Methods of labeling of nucleic acid that enable their detection
Nucleic acids (i.e., DNA and RNA) can be readily marked with the various tags that allow their detection and/or purification. These markings can then be used to recover or identify other interacting molecules. A plethora of chemical or enzymatic methods are available to produce nucleic acids, such as those marked with fluorophores, radioactive enzymes, and phosphates, or nucleotides modified with digoxygenin or biotin.
Bioconjugation methods that are employed for the generation of nucleic acid probes can also be adapted to attach nucleic acids to some other surfaces or molecules to assist immobilization or targeted delivery. The choice of the optimal method depends partially on the required degree of marking and on whether the modification can produce the desired interactions. Both chemical and enzymatic methods exist for creating that is marked at the five ′ or three ′ ends of the oligonucleotide. These methods can also be used to integrate probes throughout the sequence. When the small-scale generation of the probe is needed, enzymatic methods present an economic approach. However, chemical methods are relevant to the production of the largest scale.
Chemical methods for the labeling of nucleic acid
Periodates are anions which are formed from oxygen and iodine and are commonly found as sodium or potassium salts. The aldehyde groups that are created in solutions with periodates are spontaneously reactive towards amine-containing surfaces or molecules. Hence, oxidation of the RNA periodate is a common chemical method for labeling nucleic acid.
Carbodiimide EDC or 1-Ethyl-3- (3-dimethyl aminopropyl) is a water-soluble compound which is preferably used in aqueous reactions when the pH is between 4.0 and 6.0. EDC-mediated conjugation is an inexpensive approach for both DNA and RNA to any primary amine-containing surface or the coupled molecule. Random chemical labeling of DNA or RNA on the length of nucleic acids is also an effected labeling method that enables a higher degree of labeling when compared to labeling techniques.
However, a disadvantage of random labeling of the chemical is a direct modification of the nucleotide bases, which decreases or prevents the coupling of the bases between the complementary strands while hybridization experiments are underway. Hence, soldering the degree of the mark with the probe hybridization savings in some specific experiments is definitely authorized.
Enzymatic methods for nucleic acid labeling
DNA polymerase is an enzyme used to create DNA polymers through DNA extension or primer extension treatments. These enzymes are used to generate nucleic acid probes at random by incorporating modified nucleotides during DNA replication, especially from simple primer extension procedures or polymerase chain reactions. These probes exhibit high specificity enabling detection of even the smallest quantities of the target.
The terminal transferase of deoxynucleotidyl (usually abbreviated as TdT) is a DNA polymerase enzyme that is expressed in safe lymphoid cells. The usual sources of DNA models that can be modified with TdT include single-stranded and polymer-chain adenoid chain reaction (PCR) primers as well as the double-stranded restriction of endonuclease fragments with 3 'protrusions.
An enzyme known as the T4 RNA ligase (an adenosine triphosphate ligase) that originates from the T4 bacteriophage catalyzes the link between a terminal 5 '- phosphates and a terminal 3' - hydroxyl group on the RNA molecule. Despite the primary substrate for this specific ligase, which is RNA, the reaction conditions can be optimized for DNA molecules (more specifically, unique stranded DNA molecules) as well, with a somewhat lower yield.
The polynucleotide kinase T4 (abbreviated as T4 PNK), also found in bacteriophage T4, helps in transferring an organic phosphate from the adenosine triphosphate molecule into the 5 - hydroxyl group of a nucleic acid. This enzyme is independent of the model and can efficiently modify 5 ′ protrusions and single-stranded polynucleotides.
Nucleic acid labeling applications
Nucleic acids can be marked at their 3 'end, 5' end, or in the whole molecule, according to the desired application. The myriad of enzymatic or chemical methods is used to generate nucleic acids labeled with enzymes, radioactive phosphates, or fluorophores.
The techniques used are priming, nickel translation as well as in vitro or random polymerase chain reaction (PCR) transcription with the marked deoxynucleotide triphosphates (dNTPs) or nucleotide triphosphates (NTPs).
Hybridization between the bound nucleic acids and the labeled probe allows for the detection of specific DNA / RNA sequences found in a complex mixture of nucleic acids. The specific method of detection highly depends on the nature of the probe that is marked. More specifically, an enzyme-marked probe enables colorimetric or chemiluminescent detection, while radioactive probes facilitate autoradiographic detection.
Nucleic acid staining can provide valuable information on the integrity and copy number of the gene ( i.e., southern spot ) as well as means of analyzing mRNA size and gene expression (i.e., northern spot). These methods can be used to characterize cells and tissues grown in vitro. These methods often make clinical information important when used on patient samples.
The availability of different markings and a large selection of detection systems improve the sensitivity and flexibility required for in situ hybridization. The current options available from various manufacturers also allow for the simultaneous detection of multiple and differential probes marked within a single experiment.
Comparative genomic hybridization
Comparative genomic hybridization is based on in situ hybridizations of fluorescence and is used for the screening of tumors and for the detection of chromosomal imbalances. In this technique, marked DNA samples from two competitive sources are hybridized on the metaphase spreads of normal chromosomes. More specifically, one marked DNA sample is isolated from tumor tissue, and the other one is DNA differently marked from normal tissue. Differential hybridization allows the mapping of over-expressed or under-expressed chromosome regions on normal chromosomes.
The biotin mark is preferred as it is non-destructive. In this labeling technique, (larger) original DNA sequences are marked, which allows for analysis of loss or increase of DNA from the tested tissue sample of the tumor. The overrepresented chromosomal regions indicate tumor-promoting genes, while chromosomal eliminations potentially indicate the presence of suppressed genes.
Biotin-labeled nucleic acids have been used very successfully for the study of intracellular trafficking of gene delivery complexes, as well as the nuclear mechanisms of the inclusion of nucleoprotein molecules found in viruses. Since these labeling systems do not tear down nucleic acid, nor do they create nascent ones, it is possible to label and trace original DNA or RNA throughout the experiment.
Furthermore, there is a possibility for the meticulous study of the cellular interactions of the plasmid-marked gold DNA with the various gene vectors and with the cytoskeletal components of the cell, which can make understandings significant in gene publication procedures. These new directions in the development of nucleic acid analysis will open the door for novel applications in human genetic diseases and their treatment.