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Crude DNA Extractions for High-Throughput PCR Genotyping: Save Time, Resources and Budget Without Compromising Data Quality

The Challenge of DNA Extraction

SNP genotyping and molecular markers have revolutionized the fields of genomics for research, crop and livestock breed improvement, and seed purity testing, offering a fast and cost-effective approach. In the case of high-throughput plant genotyping, plants are typically genotyped from leaf tissue, necessitating the growth of the plant to the cotyledonary stage or its first true leaf. However, this process is time-consuming, space-intensive, and requires significant human resources, particularly at an industrial scale. A more efficient alternative is performing genetic screenings directly on seeds or early emerging roots (radicles), but the challenge lies in extracting DNA from seed tissue. Seeds are rich in reserve components like lipids, oils, proteins, polysaccharides, and polyphenols, which complicate DNA extraction. However, with the latest PACE 2.0 Genotyping Master Mix formulation, you can obtain superior data even from unpurified DNA samples.

While ultra-pure DNA is necessary for many genomic processes, for PCR genotyping, where only short stretches of DNA are amplified, crude DNA extractions such as the Hot Sodium Hydroxide and Tris (HotSHOT) methods are sufficient. When paired with an enhanced genotyping master mix like 3CR Bioscience’s PACE 2.0 Genotyping Master Mix, the need for extensive purification is eliminated.

Crude DNA Extraction Methods Overview

Crude DNA extraction involves collecting leaf, seed, or other tissue samples in 96-well plates and subjecting them to brief mechanical or chemical disruption to burst open the cells. In the simplest methods, cell lysates are centrifuged to pellet debris, and the remaining supernatant is diluted to reduce inhibitor compounds while retaining enough DNA for detection using genotyping assay primers. One commonly used method is the HotSHOT method, which is fast, inexpensive, and scalable. This method has been adapted for various tissues, including plants with high secondary metabolite concentrations, making it ideal for seed genotyping.

Example: Hop Plant DNA Prep Method

A modified HotSHOT DNA extraction method has been successfully adapted for hop plants (read the paper here), which are high in secondary metabolites. This custom extraction method is cost-effective, fast, and can be performed using basic laboratory equipment. The result is a reliable and efficient DNA extraction process that reduces both financial burden and time constraints, significantly increasing sample throughput.

Example: Carrot Seedling DNA Prep Method

For carrots, DNA extraction from young seedling tissue involves cell lysis using a tissue homogenizer (you can read the paper here). The crude extract, after brief centrifugation, is diluted to produce a final DNA solution suitable for PCR. This method is both efficient and cost-effective, providing a viable alternative to more time-consuming purification processes.

The Benefits of Crude DNA Preparation

High-quality DNA purification processes, while effective, are time-consuming and expensive. Crude DNA extraction methods offer a faster, more cost-effective alternative that is easily scalable. These methods are quicker, requiring fewer steps and reagents, and can be performed in high-throughput formats, making them ideal for downstream high-throughput genotyping. The primary benefits include:

Cost Savings: Significant reduction in expenses associated with DNA extraction kits and reagents.
Speed: Quicker preparation times compared to traditional purification methods.
Efficiency: Reduced manpower requirements and the ability to process large numbers of samples simultaneously.

The Drawbacks of Crude DNA Preparation

The primary drawback of crude DNA preparation is the lower quality of DNA compared to commercial extraction kits. For many applications, this lower quality is not sufficient. However, PCR genotyping is more forgiving, requiring only short DNA fragments. Crude DNA samples often contain compounds inhibitory to downstream PCR processes, such as polyphenols and polysaccharides. The challenge is to minimize and neutralize these inhibitors to ensure accurate and reliable results.

The Solution: PACE 2.0 Genotyping Master Mix

This advanced formulation of our patented PACE Genotyping Master Mix ensures consistent, accurate genotyping data without the need for extensive sample purification. By streamlining workflows and reducing costs, PACE 2.0 Genotyping Master Mix allows for high-throughput genotyping without compromising data quality.

Figure 1: SNP genotyping data generated using 1.6 μL final reaction volume on Array tape. Data from an undisclosed testing service company using crudely-extracted, PCR inhibitor-containing samples of globally-important agricultural crops, with purified control DNA samples (highlighted).

Features and Benefits

PACE 2.0 Genotyping Master Mix boasts several key features that make it ideal for use with crude DNA samples:

Inhibitor Resistance: Neutralizes PCR inhibitors present in crude DNA extracts from plant and animal samples.
Improved Signal-to-Noise Ratio: Enhanced fluorescent reporting system for better data quality.
No DNA Purification Required: Increases throughput while maintaining data integrity.
Compatibility: Works with a wide range of crude DNA extraction methods and is suitable for both endpoint and real-time PCR.

Case Studies

Case Study 1: Virginia-type Peanut
The North Carolina State University peanut breeding program has implemented a manual seed chipper and a crude DNA isolation protocol paired with PACE Genotyping Master Mix and PACE Genotyping Assays for an efficient, high-throughput workflow. This approach has enabled substantial expansion of marker-assisted selection (MAS) in their breeding program, achieving a genotyping success rate of 98.4% and significantly reducing resources required for greenhouse and field space (click here to read the paper).

Figure 2: Example marker figure produced by the SNP caller. Seeds homozygous for the HO mutation in FAD2B will produce predominantly HEX fluorescence signal and plot towards the Y-axis (blue cluster in top left corner). Seeds homozygous for the wild type NO allele in FAD2B will produce predominantly FAM fluorescence signal Page 16/16 and plot towards the X axis (green cluster in bottom right corner). Heterozygous seeds will produce an equal mix of both signals and cluster in red between the two homozygous clusters. Seeds that fail genotyping will cluster near the origin (yellow in bottom left corner).

