DNA Affinity Purification Sequencing (DAP-seq) represents a cutting-edge technology designed for the in-depth exploration of DNA-protein interactions in vitro. This innovative approach amalgamates protein overexpression methods with high-throughput sequencing, facilitating the incubation of genome-wide DNA fragments with transcription factor (TF) proteins expressed in vitro. Consequently, DAP-seq enables the precise determination of TF binding sequences, offering invaluable insights into the pivotal roles that transcription factors play in the growth, development, and responses to external environmental cues in both the animal and plant kingdoms.

The distinct advantage of DAP-seq technology lies in its ability to swiftly map TF binding sites, shedding light on the regulatory domains of DNA. At CD Genomics, we are at your service with comprehensive DAP-seq technology solutions and personalized data analysis.

Key Advantages of Our DAP-seq Service

Compatibility with Challenging Scenarios: Some species, particularly within the plant realm, pose difficulties when it comes to obtaining suitable antibodies for ChIP-level research. Moreover, limitations arise due to the absence of mature transgenic systems. The customization of antibodies, while beneficial, can be a protracted and formidable process.

Overcoming Challenges in Chromatin Extraction: Certain plants, such as fruits, flowers, and tea leaves, contain elevated levels of phenolic compounds, rendering chromatin extraction an intricate task.

Addressing Low Protein Expression in Samples: Transcription factors in plants often exhibit low expression levels and exhibit temporal and spatial expression patterns, which DAP-seq technology can effectively handle.

Support for Non-Model Organisms: DAP-seq technology transcends the constraints of antibody quality, making it suitable for research in both model organisms and non-model organisms, with particular relevance in the plant kingdom.

DAP-seq vs. ChIP-seq

DAP-seq is a versatile and cost-effective method suitable for a broad range of DNA-protein interaction studies, while ChIP-seq is more specific and powerful for studying known protein-DNA interactions but requires specific antibodies. Your choice should align with your research objectives, sample type, available resources, and budget constraints.

DAP-seq service workflow

Our DAP-seq service workflow includes DNA library construction, protein expression, protein-library binding reactions, library PCR, adapter addition, quantitative testing, sequencing, and bioinformatics analysis.

DAP-seq service workflow – CD Genomics

DAP-seq bioinformatics analysis

At CD Genomics, our DAP-seq bioinformatics analysis workflow is a critical component of our service that transforms raw sequencing data into valuable insights about DNA-protein interactions.

Here's a detailed overview of the key steps involved:

• Quality Control and Data Filtering
• Reference Genome Sequence Alignment
• Peak Calling
• Peak Analysis
• Functional Annotation and Enrichment Analysis
• Motif Analysis

DAP-seq bioinformatics analysis – CD Genomics

Sample requirements

To begin your DAP-seq research, you'll need to provide:

• The CDS sequence of the transcription factor for feasibility assessment,
• A sufficient sample quantity, plasmids containing the target transcription factor, and data relevant to sample replication.
• Genome information matched to your research species, including fasta, gff, vcf, etc.

More details, please contact our technique teams.

Transcription Factor (TF): > 5 µg; minimum concentration 20 ng/µL

Cell Samples: > 5 x 10^6 cells

Tissue Samples: > 5 mg

DNA can be submitted in DNase-free Water, Elution Buffer, or 10mM Tris pH 8.0. DNA samples require an OD260/280 as close to 1.8~2.0 as possible. All DNA should be RNase-treated and should show no degradation or contamination. Ship with ice packs. The total amount of DNA required depends on the specific application.

Case Study

Unveiling Growth Hormone Response Specificity through DAP-seq

The challenge in this study was to unravel whether different family members within the ARF group could regulate specific developmental processes by binding to distinct target genes. To address this question, the scientists embarked on a comprehensive DAP-seq analysis of ARFs from two different clades, Clade A and Clade B, encompassing a diverse set of 14 maize varieties. The primary goal was to create an extensive, genome-wide map of in vitro TF:DNA interactions.

The findings from the comparative analysis were both intriguing and enlightening. ARFs originating from the same branch exhibited a remarkable degree of overlap in their binding sites, highlighting the potential for shared regulatory mechanisms within these branches. However, a significant revelation emerged when comparing ARFs between Clade A and Clade B—substantial differences in binding sites became apparent.

ARF binding events are biologically relevant. (Galli et al., 2018)

It was not just differences that they uncovered; the data also revealed that ARFs from both branches shared numerous common binding sites. This observation suggested the possibility of synergistic interactions among these transcription factors, hinting at a complex web of regulatory relationships that underpin the growth hormone response in plants.

In summary, this large-scale comparative analysis of ARF binding sites brought forth a crucial revelation: growth hormone response specificity in plants does not hinge solely on individual ARF binding sites. Instead, it is shaped by a collective interplay of multiple factors.

Reference

1. Galli, Mary, et al. "The DNA binding landscape of the maize AUXIN RESPONSE FACTOR family." Nature communications 9.1 (2018): 4526.