This method is particularly useful when adding affinity tags or fluorophores to DNA probes that are too long for efficient automated synthesis. Klenow fragment E. Dot blot with biotin-labeled DNA using Klenow fragment. Use of this kit results in high yields of biotin-labeled DNA due to the Klenow fragment, exo-. RNA polymerases are template dependent and sequence dependent, requiring a promoter sequence within the template DNA in order to initiate binding of the enzyme and, depending on the host system, various cofactors are required for RNA transcription to proceed on the single-stranded template.
RNA polymerases are used for a variety of lab purposes, from the in vitro synthesis of mRNA to the generation of probes for hybridization and binding assays. Probes generated with the aid of RNA polymerase are made by the random incorporation of modified nucleotides during the transcription process. This is typically done by cloning a cDNA or other template sequence into a plasmid containing one or more RNA polymerase promoter sequences.
RNA probes generated in this manner have high specific activity and are most commonly made with radioactive nucleotides, although biotin and other tags are also available. T7 RNA polymerase promoter sequence. The resulting aldehyde groups are spontaneously reactive toward primary amine-containing molecules and surfaces.
Aldehydes can be used in two types of coupling reactions with either primary amine- or hydrazide-activated tags. Primary amines react with aldehydes to form Schiff bases, which readily hydrolyze and must be stabilized through their reduction to secondary amine bonds with sodium cyanoborohydride NaBH 4. Hydrazide-modified molecules also spontaneously react with aldehydes but form fairly stable hydrazone linkages, making the reaction much more efficient. The addition of sodium cyanoborohydride will increase the reaction efficiency further and increase bond stability over time and changes in pH.
Unlike most crosslinkers, carbodiimides do not become part of the final crosslink between the molecules and thus do not add any additional chemical structure to the resulting products. EDC EDAC, 1-Ethyl[3-dimethylaminopropyl]carbodiimide hydrochloride is a water-soluble carbodiimide preferred for use in aqueous reactions in the 4. The same reaction can be performed with phosphates i. With that minor exception, EDC-mediated conjugations are an economical means for coupling both RNA and DNA to nearly any other primary amine-containing molecule or surface.
Random chemical labeling of nucleic acids can be accomplished by various means. Because these methods label at random sites along the length of a DNA or RNA molecule, they allow a higher degree of labeling to be achieved than end-labeling techniques. However, one disadvantage of these methods is that the nucleotide bases are directly modified, which will reduce or prevent base-pairing between complementary strands during hybridization experiments.
Therefore, it may be necessary to balance the degree of labeling with the hybridization efficiency of the probes in certain experiments. Two types of photoreactive label reagents are used for nucleic acids: phenylazide- and psoralen-based. When the phenylazide functional group is exposed to UV light, it forms a labile nitrene that can insert nonspecifically into double bonds and C-H and N-H sites via addition reactions, provided that more reactive nucleophilic e.
The psoralen ring structure effectively intercalates into the double-stranded portions, and exposure to UV light causes a cyclo-addition product to be formed with the 5,6-double bond in thymine residues. The ULS labeling reagents contain the proprietary temperature-activated platinum-based moiety which reacts with guanine bases in RNA and DNA and the side chains of methionine, cysteine and histidine residues of proteins.
Note: You clicked on an external link, which has been disabled in order to keep your shopping session open. Search Thermo Fisher Scientific. Search All. Methods for Labeling Nucleic Acids. See Navigation. Nucleic acids may be modified with tags that enable detection or purification. The resulting nucleic acid probes can be used to identify or recover other interacting molecules. Common labels used to generate nucleic acid probes include radioactive phosphates, biotin, fluorophores and enzymes. In addition, the bioconjugation methods used for nucleic acid probe generation may be adapted for attaching nucleic acids to other molecules or surfaces to facilitate targeted delivery or immobilization, respectively.
Page contents Overview Summary of nucleic acid labeling methods Enzymatic methods for nucleic acid labeling Chemical methods for nucleic acid labeling Recommended reading.
Summary of nucleic acid labeling methods. Learn more Nucleic Acid Labeling Support. Enzymatic methods for nucleic acid labeling. Terminal deoxynucleotidyl transferase TdT.
In this section, we summarize the methods that do not need isolation and ex vivo handling steps, but that can deliver the nucleic acid directly in situ. In , we attempted to deliver plasmid DNA to oviductal epithelium by in vivo instillation into oviductal lumen ampulla at Day 0.
Metagenomic technologies, which provide the potential to capture and study the entire microbiome the predominant genomes from a complex microbial community in the rumen are described. These hallmark features, introduced by series editor Dr. Also activity-driven assays can be performed to screen the library for novel enzymes or specific activities and function. Journal of Microbiological Methods , v. UV crosslinking of RNA to nylon membrane enhances hybridization signals.
This approach was initially meant to deliver DNA to the zygotes residing at the ampulla, but we failed to accomplish this goal. In all likelihood, the presence of cumulus cells surrounding the oocytes might have acted as a barrier hindering DNA uptake by the zygotes. We then tried the technique at day 1. This, indeed, turned out to be the case: we observed that fluorescent 4—8 cell embryos that had been isolated from the oviducts dissected 1 day after GTOVE using EGFP plasmid [ 49 ]. Unfortunately, chromosomal integration of the exogenous DNA was not achieved in offspring analyzed at the mid-gestational stage.
