DNA Labeling: Overview
Nucleic acid labeling is one of the essential procedures in molecular biology labs to detect and purify nucleic acids (DNA and RNA).
Various labeling agents are available today for their conjugation with DNA fragments, such as nucleotides modified with biotin, protein complexes (such as streptavidin bound with biotin), digoxigenin, phosphates, fluorophores (such as Alexa Fluor dyes and Cy dyes), and haptens.
Based on the applications, the nucleotide or nucleoside sequences are either labeled throughout their length, at the 5´ end or 3´ end. For example:
- High-specific activity DNA probes must be generated to perform hybridization reactions, such as northern and southern blotting, with labels dispersed throughout the nucleic acid. It is done either by random priming or nick translation using PCR amplification techniques or by in vitro transcription using labeled dNTPs or NTPs.
- For protein interaction-based applications, such as pull-downs or gel-shift assays, it’s required to generate end-labeled probes. This can be done by PCR using primers.
The targeted attachment of the labeled DNA probes to other surfaces or molecules can be achieved by techniques like bioconjugation.
In this article, we will teach you how the labeling of DNA is done, how it works, and its application in lab workflows.
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DNA Labeling Techniques
Labeled oligonucleotide (oligos) probes are commercially available. However, it can be expensive depending on the label, synthesis, modification, and purification techniques. Thus, scientists usually prefer to design the labeled DNA on their own based on their applications using individual reagents or labeling kits.
The reagents involved in DNA labeling include labeled nucleic acid, dNTPs, DNA polymerase, reaction buffer, etc.
Here’re some DNA labeling methods used in labs:
- Chemical Method
Chemical methods are used to develop probes for large-scale applications. It includes:
- EDC activation of 5′ phosphate
EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) is a carbodiimide used in aqueous reactions. It facilitates the formation of phosphoramidite and amide linkages between carboxylate, phosphate, and amino groups.
- Chemical random-labeling
Random labeling is a high-yield labeling method. In this process, DNA or RNA is labeled at random locations throughout their length. However, the only limitation associated with the technique is reduced base-pairing because of the direct modification of the nucleotide bases.
- Enzymatic Method
This method is suitable for small-scale probe development. It involves four techniques:
- Terminal deoxynucleotidyl transferase (TdT)
TdT is a DNA polymerase enzyme expressed in some lymphoid cells. It’s a template-independent enzyme and is not affected by the DNA sequence. TdT is used to add deoxynucleotides to the 3′ ends of a DNA strand and label the 3′ termini of DNA probes with nonradioactive and radioactive tags.
It’s used to label DNA probes in applications like TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assays and RACE (Rapid Amplification of cDNA Ends).
- T4 polynucleotide kinase (PNK)
This enzyme is encoded from the genomic DNA of the T4 bacteriophage. It’s a template-independent enzyme. It can modify single-stranded DNA (or polynucleotide) and 5′ overhangs. The wild-type T4 PNK also possesses 3′-phosphatase activity.
T4 PNK has application in labeling 5′ ends of polynucleotides with radioactive phosphate. Moreover, it’s also used for the quantitative phosphorylation of 5′ ends using unlabeled ATP.
- DNA Polymerase
DNA polymerases are a family of enzymes involved in catalyzing the synthesis of DNA polymers using deoxynucleoside triphosphates (dNTPs). They are not sequence-dependent but template-dependent. They’re used for various lab applications, from sequencing to cloning. They have the ability to elongate the existing 3′-OH end using template DNA.
For amplification-based applications, heat stable enzymes cloned from thermophilic organisms are used in labeling procedures. However, experiments not involving the amplification step utilize either the Klenow fragment or T4 or T7 polymerases.
How Does DNA Labeling Work?
Labeling reactions involve the use of molecules, such as fluorophores, proteins, or enzymes. These are sensitive molecules that do not alter the properties of the target molecules. The labels are attached to a probe that binds to the DNA molecule.
After the binding is complete, a substrate is added based on the label attached to the DNA molecules to view them under a microscope. However, if a fluorophore molecule is the substrate attached to DNA, they need to be excited to a higher energy state to view them using fluorescence microscopy.
What is DNA Labeling Used For?
DNA labeling is mainly used for two purposes — the “detection” and “purification” of the DNA molecules for further experiments.
Labeled DNA helps to detect a particular DNA sequence from a complex mix of nucleic acids. Based on the labeling agent used in the assay, the detection of the molecule is either performed using colorimetric detection or autoradiographic detection.
Highly purified DNA is essential for DNA analysis using high-throughput workflows like real-time PCR or multiplex PCR. Labeling DNA samples or controls help in visualizing the molecules using gel electrophoresis and determining DNA yield and purity.
What Industries Use DNA Labeling?
The labeled DNA probes have a myriad of applications in lab procedures, such as DNA sequencing, microarray analysis, in situ hybridization, southern blotting, and northern blotting. The industries which extensively use these applications in their routine tasks mainly belong to the Biotechnology and Life sciences.
In biotechnology, DNA labeling is used for the detection and purification of nucleic acid molecules. The labeled DNA is used in detecting nucleotide sequences, screening gene libraries, and in gene technologies.
Labeled DNA has extensive applications in cloning, fluorescent DNA sequencing, next-generation sequencing (NGS), labeling, in vitro transcription/translation, and restriction enzyme digestion.
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DNA labeling is a technique used in molecular labs to detect and purify DNA molecules. It’s done by the introduction of a fluorescent molecule per DNA strand and a quantitative fragmentation of the DNA, followed by the incorporation of one label per DNA fragment.
The labels attached to DNA probes include fluorescent molecules, biotin, biotin complex proteins, enzymes, and phosphates. The techniques used to incorporate these labels in the DNA are categorized into two groups: chemical and enzymatic techniques.
Researchers use a colorimetric or autoradiographic detection technique based on the label attached to the DNA molecules. The DNA labeling procedures applications include next generation and nucleotide sequencing, southern and northern blotting, gene library screening, and fluorescent DNA sequencing.
A high-quality reagent combined with high-throughput equipment is necessary to perform such experiments. A poor yield of label DNA with low specificity will spoil your whole experiment with rarely any data in your hand.
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