Ligation-anchored PCR Research Articles

Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood.

BackgroundAs one of the genetic mechanisms for adaptive immunity, V(D)J recombination generates an enormous repertoire of T-cell receptors (TCRs). With the development of high-throughput sequencing techniques, systematic exploration of V(D)J recombination becomes possible. Multiplex PCR has been previously developed to assay immune repertoire; however, the use of primer pools leads to inherent biases in target amplification. In our study, we developed a “single-primer" ligation-anchored PCR method that may amplify the repertoire with much less biases.ResultsBy utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified TCR-beta (TRB) variable regions from total RNA extracted from blood. Next-generation sequencing libraries were then prepared for Illumina HiSeq 2500 sequencer, which generates 151-bp read length to cover the entire V(D)J recombination region. We evaluated this approach on blood samples from healthy donors and from patients with malignant and benign meningiomas. Mapping of sequencing data showed that 64% to 96% of mapped TCRV-containing reads belong to TRB subtype. An increased usage of specific V segments and V-J pairing were observed in malignant meningiomas samples. The CDR3 sequences of the expanded V-J pairs were distinct in each malignant individual, even for pairing of TRBV7-3 with TRBJ2-2 that showed increased usage in both cases.ConclusionsWe demonstrated the technical feasibility and effectiveness of ligation-anchored PCR approach in capturing the TCR-beta landscapes. Further development of this technology may enable a comprehensive delineation of immune repertoire, including other forms of TCRs as well as immunoglobulins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0153-9) contains supplementary material, which is available to authorized users.

Deoxyuridine Triphosphate Incorporation during Somatic Hypermutation of Mouse VkOx Genes after Immunization with Phenyloxazolone

Somatic hypermutation (SHM) of Ig genes is the result of a two-phase process initiated by activation-induced cytidine deaminase, relying on two different strategies for the introduction of mutations at CG pairs (phase I) and at AT pairs (phase II). To explain the selectivity of phase II, two mechanisms were proposed: AT-selective error-prone DNA-polymerases, deoxyuridine triphosphate (dUTP) incorporation, or both. However, there has been no experimental evidence so far of the possible involvement of the latter. We have developed a ligation-anchored PCR method based on the formation of single-strand breaks at uracils. In this study, we show the presence of uracil in hypermutating VkOx genes in wild type (AID(+/+)) mice, demonstrating that dUTP incorporation via DNA polymerases could be a major mechanism in SHM. Thus, error-prone DNA polymerases would participate in SHM via low-fidelity replication and incorporation of dUTP.

First infectious clone of the propagatively transmitted Oat blue dwarf virus

Oat blue dwarf virus (OBDV) is a small, phloem-limited marafivirus that replicates in its leafhopper vector. We have developed complete cDNA clones of OBDV from which infectious transcripts may be derived--the first such clones for any propagatively transmitted plant virus. Prior to clone construction, the reported sequences of the 5' and 3' ends were confirmed using 5' RACE, primer extension, and ligation-anchored PCR. Using vascular puncture of maize seeds with capped transcripts, multiple clones were shown to be infectious at an average rate of 24.3% (range 14-36%). Aster leafhoppers successfully transmitted OBDV to oats and barley after feeding on detached, infected maize leaves. Proteins and RNAs consistent in size with those expected in OBDV infection were detected in young leaves via western and northern blotting, respectively. One construct, pOBDV-2r, was designated as the reference clone. An infectious clone of OBDV will be valuable in examining the interaction of this virus with both its insect and plant hosts.

Nitrogen regulates chitinase gene expression in a marine bacterium

Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by beta-galactosidase assay of S91CX cultures containing different ammonium concentrations (NH4+; 0, 9.5 or 191 mM) and with different nitrogen sources (N-acetylglucosamine (GlcNAc) or glutamate (glt)). S91 chiA expression was found to depend on both the NH4+ concentration and source of nitrogen in marine minimal medium (MMM). Pseudoalteromonas sp. S91 and S91CX can use either GlcNAc or glt as a sole source of carbon in MMM containing a standard concentration of 9.5 mM NH4+. Adding excess NH4+, 20 times the standard concentration, to MMM significantly reduced chiA activity below that found in the presence of either GlcNAc or glt. When no NH4+ was added to MMM, S91CX was also able to use either GlcNAc or glt as a source of nitrogen; under these conditions chiA activity was significantly increased. Under all conditions tested, GlcNAc induced chiA activity significantly more than glt. Regulation of bacterial chitinases by nitrogen has not been previously reported. Transcriptional start point analysis of S91 chiA, using 5'RACE (ligation-anchored PCR), showed that during growth in MMM supplemented with (1) maltose (solely a carbon source for S91), chiA transcription occurred from only one putative sigma(70)-dependent promoter; (2) the chitin monomer GlcNAc, transcription initiated from two putative sigma(54)-dependent promoters and (3) glt, transcription initiated from all three putative promoters.

Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% α-helical, 26% β-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher β-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes.

