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Tri-nucleotide Threading for parallel amplification of minute amounts of genomic DNA
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-4313-1601
KTH, School of Biotechnology (BIO).
2006 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 34, no 6, 9- p.Article in journal (Refereed) Published
Abstract [en]

Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.

Place, publisher, year, edition, pages
2006. Vol. 34, no 6, 9- p.
Keyword [en]
genomic DNA; trinucleotide; cytidine triphosphate; guanosine triphosphate; pyrimidine nucleotide; thymidine 5'-triphosphate; thymidine triphosphate; article; cancer; computer program; DNA microarray; gene amplification; gene locus; genetic variability; genotype; high throughput screening; human; phenotype; priority journal; scoring system; sensitivity and specificity; single nucleotide polymorphism; chemistry; evaluation; genome; genomics; methodology; polymerase chain reaction; Cytidine Triphosphate; Genome, Human; Genomics; Genotype; Guanosine Triphosphate; Humans; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Software; Thymine Nucleotides
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-7687DOI: 10.1093/nar/gkl103ISI: 000237002900005Scopus ID: 2-s2.0-33645832164OAI: oai:DiVA.org:kth-7687DiVA: diva2:12787
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Interrogation of Nucleic Acids by Parallel Threading
Open this publication in new window or tab >>Interrogation of Nucleic Acids by Parallel Threading
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Advancements in the field of biotechnology are expanding the scientific horizon and a promising era is envisioned with personalized medicine for improved health. The amount of genetic data is growing at an ever-escalating pace due to the availability of novel technologies that allow massively parallel sequencing and whole-genome genotyping, that are supported by the advancements in computer science and information technologies. As the amount of information stored in databases throughout the world is growing and our knowledge deepens, genetic signatures with significant importance are discovered. The surface of such a set in the data mining process may include causative- or marker single nucleotide polymorphisms (SNPs), revealing predisposition to disease, or gene expression signatures, profiling a pathological state. When targeting a reduced set of signatures in a large number of samples for diagnostic- or fine-mapping purposes, efficient interrogation and scoring require appropriate preparations. These needs are met by miniaturized and parallelized platforms that allow a low sample and template consumption.

This doctoral thesis describes an attempt to tackle some of these challenges by the design and implementation of a novel assay denoted Trinucleotide Threading (TnT). The method permits multiplex amplification of a medium size set of specific loci and was adapted to genotyping, gene expression profiling and digital allelotyping. Utilizing a reduced number of nucleotides permits specific amplification of targeted loci while preventing the generation of spurious amplification products. This method was applied to genotype 96 individuals for 75 SNPs. In addition, the accuracy of genotyping from minute amounts of genomic DNA was confirmed. This procedure was performed using a robotic workstation running custom-made scripts and a software tool was implemented to facilitate the assay design. Furthermore, a statistical model was derived from the molecular principles of the genotyping assay and an Expectation-Maximization algorithm was chosen to automatically call the generated genotypes. The TnT approach was also adapted to profiling signature gene sets for the Swedish Human Protein Atlas Program. Here 18 protein epitope signature tags (PrESTs) were targeted in eight different cell lines employed in the program and the results demonstrated high concordance rates with real-time PCR approaches. Finally, an assay for digital estimation of allele frequencies in large cohorts was set up by combining the TnT approach with a second-generation sequencing system. Allelotyping was performed by targeting 147 polymorphic loci in a genomic pool of 462 individuals. Subsequent interrogation was carried out on a state-of-the-art massively parallelized Pyrosequencing instrument. The experiment generated more than 200,000 reads and with bioinformatic support, clonally amplified fragments and the corresponding sequence reads were converted to a precise set of allele frequencies.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. 46 p.
Keyword
genotyping, multiplex amplification, trinucleotide threading, single nucleotide polymorphism, genotype calling, Expectation-Maximization, protein-epitope signature tag, expression profiling, Human Protein Atlas, pooled genomic DNA, Pyrosequencing, 454, allelotyping, association studies, bioinformatics
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4546 (URN)978-91-7178-802-3 (ISBN)
Public defence
2007-12-14, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100813Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2010-08-13Bibliographically approved

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