Publications by authors named "Shundi Shi"

16 Publications

  • Page 1 of 1

A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing both Canonical and Modified Nucleotides.

Methods Mol Biol 2021 ;2298:261-277

Department of Biological and Chemical Sciences, New York Institute of Technology, New York, NY, USA.

Mass spectrometry (MS)-based sequencing has advantages in direct sequencing of RNA, compared to cDNA-based RNA sequencing methods, as it is completely independent of enzymes and base complementarity errors in sample preparation. In addition, it allows for sequencing of different RNA modifications in a single study, rather than just one specific modification type per study. However, many technical challenges remain in de novo MS sequencing of RNA, making it difficult to MS sequence mixed RNAs or to differentiate isomeric modifications such as pseudouridine (Ψ) from uridine (U). Our recent study incorporates a two-dimensional hydrophobic end labeling strategy into MS-based sequencing (2D-HELS MS Seq) to systematically address the current challenges in MS sequencing of RNA, making it possible to directly and de novo sequence purified single RNA and mixed RNA containing both canonical and modified nucleotides. Here, we describe the method to sequence representative single-RNA and mixed-RNA oligonucleotides, each with a different sequence and/or containing modified nucleotides such as Ψ and 5-methylcytosine (mC), using 2D-HELS MS Seq.
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http://dx.doi.org/10.1007/978-1-0716-1374-0_17DOI Listing
June 2021

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications.

J Vis Exp 2020 07 10(161). Epub 2020 Jul 10.

Department of Biological and Chemical Sciences, New York Institute of Technology;

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in direct sequencing RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides (pseudouridine (Ψ) and 5-methylcytosine (mC)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (<35 nt) when using a standard high-resolution LC-MS system, and can also be used for sequence verification of modified therapeutic RNA oligonucleotides. In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.
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http://dx.doi.org/10.3791/61281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398574PMC
July 2020

Direct Sequencing of tRNA by 2D-HELS-AA MS Seq Reveals Its Different Isoforms and Dynamic Base Modifications.

ACS Chem Biol 2020 06 19;15(6):1464-1472. Epub 2020 May 19.

Department of Biological and Chemical Sciences, New York Institute of Technology, New York, New York 10023, United States.

Post-transcriptional modifications are intrinsic to RNA structure and function. However, methods to sequence RNA typically require a cDNA intermediate and are either not able to sequence these modifications or are tailored to sequence one specific nucleotide modification only. Interestingly, some of these modifications occur with <100% frequency at their particular sites, and site-specific quantification of their stoichiometries is another challenge. Here, we report a direct method for sequencing tRNA without cDNA by integrating a two-dimensional hydrophobic RNA end-labeling strategy with an anchor-based algorithm in mass spectrometry-based sequencing (2D-HELS-AA MS Seq). The entire tRNA was sequenced and the identity, location, and stoichiometry of all eleven different RNA modifications was determined, five of which were not 100% modified, including a 2'-O-methylated G (Gm) in the wobble anticodon position as well as an -dimethylguanosine (mG), a 7-methylguanosine (mG), a 1-methyladenosine (mA), and a wybutosine (Y), suggesting numerous post-transcriptional regulations in tRNA. Two truncated isoforms at the 3'-CCA tail of the tRNA (75 nt with a 3'-CC tail (80% abundance) and 74 nt with a 3'-C tail (3% abundance)) were identified in addition to the full-length 3'-CCA-tailed tRNA (76 nt, 17% abundance). We discovered a new isoform with A-G transitions/editing at the 44 and 45 positions in the tRNA variable loop, and discuss possible mechanisms related to the emergence and functions of the isoforms with these base transitions or editing. Our method revealed new isoforms, base modifications, and RNA editing as well as their stoichiometries in the tRNA that cannot be determined by current cDNA-based methods, opening new opportunities in the field of epitranscriptomics.
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http://dx.doi.org/10.1021/acschembio.0c00119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902080PMC
June 2020

A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures.

