application of padlock probe based nucleic acid analysis in situ331818/fulltext01.pdf ·...

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2010

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 575

Application of Padlock ProbeBased Nucleic Acid Analysis In Situ

SARA HENRIKSSON

ISSN 1651-6206ISBN 978-91-554-7842-1urn:nbn:se:uu:diva-128446

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen,Rudbeckslaboratoriet, Dag Hammarskjölds väg 20, Uppsala, Friday, September 10, 2010 at09:15 for the degree of Doctor of Philosophy. The examination will be conducted in English.

AbstractHenriksson, S. 2010. Application of Padlock Probe Based Nucleic Acid Analysis In Situ. ActaUniversitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations fromthe Faculty of Medicine 575. 41 pp. Uppsala. ISBN 978-91-554-7842-1.

The great variation displayed by nucleic acid molecules in human cells, and the continuousdiscovery of their impact on life, consequently require continuous refinements of molecularanalysis techniques. Padlock probes and rolling circle amplification offer single nucleotidediscrimination in situ, a high signal-to-noise ratio and localized detection within cells andtissues.

In this thesis, in situ detection of nucleic acids with padlock probes and rolling circleamplification was applied for detection of DNA in the single cell gel electrophoresis assay todetect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair. The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier beencontroversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay.In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. Anew twelve gel approach was also developed with increased throughput of the single cell gelelectrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeatsafter H2O2 induced damage was further monitored. All three genes and Alu repeats were repairedfaster than total DNA. Finally, padlock probes and rolling circle amplification were applied fordetection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The viruswas detected by first reverse transcribing RNA into cDNA.. The virus RNA together with itscomplementary RNA and the nucleocapsid protein were detected in cultured cells.

The work presented here enables studies of gene specific damage and repair as well asviral infections in situ. Detection by ligation offers high specificity and makes it possible todiscriminate even between closely related molecules. Therefore, these techniques will be usefulfor a wide range of applications within research and diagnostics.

Keywords: Padlock probe, rolling circle amplification, single cell gel electrophoresis assay,comet assay, Crimean Congo hemorrhagic fever virus

Sara Henriksson, Disciplinary Domain of Medicine and Pharmacy, Box 256, UppsalaUniversity, SE-75105 Uppsala, Sweden

© Sara Henriksson 2010

ISSN 1651-6206ISBN 978-91-554-7842-1urn:nbn:se:uu:diva-128446 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-128446)

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Shaposhnikov S, Larsson C, Henriksson S, Collins A, Nilsson

M. (2006) Detection of Alu sequences and mtDNA in comets using padlock probes. Mutagenesis, 21(4):243-7

II Shaposhnikov, S., Azqueta, A., Henriksson, S., Meier, S., Gaivão, I., Huskisson, N.H., Smart, A., Brunborg, G., Nilsson, M. and Collins, A.R. (2010) Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation. Toxicol-ogy Letters, 19:195(1):31-4.

III Henriksson S, Shaposhnikov S, Nilsson M and Collins A. Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification. Manuscript

IV Andersson C*, Henriksson S*, Mirazimi A and Nilsson M. Si-multaneous detection of Crimean Congo Hemorrhagic fever vi-rus viral RNA and complementary RNA in situ by padlock probes and rolling circle amplification combined with viral pro-tein visualization. Manuscript *contributed equally

Reprints were made with permission from the respective publishers.

Related work by the author

Carolina Wählby, Patrick Karlsson, Sara Henriksson, Chatarina Larsson, Mats Nilsson, Ewert Bengtsson Finding cells, finding molecules, finding patterns. International Journal of Signal and Imaging Systems Engineering 1(1)2008 11 – 17

Contents

Introduction.....................................................................................................9

Methods for detection of nucleic acids in situ ..............................................11 In situ hybridization .................................................................................11 In situ detection of nucleic acids using polymerases................................13 Padlock probes .........................................................................................14 Rolling circle amplification......................................................................14 Rolling circle amplified padlock probes in situ........................................15

Single cell gel electrophoresis ......................................................................18

Viruses ..........................................................................................................20

Present investigation .....................................................................................21 Paper I: Detection of Alu sequences and mtDNA in comets using padlock probes..........................................................................................21

Aim of the study ..................................................................................21 Methods ...............................................................................................21 Results .................................................................................................22 Discussion............................................................................................22

