Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.
Direct Double-Stranded DNA Quantitation from PCR Reactions V.2
For convenience and for PCR products that are challenging to purify with high efficiency (e.g., chemically modified DNAs), it is often desirable to quantitate synthesized DNA directly from a PCR reaction. Here we describe the use of a high-sensitivity Quant-iT™ PicoGreen® dye-based fluorescence assay to quantitate PCR-synthesized, double-stranded, low molecular weight, 5’-modified DNA probes in the presence of single-stranded primers and deoxyribonucleotides.
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- Award ID(s):
- 1714778
- PAR ID:
- 10098109
- Date Published:
- Journal Name:
- Protocols.io
- ISSN:
- 2473-1838
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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R fluorophore-modified DNAs and IR fluorescence imaging provide a safe and effective alternative for investigating ligand-DNA interactions than those involving radioactivity. These DNAs can be synthesized by conventional phosphoramidite chemistries and are commercially available. However, they are relatively expensive, which can be prohibitive if many different DNA probes are required. Described here is the design of modular DNA probes that can be synthesized by PCR using conventional templates and a defined set of IR fluorophore-modified primers. These have been found effective for investigating protein-DNA interactions in a variety of assays, including Electrophoretic Mobility Shift Assays (EMSA), Restriction Endonuclease Protection Assays (REPA), and the iterative selection method Restriction Endonuclease Protection, Selection, and Amplification (REPSA).more » « less
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Lipid-anchored DNA can attach functional cargo to bilayer membranes in DNA nanotechnology, synthetic biology, and cell biology research. To optimize DNA anchoring, an understanding of DNA–membrane interactions in terms of binding strength, extent, and structural dynamics is required. Here we use experiments and molecular dynamics (MD) simulations to determine how the membrane binding of cholesterol-modified DNA depends on electrostatic and steric factors involving the lipid headgroup charge, duplexed or single-stranded DNA, and the buffer composition. The experiments distinguish between free and membrane vesicle-bound DNA and thereby reveal the surface density of anchored DNA and its binding affinity, something which had previously not been known. The Kd values range from 8.5 ± 4.9 to 466 ± 134 μM whereby negatively charged headgroups led to weak binding due to the electrostatic repulsion with respect to the negatively charged DNA. Atomistic MD simulations explain the findings and elucidate the dynamic nature of anchored DNA such as the mushroom-like conformation of single-stranded DNA hovering over the bilayer surface in contrast to a straight-up conformation of double-stranded DNA. The biophysical insight into the binding strength to membranes as well as the molecular accessibility of DNA for hybridization to molecular cargo is expected to facilitate the creation of biomimetic DNA versions of natural membrane nanopores and cytoskeletons for research and nanobiotechnology.more » « less
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Frappier, Lori (Ed.)ABSTRACT Two new structures of the N-terminal domain of the main replication protein, NS1, of human parvovirus B19 (B19V) are presented here. This domain (NS1-nuc) plays an important role in the “rolling hairpin” replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e., nicking) single-stranded DNA at a nearby site known as the terminal resolution site (trs). The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain; however, it is shown here at a significantly higher resolution (2.4 Å). Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented that predict residues important for DNA cleavage and for sequence-specific recognition at the viral origin of replication. IMPORTANCE The high-resolution structure of the DNA binding and cleavage domain of the main replicative protein, NS1, from the human-pathogenic virus human parvovirus B19 is presented here. Included also are predictions of how the protein recognizes important sequences in the viral DNA which are required for viral replication. These predictions can be used to further investigate the function of this protein, as well as to predict the effects on viral viability due to mutations in the viral protein and viral DNA sequences. Finally, the high-resolution structure facilitates structure-guided drug design efforts to develop antiviral compounds against this important human pathogen.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.17504/protocols.io.k5pcy5n