Nucleic Acids Res 2019 11;47(20):e125

Department of Biological and Chemical Sciences, New York Institute of Technology, New York, NY 10023, USA.

A complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and non-canonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these non-standard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via introduction of a 2-dimensional hydrophobic end-labeling strategy into traditional mass spectrometry-based sequencing (2D HELS MS Seq) to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA versus its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site in the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification type and can also be used in the quality control of modified therapeutic RNAs.
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http://dx.doi.org/10.1093/nar/gkz731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6847078PMC
November 2019

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array.

Proc Natl Acad Sci U S A 2016 11 11;113(44):E6749-E6756. Epub 2016 Oct 11.

Department of Genetics, Harvard Medical School, Boston, MA 02115;

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin-polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.
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http://dx.doi.org/10.1073/pnas.1608271113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098637PMC
November 2016

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

Proc Natl Acad Sci U S A 2016 May 18;113(19):5233-8. Epub 2016 Apr 18.

Genia Technologies, Santa Clara, CA 95050;

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.
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http://dx.doi.org/10.1073/pnas.1601782113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868432PMC
May 2016

DNA sequencing by synthesis using 3'-azidomethyl nucleotide reversible terminators and surface-enhanced Raman spectroscopic detection.

RSC Adv 2014 Jan;4(90):49342-49346

Center for Genome Technology and Biomolecular Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Chemical Engineering, Columbia University, 3000 Broadway, New York, NY 10027, USA ; Department of Pharmacology, Columbia University, 3000 Broadway, New York, NY 10027, USA.

As an alternative to fluorescence-based DNA sequencing by synthesis (SBS), we report here an approach using an azido moiety (N) that has an intense, narrow and unique Raman shift at 2125 cm, where virtually all biological molecules are transparent, as a label for SBS. We first demonstrated that the four 3'-azidomethyl nucleotide reversible terminators (3'-azidomethyl-dNTPs) displayed surface enhanced Raman scattering (SERS) at 2125 cm. Using these 4 nucleotide analogues as substrates, we then performed a complete 4-step SBS reaction. We used SERS to monitor the appearance of the azide-specific Raman peak at 2125 cm as a result of polymerase extension by a single 3'-azidomethyl-dNTP into the growing DNA strand and disappearance of this Raman peak with cleavage of the azido label to permit the next nucleotide incorporation, thereby continuously determining the DNA sequence. Due to the small size of the azido label, the 3'-azidomethyl-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for the azido moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS.
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http://dx.doi.org/10.1039/C4RA08398ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226440PMC
January 2014

Mitochondrial single nucleotide polymorphism genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using cleavable biotinylated dideoxynucleotides.

Anal Biochem 2012 Aug 10;427(2):202-10. Epub 2012 May 10.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC-SBE approach based on chemically cleavable biotinylated dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.
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http://dx.doi.org/10.1016/j.ab.2012.05.001DOI Listing
August 2012

Design and synthesis of cleavable biotinylated dideoxynucleotides for DNA sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Anal Biochem 2012 Aug 25;427(2):193-201. Epub 2012 Apr 25.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based methods have been widely explored for DNA sequencing. We report here the design, synthesis, and evaluation of a novel set of chemically cleavable biotinylated dideoxynucleotides, ddNTPs-N₃-biotin, for the DNA polymerase extension reaction and its application in DNA sequencing by mass spectrometry (MS). These nucleotide analogs have a biotin moiety attached to the 5 position of the pyrimidines (C and U) or the 7 position of the purines (A and G) via a chemically cleavable azido-based linker, with different length linker arms serving as mass tags that contribute to large mass differences among the nucleotides. We demonstrate that these modified nucleotides are efficiently incorporated by DNA polymerase, and the DNA strand bearing biotinylated nucleotides is captured by streptavidin-coated beads and efficiently released using tris(2-carboxyethyl)phosphine in aqueous solution, which is compatible with DNA and downstream procedures. We performed Sanger sequencing reactions using these nucleotides to generate DNA fragments for MALDI-TOF MS analysis. Both synthetic DNA and polymerase chain reaction (PCR) products were accurately decoded, and a read length of approximately 37 bases was achieved using these nucleotides in MS sequencing.
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http://dx.doi.org/10.1016/j.ab.2012.04.021DOI Listing
August 2012

Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides.

