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Genetic Sequence Analysis by Microarray Technology
KTH, School of Biotechnology (BIO).
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 [en]
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: urn:nbn:se:kth:diva-4330ISBN: 978-91-7178-609-8 (print)OAI: oai:DiVA.org:kth-4330DiVA: diva2:11833
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
List of papers
1. 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: 2017-12-14Bibliographically approved
2. Competitive enzymatic reaction to control allele-specific extensions
Open this publication in new window or tab >>Competitive enzymatic reaction to control allele-specific extensions
2005 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 33, no 5, e48:1-e48:10 p.Article in journal (Refereed) Published
Abstract [en]

Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian, A., Gharizadeh, B., O'Meara, D., Odeberg, J. and Lundeberg, J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 50 end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.

Keyword
DNA polymerase; proteinase; 3' untranslated region; 5' untranslated region; allele; article; bioassay; controlled study; enzyme activity; enzyme degradation; enzyme mechanism; genetic regulation; genotype; human; kinetics; normal human; polymerase chain reaction; priority journal; scoring system; single nucleotide polymorphism; Meara
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-8908 (URN)10.1093/nar/gni048 (DOI)000228080000003 ()2-s2.0-20144372764 (Scopus ID)
Note
QC 20100714Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2017-12-14Bibliographically approved
3. 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, E-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: 2017-12-14Bibliographically approved
4. Random loss of genetic segments during skin differentiation indicated by analysis of single cells
Open this publication in new window or tab >>Random loss of genetic segments during skin differentiation indicated by analysis of single cells
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(English)Article in journal (Other academic) Submitted
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6969 (URN)
Note
QS 20120327Available from: 2007-04-12 Created: 2007-04-12 Last updated: 2012-03-27Bibliographically approved

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