Determination of copy number for 5S rDNA and centromeric sequence RCS2 in rice by Fiber-FISH

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic PigsThe aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

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Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.51726 ◽

2019 ◽

Vol 24 (2) ◽

pp. 82

Author(s):

Agus Budi Setiawan ◽

Ari Wibowo ◽

Chee How Teo ◽

Shinji Kikuchi ◽

Takato Koba

Keyword(s):

Repetitive Dna ◽

Dna Sequences ◽

5S Rdna ◽

Repetitive Dna Sequences ◽

Type I ◽

Chromosome Identification ◽

Metaphase Chromosomes ◽

Homologous Chromosomes ◽

Centromeric Sequence ◽

Subtelomeric Sequence

Repetitive DNA sequences are highly abundant in plant genomes and are favorable probes for chromosome identification in plants. However, it is difficult to conduct studies on the details of metaphase chromosome structures in plants with small chromosomes due to their highly condensed status. Therefore, identification of homologous chromosomes for karyotyping and analyzing chromosome structures is a challenging issue for cytogeneticists without specific probes and precise chromosome stages. In this study, five repetitive DNA probes, i.e., 5S and 45S ribosomal DNAs (rDNAs), melon centromeric sequence (Cmcent), cucumber subtelomeric sequence (Type I), and microsatellite (CT)10 repeats, were used to identify primary constrictions and homologous chromosomes for karyotyping. Four and two loci of 45S rDNA were respectively observed on metaphase and pachytene chromosomes of Abelia × grandiflora. Cmcent was detected on both primary constrictions of melon pachytene and metaphase chromosomes. Furthermore, one pair of 5S rDNA signals were hybridized on melon metaphase chromosomes. Eight and two loci of 45S and 5S rDNA were respectively detected on cucumber chromosomes. Type I and (CT)10 probes were specifically hybridized on subtelomeric and interstitial regions on the chromosomes, respectively. These results suggest that repetitive DNA sequences are versatile probes for chromosome identification in plants with small chromosomes, particularly for karyotyping analyses.

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Prospect and Competence of Quantitative Methods via Real-time PCR in a Comparative Manner: An Experimental Review of Current Methods

The Open Bioinformatics Journal ◽

10.2174/1875036201811010001 ◽

2018 ◽

Vol 11 (1) ◽

pp. 1-11

Author(s):

Hossein Mahboudi ◽

Negin Mohammadizadeh Heidari ◽

Zahra Irani Rashidabadi ◽

Ali Houshmand Anbarestani ◽

Soroush Karimi ◽

...

Keyword(s):

Internal Control ◽

Copy Number ◽

Quantitative Methods ◽

Absolute Quantification ◽

Relative Quantification ◽

Absolute Quantitation ◽

Standard Curve ◽

Qpcr Quantification ◽

Comparative Manner

Background: There are numerous approaches dealing with relative and absolute quantitation. The methods differ in their efficiency assumption and applicability. Objective: Current methodologies and rations used in qPCR quantification were compared in an experimental study of transgenic copy number determination of a monoclonal antibody Daclizumab. Methods: With an inter and intra-methodical view, variations in relative and absolute quantification strategies were discretely extracted and compared to one another. Results: In relative quantification, six methods were studied and the ratios were computed relative to Glucagon as internal control. For Absolute quantification, the calculations were based on standard curve. Relative quantification considers the relative changes in expression levels while Absolute quantification relates the PCR signal to input copy number with a calibration curve. Conclusion: The observed unevenness of the ratios in Relative approach pointed mainly to the efficiency changes and its calculation formula. Whereas results in Absolute approach strategies showed homogeneity which indicates the consistency of the calculation method.

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Direct determination of retrotransposon transposition rates in Drosophila melanogaster

Genetics Research ◽

10.1017/s0016672300032249 ◽

1994 ◽

Vol 63 (2) ◽

pp. 139-144 ◽

Cited By ~ 34

Author(s):

Sergey V. Nuzhdin ◽

Trudy F. C. Mackay

Keyword(s):

Drosophila Melanogaster ◽

Transposable Element ◽

Inbred Line ◽

Copy Number ◽

Direct Determination ◽

Hybridization Analysis ◽

Spontaneous Mutations ◽

Insertion Sites

SummaryRates of transposition and excision of the Drosophila melanogaster retrotransposon elements mdg3, 297, Doc, roo and copia were estimated directly, by in situ hybridization analysis of their cytological insertion sites in 31 replicates of a highly inbred line that had accumulated spontaneous mutations for approximately 160generations. Estimated transposition rates of Doc, roo and copia were, respectively, 4·2 × 10−5, 3·1 × 10−3 and 1·3 − 10−3; no transpositions of 297 nor mdg3 were observed. Rates of transposition of copia varied significantly among sublines. Excisions were only observed for roo elements, at a rate of 9·0 × 10−6 per element per generation. Copy number averaged over these element families increased 5·9 %; therefore, in these lines the magnitude of the forces opposing transposable element multiplication were weaker than transposition rates.

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