期刊
ANALYTICAL BIOCHEMISTRY
卷 588, 期 -, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2019.113469
关键词
Nucleosome; DNA synthesis; Ramified RCA; Transcription factor binding
资金
- VIDI grant from Dutch Science Foundation NWO [NWO-CW VIDI 723.013.010]
- MRC career development award [MR/N024028/1]
- BBSRC-EASBIO Ph.D training program [BB/M010996/1]
- BBSRC [BB/M018040/1] Funding Source: UKRI
- MRC [MR/K017047/1, MR/N024028/1] Funding Source: UKRI
Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.
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