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Microarray-based AMASE as a novel approach for mutation detection
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4313-1601
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.

Place, publisher, year, edition, pages
2004. Vol. 554, no 1-2, 77-88 p.
Keyword [en]
allele-specific, apyrase, microarray, mutation, p53, sequencing
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-8905DOI: 10.1016/j.mrfmmm.2004.03.009ISI: 000223821800009Scopus ID: 2-s2.0-4344669178OAI: oai:DiVA.org:kth-8905DiVA: diva2:14388
Note
QC 20100922 QC 20110923Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Arrayed identification of DNA signatures
Open this publication in new window or tab >>Arrayed identification of DNA signatures
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
apyrase, allele-specific extension, competitive hybridization, DNA sequencing, genotyping, human papillomavirus (HPV), MC1R, microarray, mutation, p53, protease
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-549 (URN)91-7178-219-2 (ISBN)
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

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