Paper II: Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation ...............................................................23


Paper III: Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification .......................................25


Paper IV: Simultaneous detection of Crimean Congo Hemorrhagic fever virus viral RNA and complementary RNA in situ by padlock probes and rolling circle amplification combined with viral protein visualization .............................................................................................28


Future perspectives .......................................................................................31 Detection of PCV2 in situ ........................................................................31 Conclusions ..............................................................................................32

Acknowledgements.......................................................................................34

References.....................................................................................................36

Abbreviations

BAC Bacterial artificial chromosome cDNA CCHFV CNVs DNA cRNA FISH FITC FPG HPRT1 ISH L LNA M MELAS mRNA mtDNA NP OGG1 PCR PCV2 PNA PRINS RCA RCP

Complementary DNA Crimean Congo Hemorrhagic fever virus Copy number variations Deoxyribonucleic acid Complementary RNA Fluorescence in situ hybridization Fluorescein isothiocyanate Formamidopyrimidine DNA glycosylase Hypoxanthine phosphoribosyltransferase 1 In situ hybridization Large Locked nucleic acid Medium Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes Messenger RNA Mitochondrial DNA Nucleocapsid protein 8-oxoguanine DNA glycosylase Polymerase chain reaction Porcine circovirus type 2 Peptide nucleic acid Primed in situ labelling Rolling circle amplification Rolling circle product

RNA S

Ribonucleic acid Small

TSA vRNA XPD

Tyramide signal amplification Virus RNA Xeroderma pigmentosum group D

9

Introduction

Human cells harbor great variability on a molecular level. These variations are in deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. In DNA there are single nucleotide polymorphisms (SNPs)1, copy number variations (CNVs)2, insertions, deletions and translocations. On the RNA level there are differences in expression levels and methylations, causing for example mono-allelic expression. Thanks to recent years sequencing3 and sequence variation projects1, 2 information is now available in public data-bases e.g. http://www.ncbi.nlm.nih.gov4. This makes it possible to study variations in nucleic acids within different biological preparations but the current methods for this are far from ideal.

When analyzing nucleic acids in vitro the resulting data will show an av-erage result from the included cell population. For many applications this is satisfactory, but for others single cell analysis is necessary. When analyzing a tumor material in vitro that contains a point mutation or a copy number variation it is possible that there is also healthy material without the point mutation or copy number variation present. In vitro the true mutation may go undetected since it is averaged out by the healthy material. When analyzing metastasized tumors a certain cell type may harbor a mutation which is ab-sent in other cell types in the surrounding tissue. In vitro the cell type infor-mation would be lost. In other cases there may be a large cell-to-cell varia-tion in the presence of a nucleic acid e.g. when studying expression levels of RNA or bacterial or viral infections. By observing these molecules within the cell we can obtain information about cell-to-cell variations and sub-cellular localization of single molecules which can tell us about in which compartment certain processes are initiated and how certain pathogens enter the cell. It can also provide information about pathogens’ replication and transcription.

Methods to study genetic sequences and variations in situ should ideally have a number of characteristics. They should be sensitive enough to detect molecules with low abundance, specific enough to distinguish between closely related molecules, gentle to the cell or tissue to preserve the mor-phology, cheap, fast and not too labor intense. In reality there is always a compromise. Padlock probes in combination with rolling circle amplification (RCA) have advantages for in situ analysis such as high specificity, high signal-to-noise ratio, strand specificity, possibilities for multiplexing, no need for expensive apparatuses and it is performed under gentle conditions.

10

In this thesis, work is presented where padlock probes and RCA are ap-plied for detection of nucleic acids in situ. First, padlock probes and RCA were developed to detect specific DNA sequences within the single cell gel electrophoresis assay (comet assay). Throughput of the comet assay was then increased by a new twelve-gel approach. Padlock probes and RCA were then used to monitor DNA repair of specific genes. Finally the Crimean Congo Hemorrhagic fever virus (CCHFV), a single stranded RNA virus, and its complementary RNA (cRNA) was detected in infected cells at different time points after infection.

11

Methods for detection of nucleic acids in situ

In situ hybridization In situ hybridization (ISH) is a technique which is widely used within diag-nostics and research. Labelled DNA or RNA probes are hybridized to a tar-get nucleic acid in situ giving a localized detection of a nucleic acid.

