Anion exchange-based purification of HMW DNA from diverse samples
The NucleoBond HMW DNA kit employs gravity-flow filtration in combination with anion-exchange technology to enable efficient recovery of high molecular weight DNA up to 200 kb in length. This kit accommodates a diverse array of input material, including bacteria, yeast, plants, insects, blood, cultured cells, and mammalian tissues. The kit provides a gentler alternative to magnetic bead-based approaches, which include extensive pipetting and mixing steps that threaten DNA integrity, and offers superior performance vs. liquid-phase methods (e.g., salting out) that tend to yield DNA of lower purity. The versatile protocol enables processing of 12 samples in about two hours, accommodates large input amounts, and can be combined with additional upstream and downstream processing steps, including enzymatic or mechanical sample lysis (e.g., MN bead tubes) and DNA concentration/desalinization (e.g., NucleoSpin and NucleoSnap finishers). DNA purified with the kit is ideal for analysis by long-read sequencing technologies such as SMRT (PacBio) and Nanopore (Oxford Nanopore).
High-quality, high molecular weight (HMW) DNA up to 200 kb
Minimized DNA shearing due to established anion-exchange technology
Efficient processing of 12 samples in 2 hours (including 30-minute sample lysis)
Validated with diverse samples and sequencing platforms
Up to 200 kb with >90% depletion of fragments <10 kb
Sample material
<1.5 g plant leaves (ground under liquid nitrogen)
<107cultured cells (enzymatic lysis)
<300 mg solid tissue (cut into small pieces and lysed enzymatically with increased lysis time, ground under liquid nitrogen, or lysed by bead beating)
<30 mg yeast or bacteria (ground under liquid nitrogen)
<300 mg yeast or bacteria (lysed enzymatically or by bead beating)
<2 ml liquid sample (e.g., blood, body fluids, or liquid reactions)
Typical yield
Depends on sample type and amount
Elution volume
50–250 µl
Processing time
2 hr/12 preps (including 30-min lysis)
Procedure
Figure 1. Overview of the workflow for the NucleoBond HMW DNA kit. To accommodate a wide array of sample types and downstream applications, the NucleoBond HMW DNA kit allows researchers to incorporate a variety of different sample homogenization and lysis methods upstream of DNA purification. To maximize the integrity and size of recovered DNA, it is recommended to perform enzymatic pre-lysis of bacterial and yeast samples. For a faster procedure yielding DNA of medium integrity, samples can be homogenized using MN bead tubes prior to lysis. Some samples, such as plant tissues, can be homogenized with a mortar and pestle. After lysis, samples are filtered and loaded onto the NucleoBond HMW DNA Column. Following binding of DNA, a wash procedure ensures efficient removal of inhibitors and contaminants while avoiding any shearing of the DNA. The DNA is then eluted and can be desalted and/or concentrated using a variety of approaches (e.g., NucleoSnap/NucleoSpin Finishers). For maximum integrity of the eluted DNA, isopropanol precipitation is recommended.
Application data
Figure 2. Recovery of large DNA fragments. DNA was purified from a Brassica species using the NucleoBond HMW DNA kit with mechanical sample lysis. The purified DNA was then analyzed on a Femto Pulse System. Fragments up to 200 kb in size could be detected and a peak corresponding to a size of 117 kb was observed, demonstrating the ability of the NucleoBond HMW DNA kit to efficiently purify HMW DNA.
Read length (kb)
Number of reads
Cumulative read length (bp)
Total
130,591
1,096,733,169
>1
75,906
1,066,775,188
>10
36,755
925,569,331
>20
21,874
705,439,290
>30
10,918
435,266,462
>40
4,361
209,403,539
>50
1,328
75,094,595
>60
276
18,362,150
>70
52
4,041,525
>80
11
1,019,289
>90
6
597,205
>100
2
215,960
Table I. Optimal procedure for longer sequencing reads. DNA was purified from a Brassica species using the NucleoBond HMW DNA kit with mechanical sample lysis. The purified DNA was sequenced on a MinION device (Oxford Nanopore). More than 1,600 reads longer than 50 kb were sequenced (including some reads longer than 100 kb), indicating the excellent integrity and quality of high molecular weight DNA extracted with the kit.
Figure 3. High-quality sequencing data with market-leading results. DNA was purified from HeLa cells using the NucleoBond HMW DNA kit with enzymatic sample lysis. The purified DNA was then analyzed on the Single Molecule Real Time (SMRT) sequencing platform (PacBio RS II). Resulting read lengths obtained with the NucleoBond (MN) kit were compared with results for two commonly used competitor products (kits “P” and “M”, both from Competitor Q). As shown by the data, the NucleoBond HMW DNA kit provided a higher mean subread length than either competitor kit, enabling superior long-read sequencing results.
Liver
HeLa
Blood
E. coli
B. subtilis
Yeast
Zea mays
A. thaliana
Yield (µg DNA)
45.9
35.9
59.4
571.2
205.2
39.8
366.1
51.8
A260/A230
2.31
2.29
2.39
2.34
2.21
2.43
2.31
2.14
A260/A280
1.86
1.87
1.86
1.83
1.82
1.76
1.85
1.80
ng/µl
176.0
180.7
253.4
1,632.0
547.2
254.1
2,184.7
146.9
Table II. Efficient recovery of high-quality DNA from a wide array of samples. Popular methods for isolation of HMW DNA often pose limitations with regard to DNA purity and yield. To demonstrate the versatility and performance of the NucleoBond HMW DNA kit, DNA was purified from diverse samples. Each extraction yielded DNA of sufficient purity and yield for sequencing, with A260/A280 absorbance ratios >1.75 and A260/A230 absorbance ratios >2.00 for all samples analyzed.