Proc Natl Acad Sci U S A 2008 Jul 30;105(27):9145-50. Epub 2008 Jun 30.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3'-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3'-OH capping group from the DNA products generated by incorporating the 3'-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3'-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3'-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3'-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.
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http://dx.doi.org/10.1073/pnas.0804023105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442126PMC
July 2008

3'-O-modified nucleotides as reversible terminators for pyrosequencing.

Proc Natl Acad Sci U S A 2007 Oct 8;104(42):16462-7. Epub 2007 Oct 8.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

Pyrosequencing is a method used to sequence DNA by detecting the pyrophosphate (PPi) group that is generated when a nucleotide is incorporated into the growing DNA strand in polymerase reaction. However, this method has an inherent difficulty in accurately deciphering the homopolymeric regions of the DNA templates. We report here the development of a method to solve this problem by using nucleotide reversible terminators. These nucleotide analogues are modified with a reversible chemical moiety capping the 3'-OH group to temporarily terminate the polymerase reaction. In this way, only one nucleotide is incorporated into the growing DNA strand even in homopolymeric regions. After detection of the PPi for sequence determination, the 3'-OH of the primer extension products is regenerated through different deprotection methods. Using an allyl or a 2-nitrobenzyl group as the reversible moiety to cap the 3'-OH of the four nucleotides, we have synthesized two sets of 3'-O-modified nucleotides, 3'-O-allyl-dNTPs and 3'-O-(2-nitrobenzyl)-dNTPs as reversible terminators for pyrosequencing. The capping moiety on the 3'-OH of the DNA extension product is efficiently removed after PPi detection by either a chemical method or photolysis. To sequence DNA, templates containing homopolymeric regions are immobilized on Sepharose beads, and then extension-signal detection-deprotection cycles are conducted by using the nucleotide reversible terminators on the DNA beads to unambiguously decipher the sequence of DNA templates. Our results establish that this reversible-terminator-pyrosequencing approach can be potentially developed into a powerful methodology to accurately determine DNA sequences.
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http://dx.doi.org/10.1073/pnas.0707495104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2034218PMC
October 2007

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators.

Proc Natl Acad Sci U S A 2006 Dec 14;103(52):19635-40. Epub 2006 Dec 14.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.
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http://dx.doi.org/10.1073/pnas.0609513103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1702316PMC
December 2006

Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides.

Proc Natl Acad Sci U S A 2005 Apr 13;102(17):5926-31. Epub 2005 Apr 13.

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

We report four-color DNA sequencing by synthesis (SBS) on a chip, using four photocleavable fluorescent nucleotide analogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, and dCTP-PC-Bodipy-650) (PC, photocleavable; Bodipy, 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene; ROX, 6-carboxy-X-rhodamine; R6G, 6-carboxyrhodamine-6G). Each nucleotide analogue consists of a different fluorophore attached to the 5 position of the pyrimidines and the 7 position of the purines through a photocleavable 2-nitrobenzyl linker. After verifying that these nucleotides could be successfully incorporated into a growing DNA strand in a solution-phase polymerase reaction and the fluorophore could be cleaved using laser irradiation ( approximately 355 nm) in 10 sec, we then performed an SBS reaction on a chip that contains a self-priming DNA template covalently immobilized by using 1,3-dipolar azide-alkyne cycloaddition. The DNA template was produced by PCR, using an azido-labeled primer, and the self-priming moiety was attached to the immobilized DNA template by enzymatic ligation. Each cycle of SBS consists of the incorporation of the photocleavable fluorescent nucleotide into the DNA, detection of the fluorescent signal, and photocleavage of the fluorophore. The entire process was repeated to identify 12 continuous bases in the DNA template. These results demonstrate that photocleavable fluorescent nucleotide analogues can be incorporated accurately into a growing DNA strand during a polymerase reaction in solution and on a chip. Moreover, all four fluorophores can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing continuous identification of the DNA template sequence. Optimization of the steps involved in this SBS approach will further increase the read-length.
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http://dx.doi.org/10.1073/pnas.0501965102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087949PMC
April 2005

