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Arrayed identification of DNA signatures
KTH, School of Biotechnology (BIO).
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping.

P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools.

The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples.

To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension.

The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants.

Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , 67 p.
Keyword [en]
apyrase, allele-specific extension, competitive hybridization, DNA sequencing, genotyping, human papillomavirus (HPV), MC1R, microarray, mutation, p53, protease
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-549ISBN: 91-7178-219-2 (print)OAI: oai:DiVA.org:kth-549DiVA: diva2:14395
Public defence
2005-12-16, Sal D2, Lindstedtsvägen 5, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101028Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2011-09-06Bibliographically approved
List of papers
1. Microarray-based AMASE as a novel approach for mutation detection
Open this publication in new window or tab >>Microarray-based AMASE as a novel approach for mutation detection
2004 (English)In: Mutation research, ISSN 0027-5107, E-ISSN 1873-135X, Vol. 554, no 1-2, 77-88 p.Article in journal (Refereed) Published
Abstract [en]

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.

Keyword
allele-specific, apyrase, microarray, mutation, p53, sequencing
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8905 (URN)10.1016/j.mrfmmm.2004.03.009 (DOI)000223821800009 ()2-s2.0-4344669178 (Scopus ID)
Note
QC 20100922 QC 20110923Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2011-09-23Bibliographically approved
2. Comparison of PrASE and Pyrosequencing for SNP Genotyping
Open this publication in new window or tab >>Comparison of PrASE and Pyrosequencing for SNP Genotyping
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2006 (English)In: BMC Genomics, ISSN 1471-2164, Vol. 7, 291- p.Article in journal (Refereed) Published
Abstract [en]

Background: There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers.

Results: The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated.

Conclusion: The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.

Keyword
5, 10 methylenetetrahydrofolate reductase (FADH2); beta integrin; blood clotting factor 13; blood clotting factor 5; endothelial nitric oxide synthase; fibrinogen; plasminogen activator inhibitor 1; prothrombin; stromelysin; accuracy; article; automation; controlled study; gene cluster; gene technology; genetic polymorphism; genetic procedures; genetic susceptibility; genetic variability; genotype; human; intermethod comparison; microarray analysis; mutational analysis; parallel design; polymerase chain reaction; protease mediated allele specific extension; pyrosequencing; reproducibility; single nucleotide polymorphism; thrombosis
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6968 (URN)10.1186/1471-2164-7-291 (DOI)000242193100001 ()2-s2.0-34547926324 (Scopus ID)
Note
Tidigare titel: PrASE - An Accurate and Robust Genotyping Platform as Compared to DNA Sequencing QC 20100714Available from: 2007-04-12 Created: 2007-04-12 Last updated: 2011-09-06Bibliographically approved
3. Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays
Open this publication in new window or tab >>Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays
Show others...
2003 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 31, no 22, e146- p.Article in journal (Refereed) Published
Abstract [en]

 Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.

Keyword
article, DNA microarray, female, genetics, genotype, human, methodology, nucleic acid hybridization, Papilloma virus, polymerase chain reaction, reproducibility, sensitivity and specificity, virology, virus infection
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8906 (URN)10.1093/nar/gng147 (DOI)000186590600045 ()
Note
QC 20101005. Uppdaterad från In press till Published (20101005).Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2010-10-05Bibliographically approved
4. Tag-array based HPV genotyping by competitive hybridization and extension
Open this publication in new window or tab >>Tag-array based HPV genotyping by competitive hybridization and extension
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2005 (English)In: Journal of Virological Methods, ISSN 0166-0934, E-ISSN 1879-0984, Vol. 129, no 2, 102-112 p.Article in journal (Refereed) Published
Abstract [en]

A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (< 2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.

Keyword
oligonucleotide; article; assay; genotype; hybridization; infection; molecular cloning; nonhuman; priority journal; sensitivity analysis; sequence analysis; virus typing; Wart virus; Alleles; Apyrase; DNA Primers; Female; Gene Expression Profiling; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Papillomaviridae; Papillomavirus Infections; Sensitivity and Specificity; Species Specificity; Templates, Genetic
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8907 (URN)10.1016/j.jviromet.2005.05.015 (DOI)000232522400001 ()2-s2.0-25144451949 (Scopus ID)
Note
QC 20100714. Uppdaterad från Accepted till Published 20100714.Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2010-07-14Bibliographically approved
5.
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6. Detection of MC1R Polymorphisms with Protease-Mediated Allele-Specific Extension as an Alternative to Direct Sequencing
Open this publication in new window or tab >>Detection of MC1R Polymorphisms with Protease-Mediated Allele-Specific Extension as an Alternative to Direct Sequencing
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2005 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 51, no 12, 2388-2391 p.Article in journal (Refereed) Published
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8910 (URN)10.1373/clinchem.2005.056820 (DOI)000233533300033 ()2-s2.0-28044446303 (Scopus ID)
Note
QC 20101028Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2010-10-28Bibliographically approved
7. Genetic variation of the MC1R gene and the risk for cutaneous malignant melanoma in the Swedish population
Open this publication in new window or tab >>Genetic variation of the MC1R gene and the risk for cutaneous malignant melanoma in the Swedish population
Show others...
(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8911 (URN)
Note
QC 20101028Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2010-10-28Bibliographically approved

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