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Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Apr;34(4):367-372.

[A set of methods of amplification of human monoclonal antibody heavy and light chain genes from one single B cell].

[Article in Chinese]

Author information

1
BGI-Shenzhen, Shenzhen 518083; BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China.
2
BGI-Shenzhen, Shenzhen 518083, China.
3
BGI-Shenzhen, Shenzhen 518083; Complete Genomics, Inc., San Jose, CA 95134, USA. *Corresponding author, E-mail: yangnaibo@genomics.cn.

Abstract

Objective To develop a set of methods of amplifying the natural paring heavy and light chain genes from one single B cell. Methods Yanhuang (YH) cells were the first whole genome sequenced human cells of Asian origin. With the immortalized cell lines as the raw material, single CD19+ B cells were sorted into individual PCR tubes by fluorescence-activated cell sorting (FACS). Then its total RNA was released by the lysis buffer, and reverse transcribed. With the cDNA as the templates, the pairing heavy and light chains from the same B cells were amplified by two-step nested PCRs to acquire their variable region sequences. Results Amplifying methodology has been successfully developed for acquiring single cell BCR genes, and the success rate was greater than 80%. The sorted single B cells could be saved in -80DegreesCelsius for up to two weeks, and then successfully amplified. The PCR products in the same tube were TA-cloned and identified by Sanger sequencing, including the heavy and light chain pairing information. A set of effective primers were reported and released in this study. Conclusion A set of methods were successfully developed for amplifying the natural paring heavy and light genes with the beginning of one single B cell.

PMID:
29973329
[Indexed for MEDLINE]

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