High Molecular Weight (HMW) DNA Extraction

High Molecular Weight (HMW) DNA consists of long, intact DNA molecules, important for long-read sequencing analysis in structural variation studies. Reliable isolation methods for HMW DNA are vital to minimize fragmentation, help ensure purity, and maximize yield, all important for accurate genomic analyses. This can be particularly crucial in oncology research where HMW DNA analysis is used for detecting structural variants and characterizing tumor heterogeneity in solid tumor and liquid biopsy samples.

To help advance comprehensive genomic analysis including long-read sequencing applications, the Applied Biosystems MagMAX HMW DNA Kit has been designed to isolate HMW DNA with large intact fragments utilizing Dynabeads magnetic bead technology and optimized chemistries along with automated and manual workflows to enhance efficiency, reproducibility, and productivity. High-quality large genomic DNA can be achieved consistently, helping provide accurate and reliable long-read sequencing analyses to help drive genomic studies and cancer research efforts forward.

Long-read sequencing technologies, such as those developed by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies, offer the ability to read sequences of DNA that are thousands of base pairs long. This contrasts with short-read sequencing technologies, which typically read DNA sequences that are only a few hundred base pairs long. The longer reads provided by long read sequencing are particularly beneficial for:

• Detecting structural variants—long reads enable large-scale mutation detection, which are often missed by short-read sequencing
• Improving genome assemblies—long reads provide a more contiguous view of genomes, reducing gaps and improving accuracy
• Clinical development—increasingly used in clinical settings for molecular diagnosis and therapy selection, long read sequencing provides a more complete picture of genomes which aids in the development of personalized medicines

Obtaining high-quality HMW DNA can be difficult due to the large fragment size as well as added complications that can arise from common sample types and existing isolation methods. Technical challenges scientists face require reliable methods and optimized protocols that aim to address common challenges such as:

• Shearing: HMW DNA is prone to shearing due to its length and susceptibility to break under common handing conditions. Due to the fragile nature of longer fragments, isolation methods implementing careful handling techniques are necessary to maintain the integrity of the fragments throughout the isolation process.
• Cell lysis: Issues with cell lysis often stem from incomplete reactions or improper handling. If the cell membrane is not adequately broken down or the reaction is not handled with the proper attention, low DNA yields and possible shearing can occur. Choosing optimized buffers with efficient protocols tailored to specific sample types can help ensure complete and effective lysiswhile keeping the DNA intact.
• Contamination: Contaminants, such as proteins, lipids, and other cellular debris, left behind during the isolation process can impact the integrity, quality, and yield of the extracted DNA, inhibiting downstream reactions and hindering analysis. Helping ensure high purity of intact fragments can enable accurate analysis results.
• Viscosity: HMW DNA solutions, especially those in high concentrations, can be very viscous, leading to difficulties in handling and pipetting. This can cause imprecise measurements and complications in downstream applications. Proper isolation techniques can help control these issues by reducing solution viscosity while preserving DNA integrity.

Reliable preparation of HMW DNA is important for the success of long-read sequencing analysis. Effective isolation methods enable long-read sequencing by addressing common challenges associated with HMW DNA isolation and meeting workflow needs associated with common techniques, including:

• Preserving DNA integrity and preventing degradation
• Optimizing conditions and protocols to overcome handling and analysis difficulties
• Helping ensure sequencing compatibility with sufficient quantity and DNA fragment sizes
• Abling adaptation to specific throughput and sample type needs
• Consistency of isolation results to help provide the reproducibility needed in clinical workflow development

Leveraging Dynabeads magnetic bead-based technology, the MagMAX HMW DNA Kit enables effective HMW DNA isolation by helping provide:

• Reproducibility and efficiency—reliable isolation of intact HMW DNA fragments >100 kb in less than 2 hours
• Flexibility—optimized protocols that accommodate varying throughput capabilities and sample types
• Automation compatibility—pair with KingFisher instruments for a streamlined workflow
• Customization—standalone reagents available for custom applications and scalability needs

Enhance the efficiency of your HMW DNA extraction workflow using the MagMAX HMW DNA Kit on Thermo Scientific KingFisher instruments. Automated protocols are available for Duo Prime, Flex, and Apex instrument models.

