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

Abstract

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.

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

“…The resolution and production of RAPD are greatly increased by prolonging the RAMP time from the stage of annealing to extension in PCR [ 18 , 19 ]. Recently, the technique was modified using high-GC content primers to improve the effectiveness of the RAPD method (high-GC RAMP-PCR technique) [ 20 ]. This improved RAPD successfully increased the number of RAPD bands produced from a given DNA sample.…”

“…Using improved RAPD with high-GC content primers, we found that the average number of bands per primer increased from 6.41 to 15.04 in comparison to regular RAPD with standard primers. The average number of polymorphisms per primer was also increased from 4.647 to 10.22 [ 20 ]. Thus, we can produce more polymorphic bands using less RAPD primers.…”

“…The 10-bp length oligos with high-GC contents (90% of G+C) were designed and synthesized at Beijing DNA chem. Biotechnology Co., Ltd (Beijing, China) and were as previously described [ 20 ]. The sequences of high-GC primers FY-10, FY-13 and FY-31 are presented in Table 2 .…”

“…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.…”

Evaluation of genome similarities using the non-decimated wavelet transform

“…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.…”