ISH was first described using radio labelled ribosomal RNA for detection of the amplified ribosomal DNA in oocytes of Xenopus laevis5. Later fluo-rescence was used for detecting the ISH probe6 thus eliminating the use of radioactive material. Fluorescence in situ hybridization (FISH) made it pos-sible to detect multiple targets simultaneously since different fluorophores could be used for different targets7. In whole chromosome paint whole chro-mosomes can be labelled with a FISH probe for chromosome analysis mak-ing translocations easy to detect8. All chromosomes can be differentially labelled with multicolour-FISH by combining different colours for chromo-some staining9.

The FISH probe can be synthetically made oligonucleotides10, 11 but is of-ten amplified in a vector, such as a plasmid, cosmid or a Bacterial artificial chromosome (BAC)12. The probe is often made with nick translation with modified nucleotides13, 14. The probe can also be made with random primed labelling15. The FISH probe is broken into a number of smaller pieces that hybridize to a part of a chromosome or a whole chromosome. The probe can be double- or single stranded. Double stranded probes have the advantage of being easily produced. The drawback is that both the probe and the target DNA need to be denatured. Single stranded probes on the other hand are more technically difficult to produce but there is no risk that the probe re-anneals.

FISH has the limitation that very long target sequences are necessary. Short sequences reduce sensitivity. As a consequence SNPs, short insertions, deletions or translocations cannot be analyzed with FISH. Besides having low resolution long FISH probes have another disadvantage. They give high background since the probes often contain repeated sequences. To avoid the background from repeated sequences unlabelled genomic DNA or Cot-1 DNA is hybridized to the DNA before or together with the FISH probe16. There has also been an attempt to exclude repeated sequences from the probes and thereby reducing background17. Using shorter oligonucleotide probes has the advantage that the shorter probes can more easily enter the

12

nuclei. The drawback is that oligonucleotide probes have lower sensitivity and signal strength which results in that oligonucleotide probes are mostly used on repeated sequences, which limit the possible targets.

RNA is often detected with RNA probes which give more stable duplexes than DNA-RNA18 but oligonucleotide probes have been used for FISH for detection of single RNA transcripts in situ with oligonucleotides labelled with multiple fluorophores19 or with multiple singly labelled probes20. Synthesis of heavily labelled oligonucleotides is difficult and using multiple probes is expensive and excludes the possibility to genotype or study spliceotypes.

To be able to use single oligonucleotide probes with high sensitivity there are different approaches for signal amplification. Tyramide signal amplifica-tion (TSA) which was first developed for ELISA and Western blot is used in immunoassays and ISH for signal amplification. The procedure is based on that haptenized tyramide molecules are deposited close to the hybridized probes catalyzed by horseradish peroxidase21, 22, 23. Another approach for signal amplification is using branched DNA. It has been used for amplifica-tion of ISH on RNA with oligonucleotide probes24, 25 and for detection of single copy genes26. The oligonucleotide probes contain a flanking sequence where branched DNA can hybridize. Fluorescently labeled oligonucleotides are then hybridized to the branched DNA resulting in a strong signal.

Besides signal amplification to improve signal intensity oligonucleotide probes also have lower sensitivity than longer probes. Synthetic FISH probes with modified nucleic acids such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) can be used to increase hybridization sensitivity and specificity. PNA has a peptide like backbone which unlike the backbone in DNA is uncharged. PNA therefore hybridizes more stably to DNA and RNA than DNA oligonucleotides27. Because of the neutrally charged backbone in PNA, the PNA-DNA hybrid has higher melting temperature than double stranded DNA28 and can therefore hybridize in low salt conditions where native nucleic acids would be unstable29. The PNA probes have some other advantages to oligonucleotide probes. They are more stable and as a result of the higher melting temperature they are more significantly affected by mis-matches and thus have a higher specificity than oligonucleotide probes30. PNA probes have been used for FISH and the higher specificity of the PNA probes has made it possible to discriminate between a single base pair in repeated sequences using a 17-mer oligonucleotide31, 32.

Another modified nucleic acid that has been used for FISH is LNA, which is a 2′-O, 4′-C-methylene-linked ribonucleotide derivative of RNA. LNA probes have a higher melting temperature in duplexes with DNA and RNA and are more sensitive for mismatches than dsDNA or dsRNA33, 34. LNA probes have also been used for FISH with higher resolution and sensitivity than oligonucleotide probes35. The high resolution and sensitivity of LNA has made detection of different splicing of mRNA possible using short oli-gonucleotide probes36.

