Primer designing in Primer-BLAST

The polymerase chain reaction (PCR) is widely used in different areas. For example, in laboratory diagnostics, PCR is used to detect bacterial and viral pathogens, in the diagnosis of hereditary diseases, and to identify paternity and many other. There are three types of PCR — qualitative, semi-quantitative, and quantitative. The method is based on the ability of DNA polymerase to double the existing DNA strand, thus resulting in multiplication the number of copies of the region of interest. Necessary components of the reaction are DNA or RNA molecules, serving as a template for the of new molecules; polymerase — an enzyme synthesizes new DNA strands; short oligonucleotides – primers that are required for the enzyme work and are specific to the fragment of interest. Currently, there are not many primer designing tools that are easy-to-use and free. One of these tools is the Primer-BLAST online resource, which is integrated with the NCBI database. It is user-friendly and easyto-use effective tool that is integrated with GenBank and RefSeq, and always is up-to-dated. However, even for this tool, there are no detailed instructions for the primers designing. This work is an update for a previously published tutorial and provides a step-by-step guide to find target DNA regions, primers designing, and performing primer quality control. The paper is largely based on the personal experience of the authors and is intended for researchers working with PCR.

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Mechanistic Insights into theCis-andTrans-acting Deoxyribonuclease Activities of Cas12a

10.1101/353748 ◽

2018 ◽

Cited By ~ 2

Author(s):

Daan C. Swarts ◽

Martin Jinek

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Cleavage Product ◽

List Type ◽

Francisella Novicida ◽

Ssdna Binding ◽

Double Stranded Dna ◽

Target Dna ◽

Dna Strands ◽

Dna Strand

HIGHLIGHTSTarget ssDNA binding allosterically induces unblocking of the RuvC active sitePAM binding facilitates unwinding of dsDNA targetsNon-target DNA strand cleavage is prerequisite for target DNA strand cleavageAfter DNA cleavage, Cas12a releases the PAM-distal DNA productSUMMARYCRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA incisand non-target single stranded DNAs (ssDNA) intrans.To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies onFrancisella novicidaCas12a. We show how crRNA-target DNA strand hybridization conformationally activates Cas12a, triggering itstrans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn,cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent,trans-active state. Together, these results provide a revised model for the molecular mechanism of Cas12a enzymes that explains theircis- andtrans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.

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Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1700721114 ◽

2017 ◽

Vol 114 (22) ◽

pp. E4492-E4500 ◽

Cited By ~ 23

Author(s):

Pan F. Chan ◽

Thomas Germe ◽

Benjamin D. Bax ◽

Jianzhong Huang ◽

Reema K. Thalji ◽

...

Keyword(s):

Dna Cleavage ◽

Dna Gyrase ◽

Dna Breaks ◽

Antibiotic Development ◽

X Ray Crystallography ◽

Dna Mutations ◽

Target Dna ◽

Antibiotic Drug ◽

Dna Strands ◽

Dna Strand

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase–DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

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3′ → 5′ Exonucleases of DNA Polymerases ϵ and δ Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.3.717 ◽

1996 ◽

Vol 142 (3) ◽

pp. 717-726 ◽

Cited By ~ 11

Author(s):

Polina V Shcherbakova ◽

Youri I Pavlov

Keyword(s):

Dna Replication ◽

Dna Polymerases ◽

Replication Errors ◽

Dna Strands ◽

Base Analog ◽

Chromosome Iii ◽

Dna Strand ◽

Dna Replication Errors ◽

Yeast Mutants ◽

Lagging Strand

Abstract The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC → AT and AT → GC transitions that are enhanced in DNA polymerase ϵ and δ 3′ → 5′ exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ϵ contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC → AT and AT → GC transitions in a Pol→, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ϵ and δ correct HAP-induced DNA replication errors on opposite DNA strands.

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Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

Nature Communications ◽

10.1038/s41467-021-23876-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bo Zhang ◽

Diyin Luo ◽

Yu Li ◽

Vanja Perčulija ◽

Jing Chen ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Binary Complex ◽

Loop Formation ◽

Dna Duplex ◽

Target Dna ◽

Before And After ◽

Dna Strand ◽

Type V ◽

Functionally Diverse

AbstractCas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.

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Staring at the Naked Goddess: Unraveling the Structure and Reactivity of Artemis Endonuclease Interacting with a DNA Double Strand

Molecules ◽

10.3390/molecules26133986 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3986

Author(s):

Cécilia Hognon ◽

Antonio Monari

Keyword(s):

Dna Cleavage ◽

Catalytic Mechanism ◽

Dna Lesions ◽

Cleavage Reaction ◽

Dynamic Simulations ◽

Important Target ◽

System Adaptation ◽

Dna Strands ◽

Dna Strand ◽

Dna Substrate

Artemis is an endonuclease responsible for breaking hairpin DNA strands during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance, its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, and the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulations, both classically and at the hybrid quantum mechanics/molecular mechanics level, we evidenced the stable interaction modes of Artemis with a model DNA strand. We also analyzed the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.

