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Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution.

Wysoczynski CL, Roemer SC, Dostal V, Barkley RM, Churchill ME, Malarkey CS - Nucleic Acids Res. (2013)

Bottom Line: However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments.Both palindromic and non-palindromic DNA can be readily purified.Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USA and Program in Structural Biology and Biochemistry, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USA.

ABSTRACT
Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein-DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein-DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.

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Separation of 5′ single base pair overhang palindromic DNA. (a) Top: 5′-forward palindromic strand annealed to itself with the adenine overhang base highlighted in bold italics. Middle: 5′-reverse palindromic strand annealed to itself with the thymine overhang base highlighted in bold italics. Bottom: Blunt DNA sequence of the 5′-forward and reverse palindromic strands. (b) Top: DNA elution profile of 0.25 mg of 5′-forward and 5′-reverse DNA after annealing. Blunt dsDNA can be separated from overhang dsDNA as well as ssDNA. The left overhang peak corresponds to 5′-forward self-annealed palidromic DNA, whereas the right overhang peak corresponds to 5′-reverse self-annealed palindromic DNA. Middle: DNA elution profile of 0.25 mg of 5′-reverse self-annealed palindromic DNA alone. Bottom: the DNA elution profile of 0.25 mg 5′-forward self-annealed palindromic DNA alone. The acetonitrile gradient for all spectra was 5–12% over the course of 40 min.
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gkt815-F3: Separation of 5′ single base pair overhang palindromic DNA. (a) Top: 5′-forward palindromic strand annealed to itself with the adenine overhang base highlighted in bold italics. Middle: 5′-reverse palindromic strand annealed to itself with the thymine overhang base highlighted in bold italics. Bottom: Blunt DNA sequence of the 5′-forward and reverse palindromic strands. (b) Top: DNA elution profile of 0.25 mg of 5′-forward and 5′-reverse DNA after annealing. Blunt dsDNA can be separated from overhang dsDNA as well as ssDNA. The left overhang peak corresponds to 5′-forward self-annealed palidromic DNA, whereas the right overhang peak corresponds to 5′-reverse self-annealed palindromic DNA. Middle: DNA elution profile of 0.25 mg of 5′-reverse self-annealed palindromic DNA alone. Bottom: the DNA elution profile of 0.25 mg 5′-forward self-annealed palindromic DNA alone. The acetonitrile gradient for all spectra was 5–12% over the course of 40 min.

Mentions: We further investigated the potential of this dsDNA purification method to discriminate between duplex DNA species that differed by only a single base pair. The pseudopalindromic DNA sequence has an additional adenine and thymine base pair at the 5′end of the sequence (Figure 3a), so we have named this DNA sample ATPal. Thus, the palindromic 25mer has the potential to form three distinct duplex DNA products as depicted in Figure 3a: two duplex DNA species will have 5′-A or 3′-T overhangs (5′for and 3′rev), as denoted by the top and bottom strands illustrated in Figure 3a) and one will be a blunt-ended dsDNA (blunt). After loading 0.25 mg of the annealed DNA onto the Waters C18 XBridge column and applying a gradient of 5–12% acetonitrile over 40 min at room temperature, we obtained five peaks on the chromatogram (Figure 3b, top). ssDNA eluted first, followed by the ds overhang DNA corresponding to the 5′for annealed strand and then the 5′rev annealed strand (Figure 3b, top). The blunt-ended DNA was the last species to elute off the column (Figure 3b, top). To confirm the identities of the DNA within these peaks, we loaded 0.25 mg of the 5′for top strand and the 5′rev bottom strand individually and repurified the dsDNA. These DNA oligonucleotides, when annealed individually, generated dsDNA species with overhangs (Figure 3a). When these were purified, the ssDNA peak, the dsDNA peak of the 5′rev dsDNA (Figure 3b, middle) and the 5′for dsDNA (Figure 3b, bottom), respectively, were the major peaks. These peaks obtained from purifying the 5′for top strand and 5′rev bottom strand DNA sequences alone have identical retention times to the overhang DNA peaks presented in Figure 3b (top), confirming the identities of all DNA species in this purification. These experiments demonstrate that this method is able to separate DNA fragments that are highly similar in length and sequence, even DNA with slightly different overhangs, on an otherwise palindromic DNA sequence.Figure 3.


Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution.

