Preparation of Labeled DNA, RNA, and Oligonucleotide Probes

Labeled nucleic acids and oligonucleotides are typically generated by enzymatic methods such as end-labeling, random priming, nick translation, in vitro transcription, and variations of the polymerase chain reaction (PCR). Some of these methods place the label in specific locations within the nucleic acid (e.g., at the 5′ or 3′ terminus); others generate molecules that are labeled internally at multiple sites. Some methods yield labeled single-stranded products, whereas others generate double-stranded nucleic acids. Finally, some generate probes of defined length, whereas others yield a heterogeneous population of labeled molecules. Options available for generating and detecting labeled nucleic acids, as well as advice on designing oligonucleotides for use as probes, is included here.

Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae

During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, produces a nick-translation reaction that replaces the DNA at the 5’ end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous S. cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1 binding sites. However protracted ligase depletion leads to a global change in the location of these termini, moving them towards nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1 binding sites. Additionally, we observe an under-representation of DNA derived from DNA polymerase alpha – the polymerase that initiates Okazaki fragment synthesis – around the sites of Okazaki termini obtained from very brief ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis.

Development of coupling controlled polymerizations by adapter-ligation in mate-pair sequencing for detection of various genomic variants in one single assay

The diversity of disease presentations warrants one single assay for detection and delineation of various genomic disorders. Herein, we describe a gel-free and biotin-capture-free mate-pair method through coupling Controlled Polymerizations by Adapter-Ligation (CP-AL). We first demonstrated the feasibility and ease-of-use in monitoring DNA nick translation and primer extension by limiting the nucleotide input. By coupling these two controlled polymerizations by a reported non-conventional adapter-ligation reaction 3′ branch ligation, we evidenced that CP-AL significantly increased DNA circularization efficiency (by 4-fold) and was applicable for different sequencing methods but at a faction of current cost. Its advantages were further demonstrated by fully elimination of small-insert-contaminated (by 39.3-fold) with a ∼50% increment of physical coverage, and producing uniform genome/exome coverage and the lowest chimeric rate. It achieved single-nucleotide variants detection with sensitivity and specificity up to 97.3 and 99.7%, respectively, compared with data from small-insert libraries. In addition, this method can provide a comprehensive delineation of structural rearrangements, evidenced by a potential diagnosis in a patient with oligo-atheno-terato-spermia. Moreover, it enables accurate mutation identification by integration of genomic variants from different aberration types. Overall, it provides a potential single-integrated solution for detecting various genomic variants, facilitating a genetic diagnosis in human diseases.

Development of Coupling Controlled Polymerizations by Adapter-ligation in Mate-pair Sequencing for Detection of Various Genomic Variants in One Single Assay

The diversity of disease presentations warrants one single assay for detection and delineation of various genomic disorders. Herein, we describe a gel-free and biotin-capture-free mate-pair method through coupling Controlled Polymerizations by Adapter-Ligation (CP-AL). We first demonstrated the feasibility and ease-of-use in monitoring DNA nick-translation and primer extension by limiting the nucleotide input. By coupling these two controlled polymerizations by a reported non-conventional adapter ligation reaction 3’ branch ligation, we evidenced that CP-AL significantly increased DNA-circularization efficiency (by 4-fold) and was applicable for different sequencing methods but at a faction of current cost. Its advantages were further demonstrated by fully elimination of small-insert-contaminated (by 39.3-fold) with a ~50% increment of physical coverage, and producing uniform genome/exome coverage and the lowest chimeric rate. It achieved single-nucleotide variants detection with sensitivity and specificity up to 97.3 and 99.7%, respectively, compared with data from small-insert libraries. In addition, this method can provide a comprehensive delineation of structural rearrangements, evidenced by a potential diagnosis in a patient with oligo-atheno-terato-spermia. Moreover, it enables accurate mutation identification by integration of genomic variants from different aberration types. Overall, it provides a potential single-integrated solution for detecting various genomic variants, facilitating a genetic diagnosis in human diseases.