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Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1) to nick the target DNA strand

Science China Life Sciences ◽

10.1007/s11427-020-1722-0 ◽

2020 ◽

Vol 63 (11) ◽

pp. 1619-1630 ◽

Cited By ~ 2

Author(s):

Rong Fan ◽

Zhuangzhuang Chai ◽

Sinian Xing ◽

Kunling Chen ◽

Fengti Qiu ◽

...

Keyword(s):

Target Dna ◽

Dna Strand

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BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors

Nature Communications ◽

10.1038/s41467-021-26461-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

András Tálas ◽

Dorottya A. Simon ◽

Péter I. Kulcsár ◽

Éva Varga ◽

Sarah L. Krausz ◽

...

Keyword(s):

High Throughput ◽

Genome Engineering ◽

Sequence Context ◽

Base Editing ◽

Target Dna ◽

Dna Strands ◽

Deaminase Domain ◽

Target Effects ◽

Target Sets

AbstractAdenine and cytosine base editors (ABE, CBE) allow for precision genome engineering. Here, Base Editor Activity Reporter (BEAR), a plasmid-based fluorescent tool is introduced, which can be applied to report on ABE and CBE editing in a virtually unrestricted sequence context or to label base edited cells for enrichment. Using BEAR-enrichment, we increase the yield of base editing performed by nuclease inactive base editors to the level of the nickase versions while maintaining significantly lower indel background. Furthermore, by exploiting the semi-high-throughput potential of BEAR, we examine whether increased fidelity SpCas9 variants can be used to decrease SpCas9-dependent off-target effects of ABE and CBE. Comparing them on the same target sets reveals that CBE remains active on sequences, where increased fidelity mutations and/or mismatches decrease the activity of ABE. Our results suggest that the deaminase domain of ABE is less effective to act on rather transiently separated target DNA strands, than that of CBE explaining its lower mismatch tolerance.

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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

10.1101/500298 ◽

2018 ◽

Cited By ~ 1

Author(s):

Shama Sograte-Idrissi ◽

Nazar Oleksiievets ◽

Sebastian Isbaner ◽

Mariana Eggert-Martinez ◽

Jörg Enderlein ◽

...

Keyword(s):

Fluorescent Proteins ◽

Super Resolution ◽

Resolving Power ◽

Acquisition Time ◽

Efficient Manner ◽

Target Dna ◽

Multiplexed Imaging ◽

Dna Strands ◽

Linkage Error ◽

20 Nm

AbstractDNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.

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Cockayne Syndrome B protein selectively interacts and resolves intermolecular DNA G-quadruplex structures

10.1101/2021.03.25.436565 ◽

2021 ◽

Author(s):

Denise Liano ◽

Marco Di Antonio

Keyword(s):

Ribosomal Dna ◽

Living Cells ◽

Cockayne Syndrome ◽

Biological Functions ◽

Loss Of Function ◽

Endogenous Protein ◽

G Quadruplex ◽

Dna Strands ◽

Low Probability ◽

Dna Strand

AbstractGuanine-rich DNA can fold into secondary structures known as G-quadruplexes (G4s). G4s can form from a single DNA-strand (intramolecular) or from multiple DNA-strands (intermolecular), but studies on their biological functions have been often limited to intramolecular G4s, owing to the low probability of intermolecular G4s to form within genomic DNA. Herein, we report that the endogenous protein Cockayne Syndrome B (CSB) binds with picomolar affinity to intermolecular G4s, whilst displaying negligible binding towards intramolecular structures. We also observed that CSB can selectively resolve intermolecular G4s in an ATP independent fashion. Our study demonstrates that intermolecular G4s formed within ribosomal DNA are natural substrates for CSB, strongly suggesting that these structures might be formed in the nucleolus of living cells. Given that CSB loss of function elicits premature ageing phenotypes, our findings indicate that the interaction between CSB and ribosomal DNA intermolecular G4s is essential to maintain cellular homeostasis.

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Mechanism of R-loop formation and conformational activation of Cas9

10.1101/2021.09.16.460614 ◽

2021 ◽

Author(s):

Martin Pacesa ◽

Martin Jinek

Keyword(s):

Dna Cleavage ◽

Structural Basis ◽

Seed Region ◽

Loop Formation ◽

Base Pairs ◽

Guide Rna ◽

Target Strand ◽

Target Dna ◽

Dna Strand ◽

Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

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