Nevertheless, these data clearly indicate that in vivo genome editing by direct delivery of genome editing components to preimplantation embryos is possible. GONAD technology is the simplest and most convenient method among all the existing gene delivery systems targeted to pre-implantation embryos.
It does not require many complex steps for animal transgenesis, such as: i isolation of embryos; ii ex vivo handling and culturing of embryos; iii manipulation of embryos such as microinjection; or iv microsurgery to transfer embryos to recipient females.
GONAD can be used for genome editing in other species where ex vivo handling of isolated zygotes is considered difficult for example, pigs and calves. The original GONAD was performed at the 2-cell stage, where it often caused mosaic patterns of gene expression [ 50 ]. One of the obvious reasons for such mosaicism could be that the components introduced by GONAD such as EGFP or Cas9 mRNA and sgRNA might have differed between the two blastomeres at the 2-cell embryo stage, causing differential genome editing activity in the offspring of each blastomere.
Tsukamoto et al. Since then, several laboratories, including ours, have succeeded in delivering nucleic acids to post-implantation embryos and fetuses [ 52 , 53 ]. The authors concluded that the systemic delivery of shRNAs is a feasible approach to gene silencing in the embryo. To date, however, there are no reports of successful germ-line transmission of the transferred gene using these approaches.
Although a thorough analysis of the distribution of introduced DNA among fetal tissues has not been performed, delivery to organs in the circulatory system, such as the heart and blood vessels, may be readily achieved, since the exogenous DNA is delivered via the placenta through fetal vessels.
It is likely that if guide RNA and Cas9 are expressed under a tissue-specific promoter, it could be possible to generate animals with genetic mutations in the target organs or tissues. Direct delivery to post-implantation embryos and fetuses is another approach for achieving expression of the gene in specific target tissues.
For example, Tabata and Nakajima, reported an efficient in utero gene transfer system to the developing mouse brain using electroporation [ 54 ]. The method is performed by surgical exposure of the uterus carrying the fetuses, direct injection of the exogenous DNA into fetal tissues, and in vivo electroporation at the injected sites shown in Fig. The in utero delivery approach has been successfully achieved for various tissues, including those involving the skin [ 55 ], lungs [ 56 ], and brain [ 57 ]. However, examples of targeting to germ cells at the embryonic stage do not exist because the germ cell tissue and lineage is very poorly developed at this stage.
Even though such targeted gene delivery approaches to fetal tissues are useful for studies intended for fetal genome editing, they cannot be used for achieving germ-line transmittable mutations. There are a few examples of delivering exogenous DNA to ovarian cells either through viral vectors Gordon [ 58 ] or liposomal delivery systems Shimizu et al.
Gordon [ 58 ] reported infecting ovarian cells and oocytes by direct injection of adenoviral vectors into the medulla of an ovary. However, no transduction of oocytes was observed, despite administering high doses of viral particles. Shimizu et al. The injection resulted in an increased growth of immature follicles and led to an increase in the number of secondary and tertiary follicles [ 59 ]. The authors attributed this phenomenon to an increased production of GDF9 in the transfected ovaries; however, they did not report the detailed localization of gene products in the ovary expressed from the introduced DNA.
We explored another strategy, delivering plasmid DNA to ovarian cells including oocytes by intraovarian injection, followed by in vivo electroporation [ 60 ] shown in Fig. Some oocytes that were surrounded by one or two follicular layers but not those surrounded by multi-layered follicles were also positive for staining for lacZ activity. We noted exogenous gene expression up to about 1 week after the procedure [ 60 ]. Interestingly, Yang et al. They further observed that the ovulated oocytes resulted in transmission of exogenous DNA to the F0 offspring derived from them.
This illustrates the possibility of Tg mouse production with relative ease by ovarian delivery, although it remains unclear how the introduced exogenous DNA is incorporated into the oocytes within an ovary, or how it is transmitted to the F0 offspring via fertilization. It will be interesting to test whether direct delivery of genome-editing components to ovaries in situ can edit the genome of immature oocytes, and whether they develop further into oocytes capable of generating genetically modified animals.
We demonstrated for the first time that the plasmid DNA injected into the interstitial space of testis was detected in the cauda epididymis, up to 1 week after DNA injection [ 62 ]. This suggests that testicular spermatozoa can receive exogenous DNA and can then mature and be transferred to the cauda epididymis shown in Fig. Others have also reported injecting plasmid DNAs to testicular spermatozoa using a glass pipette or G needle [ 63 — 65 ].
Blanchard and Boekelheide, delivered an adenovirus vector to the interstitial compartment of adult rat testes by intratesticular injection and found transgene expression in Leydig cells [ 65 ]. Muramatsu, demonstrated that the intratesticular injection of DNA and subsequent in vivo electroporation resulted in the delivery of DNA to testicular spermatozoa as well as to interstitial cells [ 66 ]. When males injected with exogenous DNA using the intratesticular injection methods were mated to superovulated females, transmission and expression of the DNA was verified at the blastocysts and fetuses [ 67 — 71 ].
However, the technology has been debated for its suitability as a commonly-usable method for generating transgenic models because, first, the transgene insertion is not guaranteed in every sperm.