13

In situ detection of nucleic acids using polymerases The polymerase chain reaction (PCR) has revolutionized the world of bio-technology. PCR is sensitive enough to detect single copies of target se-quences and can produce DNA fragments that can be used for a number of applications i.e. cloning. The principle for PCR is that two oligonucleotide primers are hybridized on opposite strands of a target sequence. The primers are the starting point for cyclic enzymatic amplification. In each round of amplification the DNA is denatured, primers are annealed and then the po-lymerase elongates the primers. This results in an exponential amplification of the target sequence37. By cloning of thermo stable polymerases this reac-tion can be performed in a closed tube and as higher temperatures can be used it also results in increased specificity38.

The PCR technique was further developed for in situ detection of DNA and RNA sequences. The in situ PCR technique allows for detection of less abundant sequences than what can be detected with in situ hybridization39. However, the limit of detection is often higher than a single copy resulting in that in situ PCR mostly is used for detection of multi-copy targets such as mRNA or viral infections. For detection of RNA the procedure is combined with an in situ reverse transcriptase step40. Sometimes it is also combined with in situ hybridization.

In situ PCR has recently been used for detection of the Phosphoglycerate Kinase-1 gene in mouse41 and for mycobacterium tuberculosis42; however extensive optimizations are needed when using different tissues. That could be a reason why this method has never been widely used. In situ PCR also has the disadvantage of loosing localization of sequences due to the diffusion of the PCR product. The localization of the PCR product can however some-times be limited to certain cellular compartments43. Another result of the diffusion of the PCR product is that copy numbers of target sequences can not be determined since there are no distinct spots from each detected target. This makes the method unsuitable for quantification.

In the Primed in situ labelling (PRINS) technique synthetic oligonucleo-tides are used as primers for in situ incorporation of labelled nucleotides using a DNA polymerase44. Compared to cytological methods PRINS is rapid and sensitive and unlike in situ PCR it results in specific signals with sub-cellular localization.

PRINS was used to distinguish among closely related alpha satellite se-quences on different chromosomes45, 46. Through incorporation of differently labelled nucleotides detection of three different targets was done simultane-ously47. Though this method can discriminate between single nucleotide differences in repeated sequences it can not be used for lower copy number sequences due to unspecific incorporation of nucleotides at other sites in the DNA.

14

Padlock probes The padlock probe is a development of the oligonucleotide ligation assay (OLA)48. In the OLA, two oligonucleotide probes were hybridized juxta-posed on a target DNA molecule. Upon perfect hybridization at the junction the two probes were ligated with T4 DNA ligase. By using a ligase OLA allowed distinction of single nucleotide differences at the ligation junction.

In padlock probes, instead of having two separate oligonucleotide probes as in OLA, the two probes were joined together with a DNA backbone. This should increase the possibility of both DNA oligonucleotides hybridizing simultaneously since the other oligonucleotide always is in close proximity.

Padlock probes are linear oligonucleotide probes that can be circularized in a target dependant matter. The two ends of the probe are hybridized juxta-posed on a target molecule and when correctly hybridized at the junction the two ends can be enzymatically ligated forming a circular DNA molecule. The ligated padlock probe is wound around the target molecule because of the helical nature of DNA molecule, thus resembling a padlock49. Besides having target complementary ends the padlock probe has a unique sequence in the backbone which can be used for recognition of the padlock probe.

Padlock probes were first used in situ to study repeated sequences on metaphase spreads. The segregation of centromeric sequences in a family was studied by targeting single nucleotide differences in alpha satellite se-quences on chromosome 13 and chromosome 2150.

Rolling circle amplification Padlock probes offer a very specific detection of nucleic acid molecules. However, to be able to detect the padlock probes in situ signal amplification is necessary.

RCA creates a long single stranded DNA molecule with tandem repeats of the complementary sequence to a single stranded circle51. Padlock probes can be used as templates for RCA. This is a specific way to detect padlock probes since only ligated probes can serve as templates for RCA52 (see Figure 1). RCA of a ligated padlock probe can be initiated either by an ex-ternal primer or by the target DNA. RCA with an external primer requires a nearby DNA break for displacements of the target strand; otherwise the RCA is hindered by topological constraints. The target strand can be used as a primer for RCA after the removal of non-base-paired nucleotides down-stream of the target sequence53.