Wysoczynski CL, Roemer SC, Dostal V, Barkley RM, Churchill ME, Malarkey CS - Nucleic Acids Res. (2013)

Separation of 5′ single base pair overhang palindromic DNA. (a) Top: 5′-forward palindromic strand annealed to itself with the adenine overhang base highlighted in bold italics. Middle: 5′-reverse palindromic strand annealed to itself with the thymine overhang base highlighted in bold italics. Bottom: Blunt DNA sequence of the 5′-forward and reverse palindromic strands. (b) Top: DNA elution profile of 0.25 mg of 5′-forward and 5′-reverse DNA after annealing. Blunt dsDNA can be separated from overhang dsDNA as well as ssDNA. The left overhang peak corresponds to 5′-forward self-annealed palidromic DNA, whereas the right overhang peak corresponds to 5′-reverse self-annealed palindromic DNA. Middle: DNA elution profile of 0.25 mg of 5′-reverse self-annealed palindromic DNA alone. Bottom: the DNA elution profile of 0.25 mg 5′-forward self-annealed palindromic DNA alone. The acetonitrile gradient for all spectra was 5–12% over the course of 40 min.
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gkt815-F3: Separation of 5′ single base pair overhang palindromic DNA. (a) Top: 5′-forward palindromic strand annealed to itself with the adenine overhang base highlighted in bold italics. Middle: 5′-reverse palindromic strand annealed to itself with the thymine overhang base highlighted in bold italics. Bottom: Blunt DNA sequence of the 5′-forward and reverse palindromic strands. (b) Top: DNA elution profile of 0.25 mg of 5′-forward and 5′-reverse DNA after annealing. Blunt dsDNA can be separated from overhang dsDNA as well as ssDNA. The left overhang peak corresponds to 5′-forward self-annealed palidromic DNA, whereas the right overhang peak corresponds to 5′-reverse self-annealed palindromic DNA. Middle: DNA elution profile of 0.25 mg of 5′-reverse self-annealed palindromic DNA alone. Bottom: the DNA elution profile of 0.25 mg 5′-forward self-annealed palindromic DNA alone. The acetonitrile gradient for all spectra was 5–12% over the course of 40 min.
Mentions: We further investigated the potential of this dsDNA purification method to discriminate between duplex DNA species that differed by only a single base pair. The pseudopalindromic DNA sequence has an additional adenine and thymine base pair at the 5′end of the sequence (Figure 3a), so we have named this DNA sample ATPal. Thus, the palindromic 25mer has the potential to form three distinct duplex DNA products as depicted in Figure 3a: two duplex DNA species will have 5′-A or 3′-T overhangs (5′for and 3′rev), as denoted by the top and bottom strands illustrated in Figure 3a) and one will be a blunt-ended dsDNA (blunt). After loading 0.25 mg of the annealed DNA onto the Waters C18 XBridge column and applying a gradient of 5–12% acetonitrile over 40 min at room temperature, we obtained five peaks on the chromatogram (Figure 3b, top). ssDNA eluted first, followed by the ds overhang DNA corresponding to the 5′for annealed strand and then the 5′rev annealed strand (Figure 3b, top). The blunt-ended DNA was the last species to elute off the column (Figure 3b, top). To confirm the identities of the DNA within these peaks, we loaded 0.25 mg of the 5′for top strand and the 5′rev bottom strand individually and repurified the dsDNA. These DNA oligonucleotides, when annealed individually, generated dsDNA species with overhangs (Figure 3a). When these were purified, the ssDNA peak, the dsDNA peak of the 5′rev dsDNA (Figure 3b, middle) and the 5′for dsDNA (Figure 3b, bottom), respectively, were the major peaks. These peaks obtained from purifying the 5′for top strand and 5′rev bottom strand DNA sequences alone have identical retention times to the overhang DNA peaks presented in Figure 3b (top), confirming the identities of all DNA species in this purification. These experiments demonstrate that this method is able to separate DNA fragments that are highly similar in length and sequence, even DNA with slightly different overhangs, on an otherwise palindromic DNA sequence.Figure 3.

Bottom Line: However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments.Both palindromic and non-palindromic DNA can be readily purified.Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USA and Program in Structural Biology and Biochemistry, University of Colorado Denver, School of Medicine, Aurora, CO 80045, USA.

ABSTRACT
Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein-DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein-DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.

Show MeSH
Related in: MedlinePlus