ReadiLink™ Biotin Nick Translation dsDNA Labeling Kit

Nick translation labeling of DNA starts with the creation of defects within the sequence of existing DNA double-helix molecules by cleavage of phosphodiester bonds with DNase along the backbone of one strand. Polymerase then repairs these nicks beginning with the removal of the adjacent nucleotide and the immediate filling back in of those gaps with new nucleotides from the added dNTP pool. As each new nucleotide is added, the polymerase leaves the 3′ OH group open, thus translating the nick toward the 5′ end. As the reaction sequence is repeated, the polymerase enzyme continues to remove existing nucleotides and replace them with new ones at the site of the new nick. The result of these reactions is numerous labeled and unlabeled nucleotides being incorporated as a complementary sequence along the length of each DNA strand, starting at the site of the original nick.

Verification of double stranded DNA labeling with ReadiLink™ Biotin Nick Translation dsDNA Labeling Kit. Double stranded DNA were labeled with ReadiLink™ Biotin Nick Translation dsDNA Labeling Kit with suggested ratios of Biotin-dUTP:dTTP, purified and then incubated with or without streptavidin before applied to agarose gel electrophoresis. Gel was stained with Gelite™ Safe DNA Gel Stain. Streptavidin caused a supershift of Biotin-labeled dsDNA while in the absence of streptavidin, shift was not obseved. Control samples (dsDNA) were also ran in the absence and presence of Streptavidin (SA).

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

ReadiLink™ Biotin Nick Translation dsDNA Labelling Kit provides a simple and efficient way to label a double stranded DNA sample with Biotin tag. The labelling kit provides all necessary reagents for a complete workflow required for DNA labelling. This method utilizes a combination of DNAse and DNA polymerase to nick one strand of the DNA helix, to which Biotin is conjugated. In addition, the kit allows the user to optimize incorporation and product size by adjusting the ratio of Biotin-dUTP conjugate to dTTP. It is compatible with a wide variety of sample materials, including bacterial artificial chromosome (BAC) DNA, human genomic DNA, purified PCR products, supercoiled and linearized plasmid DNA. The resulted Biotin-labeled DNAs can be used in a variety of molecular biology techniques such as fluorescence in situ hybridization (FISH).

Platform

Thermal Cycler

Instrument specification(s)

N/A

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare DNA samples
Add reagents to tube
Mix and centrifuge briefly
Incubate at 15 °C for 60 minutes
Place the reaction on ice followed by addition of Stop Solution and heating at 65 °C
Place on ice for 5 minutes before using or store at 4 °C
Purify the labelled DNA

Important

Thaw all the kit components on ice before starting the experiment. Briefly vortex all the reagents to the bottom before starting the labelling process.

SAMPLE EXPERIMENTAL PROTOCOL

The following protocol can be used as a guideline.
Table 1.Reagents composition per tube for each reaction

Components	Amount
DNA sample	1 µg DNA diluted in Nuclease-free water to final volume of 34 µL
Nick Translation Buffer	5 µL
dNTP mix	5 µL
dTTP	2 µL
Biotin-dUTP working solution	2 µL
DNA Polymerase I	1 µL
DNase I	1 µL
Total Volume	50 µL

The ratio of Biotin-dUTP (Component A): dTTP (Component E) can be optimized to achieve the best labelling conditions.
Incubation time can be optimized for better labelling. Longer incubation time will help with more labelling but may shorten the size of the end product.

To a clean (Nuclease-free) 0.5 mL micro centrifuge tube or 0.2 mL PCR tube, add the reagents in the order indicated in Table 1.
Carefully mix the reagents by a brief vortex followed by brief centrifuge.
Incubate the reaction at 15 °C for 60 minutes.
After incubation, place the reaction on ice.
To terminate the reaction, add 5 µL of Stop Solution and heat the sample at 65 °C.
Place on ice for 5 minutes before using or store at 4 °C.
Purify the labeled DNA.

