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Tag-array based HPV genotyping by competitive hybridization and extension
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
Department of Molecular Medicine, Karolinska Institutet.
KTH, School of Biotechnology (BIO).
Show others and affiliations
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.

Place, publisher, year, edition, pages
2005. Vol. 129, no 2, 102-112 p.
Keyword [en]
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: urn:nbn:se:kth:diva-8907DOI: 10.1016/j.jviromet.2005.05.015ISI: 000232522400001Scopus ID: 2-s2.0-25144451949OAI: oai:DiVA.org:kth-8907DiVA: diva2:14390
Note
QC 20100714. Uppdaterad från Accepted till Published 20100714.Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2010-07-14Bibliographically 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
2. Genetic Sequence Analysis by Microarray Technology
Open this publication in new window or tab >>Genetic Sequence Analysis by Microarray Technology
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Developments within the field of genetic analysis have during the last decade become enormous. Advances in DNA sequencing technology have increased throughput from a thousand bases to over a billion bases in a day and decreased the cost thousandfold per base. Nevertheless, to sequence complex genomes like the human is still very expensive and efforts to attain even higher throughputs for less money are undertaken by researchers and companies.

Genotyping systems for single nucleotide polymorphism (SNP) analysis with whole genome coverage have also been developed, with low cost per SNP. There is, however, a need for genotyping assays that are more cost efficient per sample with considerably higher accuracy. This thesis is focusing on a technology, based on competitive allele-specific extension and microarray detection, for genetic analysis. To increase specificity in allele-specific extension (ASE), a nucleotide degrading enzyme, apyrase, was introduced to compete with the polymerase, only allowing the fast, perfect matched primer extension to occur. The aim was to develop a method for analysis of around twenty loci in hundreds of samples in a high-throughput microarray format.

A genotyping method for human papillomavirus has been developed, based on a combination of multiplex competitive hybridization (MUCH) and apyrase-mediated allele-specific extension (AMASE). Human papillomavirus (HPV), which is the causative agent in cervical cancer, exists in over a hundred different types. These types need to be determined in clinical samples. The developed assay can detect the twenty-three most common high risk types, as well as semi-quantifying multiple infections, which was demonstrated by analysis of ninety-two HPV-positive clinical samples.

More stringent conditions can be obtained by increased reaction temperature. To further improve the genotyping assay, a thermostable enzyme, protease, was introduced into the allele-specific extension reaction, denoted PrASE. Increased sensitivity was achieved with an automated magnetic system that facilitates washing. The PrASE genotyping of thirteen SNPs yielded higher conversion rates, as well as more robust genotype scoring, compared to ASE. Furthermore, a comparison with pyrosequencing, where 99.8 % of the 4,420 analyzed genotypes were in concordance, indicates high accuracy and robustness of the PrASE technology.

Single cells have also been analyzed by the PrASE assay to investigate loss of alleles during skin differentiation. Single cell analysis is very demanding due to the limited amounts of DNA. The multiplex PCR and the PrASE assay were optimized for single cell analysis. Twenty-four SNPs were genotyped and an increased loss of genetic material was seen in cells from the more differentiated suprabasal layers compared to the basal layer.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. 75 p.
Keyword
Genotyping, single nucleotide polymorphism (SNP), protease-mediated allele-specific extension (PrASE), microarray, tag-array, competitive hybridization, human papillomavirus (HPV), single cell, loss of alleles, differentiation, epidermis.
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4330 (URN)978-91-7178-609-8 (ISBN)
Public defence
2007-04-27, F3, Lindstedtsvägen 26, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100714Available from: 2007-04-12 Created: 2007-04-12 Last updated: 2010-07-14Bibliographically approved

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