Case Study 2: Hop Plants
In a recent hop plant study, researchers from the National Clonal Germplasm Repository, OR and Forage Seed and Cereal Unit, WA of the USDA-ARS used a modified HotSHOT DNA extraction method, combined with PACE 2.0 genotyping to enable rapid, cost-effective genotyping with high accuracy. This method has the potential to significantly reduce cultivar release timelines, reallocating resources to other areas of crop improvement. Find the article here.

Case Study 3: Carrot Seedlings
For carrot seedlings, DNA extraction using a tissue homogenizer followed by PACE 2.0 genotyping provided an efficient, cost-effective solution for researchers at the Department of Plant and Agroecosystem Sciences, University of Wisconsin-Madison. This method facilitated high-throughput genotyping, crucial for large-scale breeding programs (read the paper here).

Conclusion

Crude DNA extraction methods, paired with advanced solutions like PACE 2.0 Genotyping Master Mix and PACE Genotyping Assays, offer a powerful approach for high-throughput genotyping. By reducing costs and preparation times, these methods enable efficient and reliable genetic screenings, even from challenging starting materials. As demonstrated in various user case studies, the combination of crude DNA extraction and PACE® genotyping reagents can streamline workflows, increase sample throughput, and maintain high data quality, making them invaluable tools for modern genomics applications.

For more information on how PACE 2.0 Genotyping Master Mix can enhance your DNA extraction and genotyping workflows, email support@3crbio.com or visit our Applications page.

Crude DNA extractions for high-throughput PCR genotyping
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Steve Asquith Managing Director
Steve began his career in the Genetics Division of GlaxoSmithKline, as part of the team establishing GSK’s high-throughput core genotyping laboratory. Steve joined KBioscience when it was first founded in 2002 and was a key driver in taking the company from a small start-up to a multi-national service laboratory, quickly growing the company’s revenue to over $7.5M p.a. Following the acquisition of Kbioscience by LGC in 2011, Steve was appointed Global Director of Operations for LGC Genomics, responsible for over 100 staff in Europe and N. America, successfully elevating the genotyping products and service business. Steve held a crucial leadership role until he left in 2016. In 2017 Steve joined forces with John Holme to create 3CR Bioscience, a new company with a mission to deliver outstanding, customer-focused genotyping products with innovation and affordability at its core.
Dr. John Holme Technical Director
John joined KBioscience shortly after it was founded, in 2003, and became Head of Technical Development, building the company’s genotyping and DNA extraction product portfolio and service delivery until 2011 when it was acquired by LGC. Post-acquisition, John was appointed Head of Technical Group for LGC Genomics, in charge of all Research & Development and Technical Support activities for the company. In this role John continued to build on the high-quality products and services provided to the companies growing customer base. During the 19 years John has worked in commercial R&D, he has co-invented numerous highly successful products including PACE®, ProbeSure, KASP™, KlearKall, KlearGene, KlearAmp and KlearTaq™, creating breakthrough offerings in genotyping and extraction and generating huge revenues for the companies he has worked in. In 2017, he joined forces with Steve Asquith and started 3CR Bioscience. John is dedicated to developing outstanding, innovative genotyping products and providing the very best technical support to customers globally.
Dr. Nisha JainOperations Director
Nisha has been innovating since the start of her career at Geneform Technologies developing Iso-thermal Genotyping Technologies. Nisha joined KBioscience in 2008, as Senior R&D Scientist and key account Technical Support Scientist, developing KASP and Klearkall performance and coinventing two further versions of KASP. Nisha has more than 15 years’ experience working in molecular biology and genotyping technologies, with extensive experience in the areas of R&D, Quality Assurance and Customer Technical Support. She has technically assisted many giants of the industry with their protocol development and troubleshooting and continues to deliver high-quality support and guidance. In 2018, Nisha joined 3CR Bioscience as Operations Director where she continues to develop PACE and ProbeSure for an increasing range of applications, and to grow 3CR Bioscience’s new product pipeline. Nisha is dedicated to developing outstanding, innovative genotyping products and providing the very best technical support to customers globally.
Nazma Saffin General Manager
For 20 years Nazma Saffin has worked and gained extensive expertise within the genotyping sector. Working at Kbioscience and then LGC, she has held operational leadership posts responsible for manufacturing and laboratory services. With experience of ISO 9001 implementation, production scale up and LEAN operations, Nazma has successfully led highly profitable production departments. Joining 3CR Bioscience in 2022, Nazma is committed to delivering operational excellence.
Jon Curtis Non-Executive Chair
After 8 years in The Royal Air Force, Jon moved to the Imperial Cancer Research Fund where he pioneered the use of ultra high-throughput genomic automation, capable of 46,000 PCRs per hour. In the 1990’s Jon joined GlaxoSmithKline, implementing a high-throughput genomics platform into their drug discovery pipeline. Whilst there he also developed acoustic mixing into compound management, becoming the gold standard across pharma. Jon developed the world’s first commercially viable 1536-well PCR plates, automated thermal & laser plate-sealer, plus automated liquid-handling & tip washing tools to reduce waste and costs. In 2002 Jon co-founded KBioscience with Phil Robinson, utilising ultra high-throughput PCR instrumentation & a suite of automation tools to create the company’s SNPline robotic platform, with a capacity of 250,000 PCRs/day. The business was underpinned by their ground-breaking patented genotyping chemistry, KASP™, which has over 10,000 scientific papers to date. In November 2022 Jon joined 3CR Bioscience acting as an advisor bringing his commercial and scientific experience to the company.