By Ben Butkus
A team led by scientists from France's Institute of Genetics and Molecular and Cellular Biology has developed a method of amplifying picogram-scale amounts of DNA in a single tube with high fidelity for massively parallel sequencing of immunoprecipitated chromatin, or ChIP-seq, studies, according to a paper published this week.
The method, dubbed single-tube linear DNA amplification, or LinDA, is expected to facilitate genome-wide profiling of transcription factors and chromatin analyses using DNA obtained from just a few hundred cells, as is often the case when working with stem cells or cancer-initiating cells, the researchers claim.
The ChIP technique has become an important tool for genome-wide analyses of chromatin modification and dynamics, as well as transcription-modulating complexes.
However, a limitation of the method is that it requires the recovery of nanogram or smaller amounts of immunoprecipitated DNA, particularly when conducting ChIP studies in very small cell populations, thus necessitating a method of pre-amplifying DNA enriched in ChIP experiments prior to sequencing.
In particular, the research team from IGMCB, a joint research unit of France's CNRS, INSERM, and the University of Strasbourg, were planning to conduct epigenomics and transcription factor profiling of patient samples from biobanks in order to compare them to their normal cell counterparts, Hinrich Gronemeyer, a research director at INSERM and an investigator at IGMCB, told PCR Insider this week.
"We realized the urgent need to develop a technology by which a few hundred cells can be studied in a genome-wide manner by ChIP-seq," Gronemeyer said. "A second project comprises the profiling of cancer stem [cells] and cancer-initiating cells, which are also highly limiting."
As the scientists wrote in their research paper, published online this week in Nature Methods, "to date, no versatile technique has been described that (i) demonstrates reliable amplification of picogram DNA quantities of complex DNA samples corresponding to transcription-factor binding sites to chromatin; and (ii) can be used for high-throughput sequencing or the analysis of ultra-small amounts from forensic or archeological specimens."
Gronemeyer told PCR Insider that to his knowledge, only the laboratory of Bradley Bernstein of the Massachusetts General Hospital, Harvard Medical School, and the Broad Institute, has developed an adequate method for low cell numbers, a technique that was described in a Nature Methods publication last year.
"However, this technique involves PCR amplification, which has the risk of biased amplification of GC-rich sequences," Gronemeyer said.
In contrast, the French researchers' LinDA method, which uses T7 RNA polymerase, is similar to a previously described method called T7-based linear amplification of DNA, or TLAD, which has been shown in the past to have high fidelity and low bias. However, products from TLAD can't be directly used for high-throughput sequencing, and "the complex handing steps of these protocols are incompatible with ultra-small amounts of DNA," the researchers wrote.
To address these issues, the French scientists developed a protocol that uses a single buffer during amplification. In addition, "consecutive steps are performed in the same tube, thus eliminating the need for column purification and minimizing the risk of sample loss," the researchers wrote, adding that this feature makes the protocol amenable to process automation.
To validate the single-tube LinDA method, the researchers amplified a 404-bp luciferase gene fragment, generating a predicted 527-bp DNA fragment with the group's custom T7 promoter at both ends, as confirmed by sequencing. In addition, they showed that the method could amplify small amounts of DNA spiked with various amounts of luciferase DNA from a large excess of heterologous genomic DNA.
Next, the researchers tested whether the single-tube LinDA method could reliably amplify ChIPed DNA by comparing estrogen-induced target gene binding of estrogen receptor α by qPCR analysis of nine different target loci that had been identified in a separate ChIP-seq study using human breast cancer cells. They found that amplified products using LinDA were "virtually indistinguishable" from the initial ChIPed DNA.
The researchers also compared profiles generated by Illumina sequencing from 3.5 nanograms of retinoid X receptor α-specific ChIP from F9 cells with profiles generated from 35 picograms of the same genetic materials using LinDA, and found the results to be highly concordant.
In general, the researcher said, their data demonstrated that LinDA permitted 100 percent reliable retrieval of genome-wide transcription factor binding sites from picogram amounts of ChIPed DNA. The researchers also noted that using longer reads and increasing the number of mappable reads would likely boost the sensitivity of the method.
Gronemeyer also noted that the group believes it can up the sensitivity of the method by making improvements to the ChIP-seq method itself.
The researchers concluded that LinDA should be able to be applied to just a few hundred cells, and that it will facilitate chromatin conformation capture-based technologies for the mapping of long-range interaction.
"Although LinDA can be used to amplify any source of DNA, it will be particularly useful to analyze transcription factor complexes, histone modification, and chromatin remodeling in very small compartments, such as stem and cancer-initiating cells," the researchers wrote.
Gronemeyer said that the group has applied for patents on its method.
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