The genomic sequence of the accidental pathogen Legionella pneumophila.

Science 2004 Sep;305(5692):1966-8

Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water. The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants. We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets.
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http://dx.doi.org/10.1126/science.1099776DOI Listing
September 2004

Comparative sequence analysis of the icm/dot genes in Legionella.

Plasmid 2004 Mar;51(2):127-47

Columbia Genome Center, Columbia University College of Physicians and Surgeons, 1150 St. Nicholas Avenue, New York, NY 10032, USA.

The icm/dot genes in Legionella pneumophila are essential for the ability of the bacteria to survive within macrophages in lung infections such as Legionnaires' disease, or amoebae in nature. The 22 genes of the complex, thought to encode a transport apparatus for transfer of effector molecules into the host cell cytoplasm, are located in two chromosomal loci. We demonstrate that these genes are present in all the L. pneumophila strains examined herein, but display a wide range of sequence variation among the different strains, none of which are clearly associated with virulence potential. The strains fall within seven phylogenetic groups, but discrepancies among the gene trees indicate a complicated evolutionary history for the icm/dot loci, with perhaps two independent gene acquisition events and subsequent genomic rearrangements. Significant findings include a probable t-SNARE domain in IcmG that may indicate a direct role for this putative inner membrane protein in altering the host's membrane fusion machinery, a potential functional domain in the central hydrophobic portion of IcmK that may allow it to participate in forming the pore of the secretion complex, and strict conservation of the amino acid physicochemical characteristics in the IcmP region corresponding to the trbA domain that could play a role in molecular transfer.
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http://dx.doi.org/10.1016/j.plasmid.2003.12.004DOI Listing
March 2004

Multiplex genotyping of the human beta2-adrenergic receptor gene using solid-phase capturable dideoxynucleotides and mass spectrometry.

Anal Biochem 2003 May;316(2):251-8

Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

Previously, we established the feasibility of using solid phase capturable (SPC) dideoxynucleotides to generate single base extension (SBE) products which were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for multiplex genotyping, an approach that we refer to as SPC-SBE. We report here the expanding of the SPC-SBE method as a single-tube assay to simultaneously detect 20 single nucleotide variations in a model system and 3 single nucleotide polymorphisms (SNPs) in the human beta2-adrenergic receptor (beta2AR) gene. Twenty primers were designed to have a sufficient mass difference between all extension products for accurate detection of nucleotide variants of the synthetic templates related to the p53 gene. These primers were extended simultaneously in a single tube with biotin-ddNTPs to generate 3(')-biotinylated DNA products, which were first captured by streptavidin-coated magnetic beads and then released from the beads and analyzed with MALDI-TOF MS. This approach generates a mass spectrum free of primer peaks and their associated dimers, increasing the scope of multiplexing SNPs. We also simultaneously genotyped 3 SNPs in the beta2AR gene (5(')LC-Cys19Arg, Gly16Arg, and Gln27Glu) from the genomic DNA of 20 individuals. Comparison of this approach with direct sequencing and the restriction fragment length polymorphism method indicated that the SPC-SBE method is superior for detecting nucleotide variations at known SNP sites.
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http://dx.doi.org/10.1016/s0003-2697(03)00080-0DOI Listing
May 2003
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