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Competitive enzymatic reaction to control allele-specific extensions
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.ORCID iD: 0000-0003-4313-1601
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.

Place, publisher, year, edition, pages
2005. Vol. 33, no 5, e48:1-e48:10 p.
Keyword [en]
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: urn:nbn:se:kth:diva-8908DOI: 10.1093/nar/gni048ISI: 000228080000003Scopus ID: 2-s2.0-20144372764OAI: oai:DiVA.org:kth-8908DiVA: diva2:14391
Note
QC 20100714Available from: 2005-12-09 Created: 2005-12-09 Last updated: 2017-12-14Bibliographically approved
In thesis
1. 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
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Note
QC 20100714Available from: 2007-04-12 Created: 2007-04-12 Last updated: 2010-07-14Bibliographically approved

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