Random primer | ScienceGate

ABSTRACT Dangling ends and surface-proximal tails of gene targets influence probe-target duplex formation and affect the signal intensity of probes on diagnostic microarrays. This phenomenon was evaluated using an oligonucleotide microarray containing 18-mer probes corresponding to the 16S rRNA genes of 10 waterborne pathogens and a number of synthetic and PCR-amplified gene targets. Signal intensities for Klenow/random primer-labeled 16S rRNA gene targets were dissimilar from those for 45-mer synthetic targets for nearly 73% of the probes tested. Klenow/random primer-labeled targets resulted in an interaction with a complex mixture of 16S rRNA genes (used as the background) 3.7 times higher than the interaction of 45-mer targets with the same mixture. A 7-base-long dangling end sequence with perfect homology to another single-stranded background DNA sequence was sufficient to produce a cross-hybridization signal that was as strong as the signal obtained by the probe-target duplex itself. Gibbs free energy between the target and a well-defined background was found to be a better indicator of hybridization signal intensity than the sequence or length of the dangling end alone. The dangling end (Gibbs free energy of −7.6 kcal/mol) was found to be significantly more prone to target-background interaction than the surface-proximal tail (Gibbs free energy of− 64.5 kcal/mol). This study underlines the need for careful target preparation and evaluation of signal intensities for diagnostic arrays using 16S rRNA and other gene targets due to the potential for target interaction with a complex background.

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First Report of Citrus exocortis viroid from Grapevine in China

Plant Disease ◽

10.1094/pdis-94-8-1071a ◽

2010 ◽

Vol 94 (8) ◽

pp. 1071-1071 ◽

Cited By ~ 4

Author(s):

J. Shu ◽

G. P. Wang ◽

W. X. Xu ◽

N. Hong

Keyword(s):

Reverse Transcription ◽

Yield Loss ◽

Broad Bean ◽

Double Stranded Rna ◽

Random Primer ◽

Total Rna ◽

First Report ◽

Citrus Exocortis Viroid ◽

Target Dna ◽

The World

Citrus exocortis viroid (CEVd) can induce bark scaling, dwarfing, leaf epinasty, and fruit yield loss in susceptible hosts. In citrus, CEVd is reported from around the world, but in grape, it is reported from fewer locations (Australia, Brazil, California, and Spain [1]). In 2009, leaves were collected from 40 grapevines (of several different cultivars and species) from Henan, Hubei, Shandong, and Liaoning provinces, China. Total RNA or double-stranded RNA was extracted from the leaves by a described method (3) and subjected to reverse transcription with a random primer (Takara, Dalian, China) and then PCR with primer CEV-AM3 and CEV-AP3 (2). Results showed that the target DNA fragments of 372 bp long were amplified only from the symptomless leaves collected from two grapevines of cv. White Rose grown for approximately 26 years within a small garden in Hubei Province. Amplified products were recovered and cloned into pMD18-T (Takara) and 10 positive clones of each isolate were sequenced and aligned. For both isolates, 20% of the clones represented the same variant (CEVd-hn-g-1; GenBank Accession No. GU592444). It showed a max identity of 94 to 99% with the variants (GenBank Accession Nos. Y00328.1 and DQ471996.1) from grape registered in NCBI, 91 to 100% (GenBank Accession Nos. DQ431993.1 and DQ831485.1) from citrus, 91 to 98% (GenBank Accession Nos. EF488068.1 and EF488050.1) from broad bean, and 89 to 94% (GenBank Accession Nos. AY671953.1 and S67446.1) from tomato. To our knowledge, this is the first report of CEVd from grape in China. References: (1) M. Eiras et al. Fitopatol. Bras. 31:440, 2006. (2) H. J. Gross et al. Eur. J. Biochem. 121:249, 1982. (3) W. X. Xu et al. J. Virol. Methods 135:276, 2006.

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Kemiripan genetik wereng coklat, Nilaparvata lugens Stål. (Homoptera: Delphacidae) populasi Klaten dan Yogyakarta berdasarkan penanda RAPD-PCR

Jurnal Entomologi Indonesia ◽

10.5994/jei.15.2.79 ◽

2018 ◽

Vol 15 (2) ◽

pp. 85

Author(s):

Supriyadi Supriyadi ◽

Retno Wijayanti

Keyword(s):