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Figure 1. HMW DNA isolation from whole blood, cells, or tissue samples using the MagMAX HMW DNA Kit. Extract DNA manually with DynaMag magnetic separation racks or automate the workflow using a KingFisher Duo Prime, KingFisher Flex, or KingFisher Apex purification system.

Table 1. Purity and quality performance using NanoDrop instrument technologies to obtain average absorbance ratios of 260/280 and 260/230 from HMW genomic DNA isolated from cells (1 x 10⁶ input), fresh and frozen whole blood (200 µL input), and tissues (8 mg brain, 2 mg tissue) with MagMAX HMW Kit on KingFisher purification instruments.

Discover how our HMW DNA isolation solution is transforming long-read sequencing applications through in-field studies and testimonials from our customers.

Long-read sequencing applications help provide critical insights into cancer biology, identifying a wider range of genomic variation that shorter reads can miss. This more complete picture of genomic variation offers a more comprehensive understanding of cancer-causing mutations that could ultimately transform the development of cancer diagnostics and targeted therapeutics.

The new HMW DNA isolation kit supports these applications, offering high-quality intact DNA fragments essential for advancing cancer genomics research. Reliable tools like this kit are crucial for enhancing diagnostic accuracy and treatment efficacy in cancer research.

When applied to hemato-oncology, long-read sequencing can offer a more complete and accurate view of the genomic, transcriptomic, and epigenetic alterations in hematological cancers. Longer reads provide improved structural variant (SV) detection, enabling comprehensive identification of complex genomic alterations such as large insertions, deletions, inversions, and translocations. Outside of HMW DNA analysis, long-read sequencing can also enhance the understanding of gene fusions and isoforms by sequencing full-length RNA transcripts which is important for diagnosing and targeting therapies in leukemia. Long-read sequencing platforms can also directly detect epigenetic modifications, such as DNA methylation, offering valuable insights into the epigenetic landscape of hematological cancers.

Interested in hematological cancer research?
Learn more about upstream nucleic acid isolation from blood and bone marrow samples using the MagMAX Sequential DNA/RNA Kit

Long-read sequencing also allows for long-range phasing, crucial for understanding allele-specific gene expression and the impact of mutations on oncogenes and tumor suppressor genes. This can be applied to single cell isolation and analysis of rare cells, such as circulating tumor cells (CTCs), found in blood, uncovering their unique genetic and transcriptomic profiles. By identifying genomic mutations and alterations in circulating cells, the development of personalized cancer treatment strategies for targeting hematological malignancies is made possible.

Learn more about cell isolation using Invitrogen Dynabeads magnetic beads

CTCs are a component of liquid biopsies, which involve analyzing blood or other biofluids to detect and analyze circulating biomarkers. The comprehensive analysis liquid biopsy can provide contributes to an overall deeper understanding of cancer biology, facilitating in the development of personalized therapies and treatment strategies, enabling further advancements in the oncology field overall.

Want to learn more about cancer research through the lens of genetic and protein biomarkers?
Download our eBook: Synergetic biomarkers: Fueling advancements in cancer research

Effective isolation of smaller genomic DNA (gDNA) fragments is important for genomic studies outside of long read sequencing. These smaller fragment targets are often used in applications such as next generation sequencing, dPCR, and other molecular biology techniques. Reliable analyses often rely on the isolation of the DNA. MagMAX automated magnetic bead-based kits can support this upstream sample preparation with reliable and efficient extraction methods.

Product	MagMAX HMW DNA Kit	MagMAX DNA Multi-Sample Ultra 2.0 Kit	MagMAX DNA Multi-Sample Ultra Kit
Qualified sample types	Blood, cells, tissues	Blood, cells, tissues, bone marrow, saliva, buccal swabs, buffy coat, blood cards	Blood, cells, tissues, buccal swabs, saliva, blood cards, urine
DNA fragment size range	100–300 kb	1–40 kb	1–40 kb
KingFisher compatibility	Duo Prime, Flex, Apex	Duo Prime, Flex, Apex	Duo Prime, Flex
Standalone reagents available	Yes	Yes	No
Downstream applications	Long-read sequencing, microarray	PCR, NGS, microarray	PCR, NGS
Cat. No. unit size	A36626, 100 preps A36627, 100 preps*	A36570, 1 kit	A25597, 500 preps A25598, 2,500 preps A45721, 1 kit*

*with cell and tissue extraction buffer

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