Labeling of DNA Probes by Nick Translation

2020 ◽  
Vol 2020 (7) ◽  
pp. pdb.prot100602
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Dna Probes ◽  
Nick Translation

scholarly journals Fluorescence in situ hybridization in diluted and flow cytometrically sorted boar spermatozoa using specific DNA direct probes labelled by nick translation

Reproduction ◽  
2003 ◽  
pp. 317-325 ◽  
Author(s):  
I Parrilla ◽  
JM Vazquez ◽  
M Oliver-Bonet ◽  
J Navarro ◽  
J Yelamos ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Y Chromosome ◽  
Internal Control ◽  
Dna Probes ◽  
Chromosome 1 ◽  
Nick Translation ◽  
Fluorescent Signal ◽  
Sperm Separation

Successful evaluation of X- and Y-chromosome-bearing sperm separation technology using flow cytometry-cell sorter is of great importance. Fluorescence in situ hybridization (FISH), which allows for the detection of specific nucleic acid sequences on morphologically preserved spermatozoa, is an ideal method for quantitatively and qualitatively assessing the purity of sorted sperm samples. In this study specific pig DNA direct probes for small regions of chromosomes 1 and Y were used. Chromosome 1 was labelled in green and used as internal control to detect a lack of hybridization, whereas chromosome Y was labelled in red. Nick translation was used as the labelling method for the preparation of these probes. Spermatozoa, unsorted and sorted for high and low Y-chromosome purity from ejaculates of five boars, were fixed on slides and two-colour direct FISH was performed for chromosomes 1 and Y. About 500 non-sorted and 200 sorted spermatozoa per sample were scored. The proportion of Y-chromosome-bearing spermatozoa was determined by the presence of a red fluorescent signal on the sperm head and the proportion of X-chromosome-bearing spermatozoa was determined by subtraction. The efficiency of the hybridization procedure was established as near 98% on sorted and unsorted samples. The results of this study confirm that direct FISH using specific pig DNA probes labelled by nick translation provides a useful tool for laboratory validation of sperm separation by flow sorting technology. Moreover, the ease of nick translation and the quality of the fluorescent signal obtained using this method makes this procedure the most appropriate method for labelling pig DNA probes to be used for direct FISH on pig spermatozoa.

Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations

2002 ◽  
Vol 10 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Yunsheng Li ◽  
Scott Hatfield ◽  
Jing Li ◽  
Mark McMills ◽  
Yufen Zhao ◽  
...  
Keyword(s):  
Dna Probes ◽  
Nick Translation ◽  
Dna Nicking

A Simplified Combination of DNA Probe Preparation and Fluorescence in situ Hybridization

1992 ◽  
Vol 47 (9-10) ◽  
pp. 739-747 ◽  
Author(s):  
Dino Celeda ◽  
Ulrich Bettag ◽  
Christoph Cremer
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Repetitive Dna ◽  
Human Lymphocytes ◽  
Dna Probes ◽  
Dna Probe ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Human Dna

Fluorescence in situ hybridization (FISH) has found widespread applications in cytogenetics. So far the standard protocols for probe amplification (and simultaneous labeling) by PCR, nick translation and in situ hybridization involve different buffer systems leading to a number of time consuming washing steps even before hybridization. In this manuscript we show a fast technique of a close combination of DNA probe preparation and in situ hybridization (ISH). This method was applied to metaphase chromosomes from human lymphocytes fixed on slides. Two specific repetitive DNA probes, the pUC 1.77 DNA probe and the DYZ 1 repetitive DNA fraction were used, amplified and labeled in different ways. Additional experiments with total genomic male human DNA as the DNA probe suggest that this method may be extended to a large variety of other probes. Moreover the ISH technique described does not require toxic denaturing agents, such as formamide.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

A Modified Nick Translation Method Used with FISH that Produces Reliable Results with Archival Tissue Sections

2002 ◽  
Vol 20 (3) ◽  
pp. 257-260 ◽  
Author(s):  
Amanda D Watters ◽  
Michael W Stacey ◽  
John M S Bartlett
Keyword(s):  
Translation Method ◽  
Nick Translation ◽  
Archival Tissue ◽  
Tissue Sections

Development of pattern recognition based on the nanosheet-DNA probes and an extendible DNA library

The Analyst ◽  
2021 ◽  
Author(s):  
Jiawei Qi ◽  
Pinhua Rao ◽  
Lele Wang ◽  
Li Xu ◽  
Yanli Wen ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
High Throughput ◽  
Analysis Data ◽  
Dna Probes ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
Dna Library ◽  
Array Sensing

Pattern recognition, also called “array sensing” is a recognition strategy with a wide and expandable analysis range, based on the high-throughput analysis data. In this work, we constructed a sensor...

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