Gene probe.

Gene
probe is a specific
nucleotide
sequence that complements the gene of destination or
DNA
and can include the entire gene, or it can be just a part of a gene;1. There are three sources of DNA probes from which the probe comes from: a gene itself from the
genome
, called a genomic probe, and a probe called a cDNa probe, which obtains mRNA from the corresponding gene transcription and reverse transcription. Unlike genomic probes, cDNA probes do not contain containing subsequencies. In addition, a small number of DNA fragments, called oligonucleotide probes, that complement the gene sequence can be synthesized in-body.2. Probe preparation For molecular mutations requires a large number of copies of the probe, which are generally obtained through molecular cloning. Cloning is the use of asexual reproduction methods to obtain a large number of copies of the same body, cell or molecule. When preparing a genomic DNA probe, a genome library should be prepared first, i.e. the genomic DNA is interrupted, or incomplete hydrolysis is made with restrictive enzymes, and many random fragments of varying sizes are obtained, recombined in-body to the carrier (phages, protons, etc.) and then trans-dyed. Appropriate host cells such as E. coli, then on the solid
culture
base can be obtained with different DNA fragments of cloned phage spots, through in-place hybridization, from which can be screened containing the clone of the gene fragments of the purpose, and then through cell amplification, the preparation of a large number of probes.
In order to prepare a cDNA probe, the purified corresponding mRNA needs to be isolated first, which is easier to do from
tissue
, which contains a large amount of mRNA, and from cells, such as α or β globin mRNA from hematocytes. With mRNA as a template, complementary DNA (i.e. cDNA) can be synthesized under the help of reverse transcriptases, which have exactly the same base sequence as the coding area of the gene to be tested, but the inclusions have been removed during processing.
oligonucleotide probes are synthetic and complement known genetic DNA and can be as long as a dozen to dozens of nucleotide fragments. If only the
amino acid
sequence quantity of
protein
is known, the nucleotide sequence can also be derived according to the code of the amino acid and synthesized by chemical method.. 3. Probe marking In order to determine whether the probe is hybridized with the corresponding genomic DNA, it is necessary to mark the probe in order to obtain an identifiable signal at the binding site, usually using a radioactive
isotope
32P marker probe of a nucleotide α phosphoric acid. However, in recent years, some methods have been developed using non-isotopes such as biotin, geo-high-sin complexes and so on as markers. But it's not as sensitive as isotopes. The advantage of non-isotope markers is that they take longer to save and avoid contamination of isotopes. The most commonly used probe marking method is nick translation. First, the appropriate concentration of DNA enzyme I. (DNAseI.) is used to create a gap in the probe DNA double strand, and then with the help of DNA polymerase I. (DNa poly meras I.) 5'→3' exochase activity, cut off nucleotides with 5' phosphoric acid; Using the enzyme's 5'→3' polyenzyme activity, the complementary nucleotides labeled 32P are filled into the gap, and the alternating action of the two activities of dna polymerase I. makes the gap move in the direction of 3', while the nucleotides on the DNA chain are constantly replaced by the 32P labeled nucleotides.The marking of
probes can also be marked by random primer method, i.e. the random DNA fragments of 6 nucleotides are added to the denatured probe solution as primer, when the latter is complementary to single-stranded DNA, the isotope-labeled mononucleotides are constantly added to their 3'OH ends according to the principle of base complementarity, so that DNA probes that are higher than radioactivity can also be obtained..