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Zhang Y.,Shenzhen Blood Center Institute of Transfusion Medicine | Xu H.,Shaanxi Blood Center | Zhou H.,Guangzhou University of Chinese Medicine | Wu F.,Shenzhen Blood Center Institute of Transfusion Medicine | And 3 more authors.
Analytical Biochemistry | Year: 2015

Abstract The quality and yield of single-stranded DNA (ssDNA) play key roles in ssDNA aptamer selection. However, current methods for generating and purifying ssDNA provides either low yield due to ssDNA loss during the gel purification process or low specificity due to tertiary structural damage of ssDNA by alkaline or exonuclease treatment in removing dsDNA and by-products. This study developed an indirect purification method that provides a high yield and quality ssDNA sublibrary. Symmetric PCR was applied to generate a sufficient template, while asymmetric PCR using an excessive nonbiotinylated forward primer and an insufficient biotinylated reverse primer combined with a biotin-strepavidin system was applied to eliminate dsDNA, hence, leading to ssDNA purification. However, no alkaline or exonuclease were involved in treating dsDNA, so as to warrant the tertiary structure of ssDNA for potential aptamer SELEX selection. Agarose gel imaging indicated that no dsDNA or by-product contamination was detected in the ssDNA sublibrary generated by the indirect purification method. Purified ssDNA concentration reached 1020 ± 210 nM, which was much greater than previous methods. In conclusion, this novel method provided a simple and fast tool for generating and purifying a high yield and quality ssDNA sublibrary. © 2015 Elsevier Inc. All rights reserved. Source

Liu C.,Guangzhou University of Chinese Medicine | Lv P.,Guangzhou University of Chinese Medicine | Zhang Y.,Shenzhen Blood Center Institute of Transfusion Medicine | Shang J.,The Inner Mongolia Autonomous Region Blood Center | And 3 more authors.
Genetic Testing and Molecular Biomarkers | Year: 2016

Aims: This study aimed to compare the intron 4 sequence of the RHD and RHCE genes from Han Chinese, Tibetans, and Mongols, and explore its polymorphisms. Materials and Methods: To investigate the distinction in the RHD and RHCE intron 4, polymerase chain reaction (PCR) was performed by a set of primers: Intron4F and Intron4R. Primer Intron4F for a sequence located in exon 4 and primer Intron4R for a sequence located in exon 5, respectively. RHD and RHCE intron 4 of all the samples from 26 cases of random unrelated Hans (13 RhD-positive donors and 13 RhD-negative donors), 25 cases of random unrelated Tibetans (18 RhD-positive donors and 7 RhD-negative donors), and 4 cases of random unrelated Mongols (1 RhD-positive donor and 3 RhD-negative donors) were amplified with PCR. The PCR products were then sequenced. Results: A 576-bp product was detected in all the Han, Tibetan, and Mongol RhD-positive donors, whereas a 1228-bp product was detected in RhD-negative donors. The sequences of RHCE gene intron 4 were identical to each other in all Han, Tibetan, and Mongol RhD-negative donors, including 335 bp of Alu element, with a whole length of 1078 bp. By contrast, a 426-bp product was detected in all Han, Tibetan, and Mongol RhD-positive donors. Compared with the RHCE gene, a 652-bp deletion was noted in the RHD gene of Chinese, including the whole Alu element. The results were similar to the findings of Caucasians, whereas the lengths of RHD gene deletion fragments of Japanese and French were 649 and 654 or 651 bp, respectively. Conclusions: The RHCE gene intron 4 of Han Chinese, Tibetans, and Mongols differs from the RHD gene intron 4 in the presence of a 652-bp fragment. The RHCE gene intron 4 in Chinese has its own structural characteristics and differs among various ethnicities and regions. © Mary Ann Liebert, Inc. 2016. Source

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