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Parallel target selection by trinucleotide threading
KTH, School of Biotechnology (BIO), Gene Technology. (Gene technology)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , 91 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:19
Keyword [en]
trinucleotide threading, multiplex amplification, expression profiling, microarray, generic tag, short tandem repeat, microsatellite, electrophoresis, single nucleotide polymorphism, allelotyping, 454, Pyrosequencing
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11284ISBN: 978-91-7415-431-3 (print)OAI: oai:DiVA.org:kth-11284DiVA: diva2:272132
Public defence
2009-11-06, FR4 (Oscar Kleins Auditorium), AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:30 (Swedish)
Opponent
Supervisors
Note
QC 20100819Available from: 2009-10-15 Created: 2009-10-13 Last updated: 2011-11-23Bibliographically approved
List of papers
1. Expression profiling of signature gene sets with trinucleotide threading
Open this publication in new window or tab >>Expression profiling of signature gene sets with trinucleotide threading
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2008 (English)In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 9, no 2, 209-217 p.Article in journal (Refereed) Published
Abstract [en]

In recent years, studies have shown that expression profiling of carefully chosen intermediary gene sets, comprising approximately 10 to 100 genes, can convey the most relevant information compared to much more complex whole-genome studies. In this paper, we present a novel method suitable for expression profiling of moderate gene sets in a large number of samples. The assay implements the parallel amplification features of the trinucleotide threading technique (TnT), which encompasses linear transcript-based DNA thread formation in conjunction with exponential multiplexed thread amplification. The amplifications bestow the method with high sensitivity. The TnT procedure together with thread detection, relying on thread-specific primer extension followed by hybridization to universal tag arrays, allows for three distinction levels, thus offering high specificity. Additionally, the assay is easily automated and flexible. A gene set, comprising 18 protein epitope signature tags from the Swedish Human Protein Atlas program, was analyzed with the TnT-based approach and the data were compared with those generated by both real-time PCR and genome-wide cDNA arrays, with the highest correlation observed between TnT and real-time PCR. Taken together, expression profiling with trinucleotide threading represents a reliable approach for studies of intermediary gene sets.

Keyword
Gene expression profiling; HPA; Microarray; Multiplex; Real-time PCR; TnT; complementary DNA; DNA polymerase; epitope; transcriptome; trinucleotide; article; cell line; controlled study; DNA synthesis; gene expression profiling; housekeeping gene; human; human cell; microarray analysis; priority journal; real time polymerase chain reaction; RNA isolation; serial analysis of gene expression; signal noise ratio; DNA Primers; Epitopes; Gene Expression Profiling; Humans; Nucleic Acid Amplification Techniques; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7689 (URN)10.1016/j.ygeno.2007.10.012 (DOI)000252944500011 ()2-s2.0-38149086782 (Scopus ID)
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2017-12-14Bibliographically approved
2. Analysis of Short Tandem Repeats by Parallel DNA Threading
Open this publication in new window or tab >>Analysis of Short Tandem Repeats by Parallel DNA Threading
2009 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 4, no 11, e7823- p.Article in journal (Refereed) Published
Abstract [en]

The majority of studies employing short tandem repeats (STRs) require investigation of several of these genetic markers. As such, we demonstrate the feasibility of the trinucleotide threading (TnT) approach for scalable analysis of STRs. The TnT method represents a parallel amplification alternative that addresses the obstacles associated with multiplex PCR. In this study, analysis of the STR fragments was performed with capillary gel electrophoresis; however, it should be possible to combine our approach with the massive 454 sequencing platform to considerably increase the number of targeted STRs.

Keyword
eukaryotic genomes; microsatellites; evolution
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11292 (URN)10.1371/journal.pone.0007823 (DOI)000271721900014 ()2-s2.0-70649103962 (Scopus ID)
Note

QC 20100819

Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2016-12-09Bibliographically approved
3. Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads
Open this publication in new window or tab >>Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads
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2008 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 2, 323-329 p.Article in journal (Refereed) Published
Abstract [en]

Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.

Keyword
trinucleotide threading; pooled genomic DNA; association studies; multiplexing; SNP; allelotyping; cancer; obesity
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7690 (URN)10.1002/humu.20655 (DOI)000253033000016 ()2-s2.0-38949145121 (Scopus ID)
Note
QC 20100416Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2017-12-14Bibliographically approved
4. Targeted transcript profiling by sequencing
Open this publication in new window or tab >>Targeted transcript profiling by sequencing
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In recent years, second generation sequencers have been employed to study various facets of the transcriptome in a comprehensive manner. However, intermediary gene sets featuring differentially expressed genes can reduce the dimensionality of experiments while providing researchers with the most significant data. Trinucleotide threading (TnT) is a multiplex amplification method previously implemented in an assay for expression profiling of moderate gene sets. Here, two additional detection systems were evaluated with a focus on lowering the input material requirements. 32 genes were simultaneously assayed with detection either by direct hybridization of TnT products or by sequencing these using the massively parallel 454 sequencer. Both approaches produced reliable transcript abundance data starting from total RNA from about 200 cells. The direct hybridization readout is beneficial for smaller-scale studies, while more ambitious efforts employing numerous individuals are, together with a sample barcoding and pooling scheme, well suited for the second generation sequencing approach. Moreover, with protocol optimizations the starting material requirements for the sequencing strategy may be further reduced. Accordingly, this study presents a targeted RNA-Seq method.

National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11293 (URN)
Note
QC 20100819Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2011-02-04Bibliographically approved

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