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)	Correction Factor (482 nm)	Correction Factor (565 nm)
ReadiLink™ Cy3 Nick Translation dsDNA Labeling Kit	555	569	150000¹	0.15¹	0.07	0.073	-	-
ReadiLink™ Cy5 Nick Translation dsDNA Labeling Kit	651	670	250000¹	0.27¹, 0.4²	0.02	0.03	0.009	0.09

Images

Figure 1. Nick translation labeling of DNA starts with the creation of defects within the sequence of existing DNA double-helix molecules by cleavage of phosphodiester bonds with DNase along the backbone of one strand. Polymerase then repairs these nicks beginning with the removal of the adjacent nucleotide and the immediate filling back in of those gaps with new nucleotides from the added dNTP pool. As each new nucleotide is added, the polymerase leaves the 3′ OH group open, thus translating the nick toward the 5′ end. As the reaction sequence is repeated, the polymerase enzyme continues to remove existing nucleotides and replace them with new ones at the site of the new nick. The result of these reactions is numerous labeled and unlabeled nucleotides being incorporated as a complementary sequence along the length of each DNA strand, starting at the site of the original nick.

Figure 2. Verification of double stranded DNA labeling with ReadiLink™ Biotin Nick Translation dsDNA Labeling Kit. Double stranded DNA were labeled with ReadiLink™ Biotin Nick Translation dsDNA Labeling Kit with suggested ratios of Biotin-dUTP:dTTP, purified and then incubated with or without streptavidin before applied to agarose gel electrophoresis. Gel was stained with Gelite™ Safe DNA Gel Stain. Streptavidin caused a supershift of Biotin-labeled dsDNA while in the absence of streptavidin, shift was not obseved. Control samples (dsDNA) were also ran in the absence and presence of Streptavidin (SA).

References

View all 50 references: Citation Explorer

Biotin as a Reactive Handle to Selectively Label Proteins and DNA with Small Molecules.
Authors: Cotton, Adam D and Wells, James A and Seiple, Ian B
Journal: ACS chemical biology (2021)

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions.
Authors: Yu, Lina and He, Wenxiu and Xie, Jie and Guo, Rui and Ni, Juan and Zhang, Xia and Xu, Quishi and Wang, Caifeng and Yue, Qiuling and Li, Fangfang and Luo, Mengcheng and Sun, Bo and Ye, Lan and Zheng, Ke
Journal: Journal of visualized experiments : JoVE (2019)

Quantifying Activity for Repair of the DNA Lesion 8-Oxoguanine by Oxoguanine Glycosylase 1 (OGG1) in Mouse Adult and Fetal Brain Nuclear Extracts Using Biotin-Labeled DNA.
Authors: Bhatia, Shama and Wells, Peter G
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 329-349

Gold nanoparticle aggregation: Colorimetric detection of the interactions between avidin and biotin.
Authors: Shi, Dongmin and Sheng, Feifan and Zhang, Xiaojun and Wang, Guangfeng
Journal: Talanta (2018): 106-112

Sulfinate Based Selective Labeling of 5-Hydroxymethylcytosine: Application to Biotin Pull Down Assay.
Authors: Wu, Qiong and Amrutkar, Suyog Madhav and Shao, Fangwei
Journal: Bioconjugate chemistry (2018): 245-249

On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells.
Authors: Lebon, Cecile and Rodriguez, Gloria Villalpando and Zaoui, Ikram El and Jaadane, Imene and Behar-Cohen, Francine and Torriglia, Alicia
Journal: Analytical biochemistry (2015): 37-41

Re-evaluation of biotin-streptavidin conjugation in Förster resonance energy transfer applications.
Authors: Saremi, Bahar and Wei, Ming-Yuan and Liu, Yuan and Cheng, Bingbing and Yuan, Baohong
Journal: Journal of biomedical optics (2014): 085008

Biotin-conjugated anti-CD44 antibody-avidin binding system for the improvement of chondrocyte adhesion to scaffolds.
Authors: Lin, Hong and Zhou, Jian and Shen, Longxiang and Ruan, Yuhui and Dong, Jian and Guo, Changan and Chen, Zhengrong
Journal: Journal of biomedical materials research. Part A (2014): 1140-8

Electrochemical determination of microRNA-21 based on graphene, LNA integrated molecular beacon, AuNPs and biotin multifunctional bio bar codes and enzymatic assay system.
Authors: Yin, Huanshun and Zhou, Yunlei and Zhang, Haixia and Meng, Xiaomeng and Ai, Shiyun
Journal: Biosensors & bioelectronics (2012): 247-53

A novel electrochemiluminescence strategy for ultrasensitive DNA assay using luminol functionalized gold nanoparticles multi-labeling and amplification of gold nanoparticles and biotin-streptavidin system.
Authors: Chai, Ying and Tian, Dayong and Wang, Wei and Cui, Hua
Journal: Chemical communications (Cambridge, England) (2010): 7560-2