Single-nucleotide polymorphism analysis by pyrosequencing.

There is a growing demand for high-throughput methods for analysis of single-nucleotide polymorphic (SNP) positions. Here, we have evaluated a novel sequencing approach, pyrosequencing, for such purposes. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. One feature of typing SNPs with pyrosequencing is that each allelic variant will give a unique sequence compared to the two other variants. These variants can easily be distinguished by a pattern recognition software. The software displays the allelic alternatives and allows for direct comparison with the pyrosequencing raw data. For optimal determination of SNPs, various protocols of nucleotide dispensing order were investigated. Here, we demonstrate that typing of SNPs can efficiently be performed by pyrosequencing using an automated system for parallel analysis of 96 samples in approximately 5 min, suitable for large-scale screening and typing of SNPs.

Sequence analysis of genes and genomes.

A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.

Recovery of upstream cDNA sequences by a PCR-based biotin-capture method.

The world-wide, large-scale sequencing efforts have generated an abundance of partial cDNA sequences, i.e., expressed sequence tags (ESTs), accessible in the public databases. To enable functional characterization of these partial cDNA sequences, general and robust methods for recovery of upstream full-coding cDNA sequences are needed. Here, a novel biotin- and PCR-assisted capture method was used directly on poly(A)+ RNA for the purpose of generating a full-coding sequence of a gene with only partially known sequence and for which a full-length clone of the gene was not found in existing cDNA libraries. The presented method involves linear extension by reverse transciptase from a biotinylated primer annealing in a region with known sequence. After capture of the generated single-stranded cDNA onto paramagnetic beads, unspecifically annealing primers, i.e., arbitrary primers, were used to generate cDNA fragments that could be amplified by PCR and thereafter directly sequenced without subcloning. By using the presented strategy, which is to be seen as a complement to rapid amplification of cDNA ends (RACE)-related methods, we were able to recover full-coding sequence versions of two potential splice variants of the target gene. The general applicability of the novel method for recovery and sequencing of cDNA sequences is discussed.

Gene discovery in the wood-forming tissues of poplar: analysis of 5, 692 expressed sequence tags.

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as Arabidopsis, Brassica, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars, Populus tremula L. x tremuloides Michx. and Populus trichocarpa 'Trichobel.' The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Chromosomal sequencing using a PCR-based biotin-capture method allowed isolation of the complete gene for the outer membrane protein A of Klebsiella pneumoniae.

By employing a novel biotin- and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA, the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate sequences with only partly determined nt sequences. The homology of the sequenced gene to ompA of related bacteria is discussed, and the gene fragment was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed.

Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR.

Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research. We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes. The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised onto paramagnetic beads. An arbitrary primer initiates extension from the unknown region and back towards the known locus. The arbitrary primer contains a universal primer 'handle', which is utilised for subsequent amplification. The PCR products are then directly sequenced by solid-phase or cycle sequencing. The fact that BACs or bacterial chromosomes can be sequenced without prior purification or subcloning might be useful in numerous applications, such as gap-filling, sequencing of regulatory regions upstream known genes and determination of intron/exon-boundaries.

Predominance of H-2d- and H-2k-restricted T-cell epitopes in the highly repetitive Plasmodium falciparum antigen Pf332.

Genetic restriction of immune responses to malaria antigens is an important issue for a better comprehension of malaria immunity as well as for development of subunit vaccines. To experimentally define the major histocompatibility complex restriction of immune responses to the highly repetitive Plasmodium falciparum high-molecular-weight antigen Pf332, H-2-congenic mice were immunized with EB200, a recombinant fragment of Pf332 consisting of degenerate repeat motifs. Strong B- and T-cell responses were elicited in H-2d and H-2k mice whereas responses in H-2b, H-2q and H-2s mice were of lower magnitude. The T-cell specificity elicited by EB200 was defined by in vitro proliferative responses to a panel of overlapping peptides spanning EB200. Dominant epitopes were identified for H-2d and H-2k mice, respectively, and an additional epitope was recognized by all five mouse strains. Selected EB200-derived peptides were further investigated for their ability to elicit T-cell help when injected as multiple antigen peptides. Defined H-2d- and H-2k-restricted T-cell epitopes generated high antibody levels in the respective mouse strains, as did several peptides lacking defined epitopes indicating the presence of additional H-2d- and H-2k-restricted, cryptic or subdominant T-cell epitopes in EB200. The biased H-2 restriction pattern of T-cell epitopes in Pf332 and, as previously reported, in structurally related repeats in the malaria antigens Pf11.1 and Pf155/RESA may be explained by a shared motif for H-2d and H-2k class II-restricted T-cell epitopes, as revealed by alignment of these sequences.

The sequence of a 30 kb fragment on the left arm of chromosome XV from Saccharomyces cerevisiae reveals 15 open reading frames, five of which correspond to previously identified genes.

We report the sequence of a 30,469 bp long DNA fragment on the left arm of chromosome XV of Saccharomyces cerevisiae. The fragment contains 15 open reading frames (ORFs) of at least 300 bp. Five previously sequenced yeast genes, PHO80, TIR2, SLG1, the gene encoding the subtilisin-like protease III precursor and the gene coding for ATP-dependent permease, are found among these ORFs. By DNA sequence comparison, two ORFs identified previously reported expressed sequence tags from yeast. Of the proteins encoded by the remaining eight ORFs, six show similarities to proteins from different organisms and two lack detectable similarity with any amino acid sequence described in public data banks.