CRISPR-bind: a simple, custom CRISPR/dCas9-mediated labeling of genomic DNA for mapping in nanochannel arrays

Bionano genome mapping is a robust optical mapping technology used for de novo construction of whole genomes using ultra-long DNA molecules, able to efficiently interrogate genomic structural variation. It is also used for functional analysis such as epigenetic analysis and DNA replication mapping and kinetics. Genomic labeling for genome mapping is currently specified by a single strand nicking restriction enzyme followed by fluorophore incorporation by nick-translation (NLRS), or by a direct label and stain (DLS) chemistry which conjugates a fluorophore directly to an enzyme-defined recognition site. Although these methods are efficient and produce high quality whole genome mapping data, they are limited by the number of available enzymes—and thus the number of recognition sequences—to choose from. The ability to label other sequences can provide higher definition in the data and may be used for countless additional applications. Previously, custom labeling was accomplished via the nick-translation approach using CRISPR-Cas9, leveraging Cas9 mutant D10A which has one of its cleavage sites deactivated, thus effectively converting the CRISPR-Cas9 complex into a nickase with customizable target sequences. Here we have improved upon this approach by using dCas9, a nuclease-deficient double knockout Cas9 with no cutting activity, to directly label DNA with a fluorescent CRISPR-dCas9 complex (CRISPR-bind). Unlike labeling with CRISPR-Cas9 D10A nickase, in which nicking, labeling, and repair by ligation, all occur as separate steps, the new assay has the advantage of labeling DNA in one step, since the CRISPR-dCas9 complex itself is fluorescent and remains bound during imaging. CRISPR-bind can be added directly to a sample that has already been labeled using DLS or NLRS, thus overlaying additional information onto the same molecules. Using the dCas9 protein assembled with custom target crRNA and fluorescently labeled tracrRNA, we demonstrate rapid labeling of repetitive DUF1220 elements. We also combine NLRS-based whole genome mapping with CRISPR-bind labeling targeting Alu loci. This rapid, convenient, non-damaging, and cost-effective technology is a valuable tool for custom labeling of any CRISPR-Cas9 amenable target sequence.

Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

The nick translation property of DNA polymerase I (Pol I) ensures the maturation of Okazaki fragments by removing primer RNAs and facilitating ligation. However, prolonged nick translation traversing downstream DNA is an energy wasting futile process, as Pol I simultaneously polymerizes and depolymerizes at the nick sites utilizing energy-rich dNTPs. Using an in vitro assay system, we demonstrate that the β-clamp of the Escherichia coli replisome strongly inhibits nick translation on the DNA substrate. To do so, β-clamp inhibits the strand displacement activity of Pol I by interfering with the interaction between the finger subdomain of Pol I and the downstream primer-template junction. Conversely, β-clamp stimulates the 5’ exonuclease property of Pol I to cleave single nucleotides or shorter oligonucleotide flaps. This single nucleotide flap removal at high frequency increases the probability of ligation between the upstream and downstream DNA strands at an early phase, terminating nick translation. Besides β-clamp-mediated ligation helps DNA ligase to seal the nick promptly during the maturation of Okazaki fragments.

Intra- and intergenomic chromosomal pairing in artificially polyploidized elephant grass and pearl millet hybrids

The objective of this work was to evaluate, by genomic in situ hybridization (GISH), pairing configurations as potential indicators of recombination between chromosomes of different parental genomes, in two interspecific hybrids (elephant grass x pearl millet) artificially polyploidized. Anthers from young flower buds were used in the chromosomal preparations. The genomic probe was prepared with pearl millet DNA and labeled with biotin-16-dUTP by the nick translation reaction. Blocking DNA was prepared with genomic elephant grass DNA. The homoeologous intergenomic pairing, observed in the two hybrids, indicates the possibility of recombination between chromosomes of the parental genomes.