Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cancer,mutation, variation, single nucleotide polymorphism,allele-specific extension, bioluminescence, apyrase. / QC 20100415

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p>

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. / QC 20101028

Nucleic acid technology has assumed an essential role invarious areas ofin vitrodiagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis. The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products. Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection. Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy&gt;99%is required. <b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.

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. / QC 20100714

<p>Nucleic acid technology has assumed an essential role invarious areas of<i>in vitro</i>diagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis.</p><p>The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products.</p><p>Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection.</p><p>Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy>99%is required.</p><p><b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.</p>

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods. This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed. The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers. The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides. / QC 20101008