Development of novel SCAR markers for genetic characterization of Lonicera japonica from high GC-RAMP-PCR and DNA cloning

Cheng, Jingliang; Li, J; Qiu, Y M; Wei, Chunli; Yang, Luquan; Fu, Junjiang

doi:10.4238/gmr.15027737

Cited by 10 publications

(13 citation statements)

References 11 publications

(26 reference statements)

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“…PCR conditions were as follows: initial denaturation at 95°C for 90 s, followed by 40 cycles of 40 s at 94°C, 60 s at 36°C, 90 s at 72 °C, and final extension of 5 min at 72°C. PCR amplification was executed in an “Applied Biosystems Veriti ® 96-Well Thermal Cycler” (Life Technology, USA), and the RAMP time from annealing to extension with a RAMP rate for 5% (0.125 °C/s) was used [ 20 , 21 ].…”

Section: Methodsmentioning

confidence: 99%

“…Resolution and production of RAPD are greatly increased by prolonging the RAMP time from the stage of annealing to extension in PCR [ 18 , 19 ]. To improve the effectiveness of the RAPD method, we developed a highly effective DNA marker technique using improved RAPD with high-GC content primers, in which the GC content reaches 8–10 nucleotides for a 10 nucleotide primer (80~100%) [ 20 , 21 ]. The Sequence Characterized Amplified Region (SCAR) markers are very stable, sensitive, and reliable.…”

Section: Introductionmentioning

confidence: 99%

“…The Sequence Characterized Amplified Region (SCAR) markers are very stable, sensitive, and reliable. These markers are also effective for diagnostic purposes by using a simple PCR assay, which are generally derived from the molecular cloning of RAPD fragments in medicinal plants [ 21 – 28 ].…”

Section: Introductionmentioning

confidence: 99%

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Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments

Cheng

Wei

et al. 2017

Oncotarget

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Cancer is genetically heterogeneous regarding to molecular genetic characteristics and pathogenic pathways. A wide spectrum of biomarkers, including DNA markers, is used in determining genomic instability, molecular subtype determination and disease prognosis, and estimating sensitivity to different drugs in clinical practice. In a previous study, we developed highly effective DNA markers using improved random amplified polymorphic DNA (RAPD) with high-GC primers, which is a valuable approach for the genetic authentication of medicinal plants. In this study, we applied this effective DNA marker technique to generate genetic fingerprints that detect genomic alterations in human breast cancer tissues and then developed sequence-characterized amplified region (SCAR) markers. Three SCAR markers (BC10-1, BC13-4 and BC31-2) had high levels of genomic DNA amplification in breast cancer. The PHKG2 and RNF40 genes are either overlapping or close to the sequences of SCAR marker BC13-4, while SCAR marker BC10-1 is in the intron and overlap the DPEP1 gene, suggesting that alterations in the expression of these genes could contribute to cancer progression. Screening of breast cancer cell lines showed that the mRNA expression levels for the PHKG2 and DPEP1 were lower in non-tumorigenic mammary epithelial cell MCF10A, but elevated in other cell lines. The DPEP1 mRNA level in invasive ductal carcinoma specimens was significantly higher than that of the adjacent normal tissues in women. Taken together, high-GC RAMP-PCR provides greater efficacy in measuring genomic DNA amplifications, deletion or copy number variations. Furthermore, SCAR markers BC10-1 and BC13-4 might be useful diagnostic markers for breast cancer carcinomas.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments

Cheng

Wei

et al. 2017

Oncotarget

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“…These gene-rich regions bring significant biological information about the genome. Cheng et al (2016) and Wei et al (2016) worked with high GC-content, aiming the development of new molecular markers, highlighting the importance of working with gene-rich regions. Table 1 shows the information for each sequence obtained at the site of the NCBI.…”

Section: Gc-contentmentioning

confidence: 99%

Evaluation of genome similarities using the non-decimated wavelet transform

Ferreira

SÃ¡fadi

Lima

2017

gmr

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The wavelets have become increasingly popular in the field of bioinformatics due to their capacity in multiresolution analysis and space-frequency localization; the latter particularity is acquired due to a moving window that runs through the analyzed space. As a feature, they have a better ability to capture hidden components of biological data and an efficient link between biological systems and the mathematical objects used to describe them. The decomposition of signals/sequences at different levels of resolution allows obtaining distinct characteristics in each level. The energy (variance) obtained at each level provides a new set of information that can be used to search similarities between sequences. We show that the behavior of GC-content sequence can be succinctly described regarding the non-decimated wavelet transform, and we indicate how this characterization can be used to improve clustering of the similar strains of the genome of the Mycobacterium tuberculosis, having a very efficient level of detail. The clustering analysis using the energy obtained at each level of the analyzed sequences was essential to verify the dissimilarity of the sequences.

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“…Here, we refer to this effective DNA marker technique utilizing improved RAPD with high-GC content primers as "high GC-RAMP-PCR". This method may also be very useful for developing sequenced characterized amplified region marker markers using high GC-RAMP-PCR (Cheng et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

An improved DNA marker technique for genetic characterization using RAMP-PCR with high-GC primers

et al. 2016

Self Cite

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ABSTRACT. Random amplified polymorphic DNA (RAPD) is a widely used molecular marker technique. As traditional RAPD has poor reproducibility and productivity, we previously developed an improved RAPD method (termed RAMP-PCR), which increased the reproducibility, number of bands, and efficiency of studies on polymorphism. To further develop the efficiency of this method, we used high-GC content primers for improved RAMP-PCR with DNA samples from Lonicera japonica. Comparison of amplification profiles obtained by standard RAPD primers with those obtained by regular PCR and RAMP-PCR, and high-GC primers with regular PCR and RAMP-PCR showed that the average number of bands and polymorphisms per primer gradually and significantly increased (from 6.4 to 15.0 and from 4.6 to 10.2, respectively). Cluster dendrograms showed similar results, indicating that this new method is consistent and reproducible. A total of 22 samples from different species, including plants, animals, and humans, were used for RAMP-PCR with high-GC primers. Multiple bands were successfully amplified from all samples, demonstrating that this method is a reliable technique with consistent results and may be of general interest in studies on different genera and species. We developed highly effective DNA markers, which can provide a more effective and potentially valuable approach than traditional RAPD for the genetic identification of various organisms, particularly of medicinal plants.

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Development of novel SCAR markers for genetic characterization of Lonicera japonica from high GC-RAMP-PCR and DNA cloning

Cited by 10 publications

References 11 publications

Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments

Development of diagnostic SCAR markers for genomic DNA amplifications in breast carcinoma by DNA cloning of high-GC RAMP-PCR fragments

Evaluation of genome similarities using the non-decimated wavelet transform

An improved DNA marker technique for genetic characterization using RAMP-PCR with high-GC primers

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