E6AP was originally identified as the ubiquitin-protein ligase involved in human papillomavirus (HPV) E6-mediated p53 degradation and has since been shown to act as an E3 ubiquitin-protein ligase in the ubiquitination of several other protein substrates. To further define E6AP function at the molecular and cellular levels, a ribozyme-based gene inactivation approach was adopted. A library of hammerhead ribozymes, with randomized arm sequences, was used to screen active molecules along the entire E6AP transcript for ribozyme-cleavable sites. Ligation-anchored PCR was adapted to detect cleavage products, and ribozymes designed to the selected sites were characterized both in vitro and in vivo. Ribozyme-mediated reduction in E6AP expression was found to enhance the apoptotic response of HeLa cells to mitomycin C-induced DNA damage. These findings suggest that E6AP has potential as a drug target, as its suppression can potentiate apoptosis in HPV-positive cells treated with a cytotoxic drug.

Isolation and characterization of a class II alpha-mannosidase cDNA from lepidopteran insect cells.

Lepidopteran insect cells are used routinely as hosts for foreign glycoprotein expression by recombinant baculoviruses, but the precise nature of their N-glycosylation pathway remains poorly defined. These cells clearly have processing glucosidases and mannosidases that can convert precursors to Man3GlcNAc2 structures and fucosyltransferases that can add fucose to the oligosaccharide core. However, their ability to extend these structures to produce complex side chains like those found in mammalian cells remains to be determined. To begin to examine this pathway at the molecular genetic level, we isolated and characterized a class II alpha-mannosidase (alpha-mannosidase II) cDNA from Sf9, a lepidopteran insect cell line. In mammalian cells, this enzyme catalyzes the committed step in the pathway converting N-linked carbohydrates to complex forms. Degenerate primers against conserved regions in known class II alpha-mannosidase protein sequences were used to generate an alpha-mannosidase II-specific PCR product from Sf9 cell DNA. Sequence information from this product was used to isolate a partial cDNA clone, the 5' end was isolated by ligation-anchored PCR, and the full length alpha-mannosidase II cDNA was assembled. This cDNA contained a long open reading frame predicted to encode an 1130 amino acid protein with 37% identity to human Golgi alpha-mannosidase II and with a type II membrane topology, a feature of all known Golgi processing enzymes. Southern blotting indicated that alpha-mannosidase II is a single copy gene in Sf9 cells. Other Lepidoptera had related alpha-mannosidase II genes, but there was variation among different genera, and the Sf9 alpha-mannosidase II cDNA did not cross-hybridize with DNA from animals outside Lepidoptera. Steady-state levels of alpha-mannosidase II RNA were low in uninfected Sf9 cells and even lower after baculovirus infection. The in vitro-translated Sf9 alpha-mannosidase II protein had the expected size and was translocated and N-glycosylated by microsomal membranes. Expression of the Sf9 alpha-mannosidase II cDNA in the baculovirus system produced large amounts of a protein with the expected size and swainsonine-sensitive alpha-mannosidase II activity towards an aryl-alpha-mannoside substrate. These results demonstrate that Sf9 cells encode and express an alpha-mannosidase II with properties similar to those of the mammalian enzyme.

Improved ligation-anchored PCR strategy for identification of 5' ends of transcripts.

Improved ligation-anchored PCR strategy for identification of 5' ends of transcripts.

A method for accurate determination of terminal sequences of viral genomic RNA.

A combination of ligation-anchored PCR and anchored cDNA cloning techniques were used to clone the termini of the saguaro cactus virus (SCV) RNA genome. The terminal sequences of the viral genome were subsequently determined from the clones. The 5' terminus was cloned by ligation-anchored PCR, whereas the 3' terminus was obtained by a technique we term anchored cDNA cloning. In anchored cDNA cloning, an anchor oligonucleotide was prepared by phosphorylation at the 5' end, followed by addition of a dideoxynucleotide at the 3' end to block the free hydroxyl group. The 5' end of the anchor was subsequently ligated to the 3' end of SCV RNA. The anchor-ligated, chimerical viral RNA was then reverse-transcribed into cDNA using a primer complementary to the anchor. The cDNA containing the complete 3'-terminal sequence was converted into ds-cDNA, cloned, and sequenced. Two restriction sites, one within the viral sequence and one within the primer sequence, were used to facilitate cloning. The combination of these techniques proved to be an easy and accurate way to determine the terminal sequences of SCV RNA genome and should be applicable to any other RNA molecules with unknown terminal sequences.

Ligation-anchored PCR: A simple amplification technique with single-sided specificity

Ligation-anchored PCR: A simple amplification technique with single-sided specificity

Ligation-anchored PCR: a simple amplification technique with single-sided specificity.

A simple, efficient, and sensitive technique has been developed for amplification of cDNAs encoding molecules with 5' regions of unknown sequence. In this ligation-anchored PCR, T4 RNA ligase is used to covalently link an "anchor" oligonucleotide to first-strand cDNAs. These anchored cDNAs are then amplified by using one PCR primer specific for the anchor and another specific for a sequence within the molecule of interest. The anchor oligonucleotide has been especially designed to facilitate subsequent analysis and cloning of the resultant PCR products. This three-stage procedure does not require purification of product between steps and avoids many of the technical difficulties associated with established anchored PCR protocols. The efficacy of ligation-anchored PCR was demonstrated by amplification of a specific IgG1 cDNA; total RNA equivalent to as few as 100 cells yielded the expected PCR product.