Nilaparvata Lugens ◽

Random Primer ◽

Rapd Pcr ◽

Nilaparvata Lugens Stål

Wereng coklat (Nilaparvata lugens Stål.) populasi Klaten dan Yogyakarta menunjukkan kemampuan adaptasi pada varietas padi tahan lebih cepat daripada populasi asal wilayah sekitarnya, namun kajian aspek genetik terkait hal ini masih terbatas. Penelitian ini dilakukan untuk mengidentifikasi kemiripan genetik wereng coklat populasi Klaten dan Yogyakarta dengan populasi asal lokasi sekitarnya sebagai pembanding, berdasarkan penanda RAPD-PCR. Pengambilan sampel wereng coklat dilakukan di pertanaman padi di Klaten, Yogyakarta, Sukoharjo, Boyolali, Karanganyar, Sragen, dan Ngawi. Identifikasi kemiripan genetik dilakukan dengan teknik RAPD-PCR, menggunakan lima random primer, yakni OPB 01, OPB 07, OPC 04, OPC 08, dan OPN 15. Hasil pengujian menunjukkan bahwa kelima primer mampu mengamplifikasi DNA wereng coklat dengan baik, namun tidak ada primer yang mampu membedakan secara jelas populasi Klaten dan Yogyakarta dengan populasi asal wilayah sekitarnya. Populasi wereng coklat asal Klaten dan Yogyakarta juga menunjukkan kemiripan genetik dengan populasi asal wilayah sekitarnya, yakni Boyolali, Sukoharjo, dan Sragen, kecuali dengan Karanganyar dan Ngawi. Kajian genetik antar populasi, termasuk populasi asal Klaten dan Yogyakarta diperlukan untuk mengungkap perbedaan genetiknya.

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Random amplified polymorphic DNA (RAPD) analysis of Ornithobacterium rhinotracheale strains isolated from chickens in Turkey

Veterinární Medicína ◽

10.17221/5660-vetmed ◽

2012 ◽

Vol 50 (No. 12) ◽

pp. 526-530 ◽

Cited By ~ 4

Author(s):

G. Ozbey ◽

Ertas HB ◽

A. Muz

Keyword(s):

Rapd Analysis ◽

Random Amplified Polymorphic Dna ◽

Random Primer ◽

Serotype A ◽

Dna Assay ◽

Rapd Profile ◽

Rapd Pattern ◽

Serotype B ◽

Dna Profiles ◽

Ornithobacterium Rhinotracheale

Six field strains of Ornithobacterium rhinotracheale isolated from chickens in Elazig province located in the East of Turkey were typed by serotyping and random amplified polymorphic DNA assay using a random primer (OPG-11). Using the AGP test used for serotyping, serotype A was found to be the predominant serotype, only one strain was serotyped as serotype B. By RAPD assay, the tested ORT strains were found to have different RAPD profiles. In addition, the RAPD assay showed almost similar DNA profiles among the tested strains of the serotypes A, B, D and E. The strain of serotype C did give a different RAPD profile. Within strains of the same serotype (A), different profiles were found but the strain of serotype (B) had an identical profile as strains of serotype A. This study suggests that more genotypes of ORT strains are present within the same serotype and thus that no relationship exists between the RAPD pattern of ORT and their serotype.

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A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

Science Advances ◽

10.1126/sciadv.1600699 ◽

2016 ◽

Vol 2 (8) ◽

pp. e1600699 ◽

Cited By ~ 11

Author(s):

Hiroshi Arakawa

Keyword(s):

Dna Sequences ◽

Genetic Screening ◽

Restriction Enzymes ◽

Mrna Sequence ◽

Complementary Sequence ◽

Complementary Dna ◽

Random Primer ◽

Guide Rna ◽

Protospacer Adjacent Motif ◽

Target Dna

The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.

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Tagged random primerpolymerasechainreaction (tagged random primer PCR, tagged PCR, T-PCR)

The Dictionary of Genomics, Transcriptomics and Proteomics ◽

10.1002/9783527678679.dg12833 ◽

2015 ◽

pp. 1-1

Keyword(s):

Random Primer

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The random primer labeling technique applied to in situ hybridization on tissue sections.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.10.3138309 ◽

1988 ◽

Vol 36 (10) ◽

pp. 1335-1340 ◽

Cited By ~ 13

Author(s):

M Thomas-Cavallin ◽

O Aït-Ahmed

Keyword(s):

Drosophila Melanogaster ◽

In Situ Hybridization ◽

Specific Activity ◽

High Specific Activity ◽

High Sensitivity ◽

Random Primer ◽

Tissue Sections ◽

In Situ Hybridizations ◽

Labeling Method

We report an application of the random primer labeling technique to in situ hybridizations on tissue sections. The ease of the method and the high specific activity achieved make it valuable when a large number of probes must be analyzed and high sensitivity is needed. We have applied this technique to study the spatial expression of a cluster of maternally acting genes (the yema gene region of Drosophila melanogaster which encodes eleven transcripts, some of them having a very low level of expression) (Aït-Ahmed et al., 1978: Dev Biol 122:153; Aït-Ahmed et al., unpublished results). The results reported here concern one of the transcripts of the yema region, which displays a peculiar anterior localization in the oocyte. We demonstrate that the "oligo-labeling" method allows a far better level of detection of the transcript of interest.

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