design of primers and primers used in PCR. Note that, except for the area to be amplified, the 3 'end of the primer and backend is not homogeneous with other molded DNA segments.

3. Mark the sample

3.1 Types of markers

3.1.1. Radioactive labels (Radioactive Labels)

Nucleic acid probes may be marked with radioactive isotope (32P, 35S, 125I, 3H) and determined by X-ray image or Geiger-Muller system. Radioactive markers have been used extensively in the past, but with safety concerns as well as prices and the issue of radioactive waste, people today are less likely to use them. This is the type of detector that has the highest sensitivity and resolution. Only a small amount of target-target hybrid can be detected, for example: the 32P marking probes are capable of identifying copies of the gene in 0.5 μg DNA. Keller and Manak explained as follows:

32P has the strongest specific activity.
32P high beta particle dispersions.
Although this sample detects small amounts of target DNA (<1pg), their shortcomings are short half-lives (only used within 1 week after preparation) and can not be used for technical purposes. High resolution images. Compared with 32P, low energy and long half-life make 35S isomer stable and less specific. The 35S markers, though with lower sensitivity, offer higher resolution X-ray images and are particularly suitable for in-situ hybridization. Another advantage of 35S marking nucleotides is less toxic than 32P. Low energy b particles do not penetrate the upper skin and are easily captured by laboratory tubes. Similarly, 3H markers have been used in situ hybridization because they emit low energy β particles, resulting in maximum contrast and low interference, which also have time to sell. the longest (12.3 years). The use of the 125I and 131I decreased since the 1970s due to the availability of the 125I nucleoside triphosphate marker with high specificity. 125I has lower energy and longer half life

(60 days) compared to 131I, commonly used in spot hybrid. Non-radioactive Markers With radioactive markers, using non-radioactive markers has several advantages: Safety. Higher stability. Effective marking. Define in place. Need less time. Some signals in non-radioactive markers: Biotin: Discovered using avidin or streptavidin. Streptavidin, avidin has a strong affinity for biotin. Since the reporter is not directly linked to the probe but attached to it via a bridge (eg, streptavidin-biotin), this form of identification is known as an indirect system. Biotin probes work very well, but biotin is a common molecule in mammalian tissue and biotin probes tend to stick to certain types of nylon membranes. can occur during hybridization. This problem will be solved by using nucleotide derivatives, including: digoxigenen-11-UTP, -11-dUTP, -11-ddUTP, and biotin-11-dUTP or biotin-14-dATP. After crossing, identify target / target genes by antibody or avidin, followed by luminescent / color reaction catalysed by alkaline phosphatase or peroxidase linked to antibody or avidin. Enzyme: Enzyme molecule ( called the reporting group) attached to the probe and its presence is determined by the reaction with the substrate. Common enzymes include alkaline phosphatase and horseradish peroxidase (HRP). In the presence of peroxide and peroxidase, chloronaphtol - a substrate of HRP - produces a non-tanning color. HRP also catalyzes the oxidation of luminol - a luminescent molecule. Chemiluminescence: In this method, the luminous chemicals associated with the sample are determined by the light they emit. out. The chemical detector (including the enzyme probe) can be separated from the membrane, allowing the membrane to bind multiple times without resolution. Fluorescence chemicals: with fluorescent detector under UV light. This type of detector is extremely useful in the study of cell samples or microscopic samples under the microscope - this technique is called fluorescent in situ hybridization (FISH). FISH probe: Antibodies: A group of antigens associated with the probe, their presence detected by specific antibodies. At the same time, monoclonal antibodies will recognize DNA hybrid DNA. DIG system: This is the most complete, useful and efficient system in marking and identification of DNA, RNA and oligonucleotide. Like biotin, digoxigenin (DIG) can bind to the linker and nucleotide; Nucleotides are replaced by DIG combining nucleic acid probes with any enzyme method. Compared to biotin probes, these samples produce less noise. An anti-digoxigenin antibody-alkaline phosphatase complex is capable of binding to the DIG marker. The signal is determined by the colorant or luminescence of the alkaline phosphatase. If the substrate is used, the signal forms directly on the film. In case of light substrate, using X-ray film to show the signal.Table 1. Types of markers3.2. Marking method3.2.1. Nick Translation The cut-off point is a DNA marker method that uses the enzyme DNase I pancreas and Escherichia coli DNA polymerase I. In reaction, DNase I cleaves the DNA. At some point, nickel and E. coli DNA polymerase I will add the nucleotide to the 3'-OH site exposed at the cut sites, while the 5 '- 3' exonuclease activity will remove the nucleotide at the 5 ' . If the nucleotide markers are present in the reaction, these nucleotides will join the DNA. For radiolabeled DNA, [α-32P] dNTP will be added. For non-radioactive specimens, digoxigenin or a biotin tip attached to a similar dNTP molecule will be used. Nick Translation 3.2.2. Random-Primed Labeling / Primer Extension Genetic detection (PCR amplification or amplification), oligonucleotide probes may be marked by random primers with radioisotope and non-radioactive (eg DIG). The double-stranded DNA molecule undergoes denaturation and incorporates random oligonucleotide primers (6-mers). These oligonucleotides act as primers for the 5 '- 3' polymerase (Klenow fragment). The marker is synthesized by this enzyme when the nucleotide is marked. Figure A shows the steps in the DIG sign by random sampling. (A). Marking DIG with random bait. Double-stranded DNAs and random oligonucleotide primers (6-mers); This oligonucleotide acts as a primer for the Klenow fragment of E. coli DNA polymerase I, which synthesizes the DIG-dUTP marker. (B). DIG Marking by PCR: DIG-dUTP joins the DNA circuit during amplification of the target DNA template. An asterisk expression

digoxigenin molecule.3.2.3. Digitization by DIG-PCR Labeling This is an advanced method for marking gene samples with DIG using PCR. The probe was amplified using suitable primers, however, the dNTP mixture contained less dTTP because DIG-dUTP was also added to this reaction. The advantage of this approach is that it attaches multiple DIG strands along the DNA circuit during amplification. Figure B shows the steps in the DIG marking process by PCR. Photobiotin Labeling Photobiotin labeling is a chemical reaction, not simply an enzyme reaction. Biotin and DIG are linked to a nitrophenyl azide group. This group will be altered by emission with UV light or visible light, producing strong nitrous molecules. This group can make strong covalent bonds with DNA and RNA. Materials for photobiotin labeling are more durable than enzymes in cut-off point shifts or oligonucleotide markers and are cheaper than other methods. However, the amount of probe produced is large but the sensitivity is not high. Photobiotin has chemical formula C23H35N9O45 • C2H4O2. The molecule consists of a biotinyl group, a cad linker, and a nitrophenyl azide group. End Labeling This method is mainly used for marking the oligonucleotide sample. Three marker methods for oligonucleotide probes with digoxigenin: Mark the 3 'end of a 14 - 100 nucleotide oligonucleotide with a DIG- 11-ddUTP / 1 oligonucleotide molecule. The 3 'tail extension response: the terminal transferase site attaches to the unmarked nucleotide mixture and the DIG-11-dUTP is the tail containing the various DIG-11-dUTP components. 'consists of two steps: synthesizing a first 5' amino bond bridge; After purification, then a digoxigenin-N-hydroxy-succinimide ester binds to the 5'-amino-free head. Oligonucleotides may also be marked with the bacteriophage T4 polynucleotide kinase. If the reaction is successful, the activity of the specimen may be as high as [γ-32P] ATP activity.