Phi29 DNA polymerase (Bacillus subtilis) is a polymerase with two im-portant activities. It has a 3’ to 5’ exonucleolytic activity on single stranded DNA and it also has polymerase activity54. This means that it can be used to remove the non-base-paired nucleotides downstream of the padlock probe

15

and it can also RCA using the target strand as a primer. Alternatively, the DNA can be cut site-specifically before amplification by introducing an AG mismatch. The padlock probe should contain a G which is mismatched with an A in the target molecule. MutY will remove the A base at the mismatch leaving an abasic site in the target molecule. The abasic site in the phosphate backbone is then recognized and cleaved with EndoIV55.

The long molecule which is produced during RCA collapses spontaneously into a point like structure which is ~1 µm in diameter. After hybridization with short fluorescently labelled oligonucleotides, which have the same sequence as the part of the backbone of the padlock probe, the rolling circle product (RCP) can be visualized as a bright spot in a fluorescence microscope.

Figure 1.The two ends of the padlock probes are hybridized to a complementary sequence. Upon correct base pairing at the junction the two ends are enzymatically ligated forming a circular DNA molecule. The circularized padlock probe is used as a template for RCA. The RCP can be visualized by hybridization of fluorescently labelled oligonucleotides.

Rolling circle amplified padlock probes in situ Padlock probes and target primed RCA was first used in situ for genotyping of the mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) mutation in mitochondrial DNA (mtDNA). In situ the target DNA has to be made single stranded before ligation. Here the target DNA was cut with a restriction enzyme creating a double strand break downstream of the target sequence. The DNA was then exposed to an ex-

16

onuclease to make DNA single stranded before hybridization of the padlock probe. The combination of restriction digestion and exonucleases is a mild treatment for making DNA single stranded. It can be performed at 37 de-grees and should not have a severe effect on the cell morphology. The re-striction digestion also created a starting point for RCA. In this case the tar-get molecule could be used as a primer for RCA making the RCA product covalently linked to the target molecule56 (see Figure 2).

Using ligases for detection makes it possible to distinguish between single nucleotides. The amplification of the padlock probe with RCA creates a ~1 µm signal which allows for single molecule detection in situ. Signals from the padlock probes targeting mtDNA were located in the cytoplasm showing that the method has sub-cellular resolution. The RCPs are discrete signals that can be counted using programs such as Blobfinder57. This method can thus be used to do quantitative measurements of bio molecules. By using different fluorophores the method can be used for multiplexed detection of nucleic acid targets.

Padlock probes and target primed RCA was used for detection of bacterial infection of Anaplasma phagocytophilum and Anaplasma marginale within intact cultured mammalian cells58. Target primed RCA of padlock probes was also used on metaphase chromosomes for detection of repeated se-quences in the Human satellite I sequence on the Y-chromosome and the repetitive kringle domain from the apolipo protein gene on chromosome 659.

Ligation of padlock probes with T4 DNA ligase can be performed with an RNA template but with reduced sensitivity and specificity compared to DNA templated ligation60, 61. RNA has been detected in cells with RNA templated ligation but detection efficiency was low62. To overcome the problems with low sensitivity and specificity of RNA templated ligation in situ, two differ-ent approaches have been attempted. Stougaard et al. used a so called turtle probe for detection of non-polyadenylated RNA. The turtle probe is a linear probe which upon hybridization to a target RNA forms a hairpin structure and templates its own ligation. It thereby forms a circular molecule which is detected by target primed RCA initiated from the natural 3’-end of the RNA63. Since this method requires that the probe is hybridised to the 3’ end of the RNA this method cannot be used for genotyping or detection of mono-allelic expression. Also, since it templates its own ligation it will not have the specificity of the padlock probe.

Recently another approach was taken by Larsson et al. They detected various transcripts in cultured cells and in fresh frozen tissue using padlock probes and RCA. Their approach was to initially perform a reverse transcrip-tase step with a transcript specific LNA containing primer creating a com-plementary DNA (cDNA) molecule. The cDNA was then targeted with a padlock probe and detected with RCA. This method was used to distinguish between single nucleotide differences in human and mouse Β-actin and in human k-RAS transcript64.

17

Figure 2.Target primed RCA. DNA is made single stranded by a combination of restriction enzymes and exonucleases. Upon perfect hybridization at the junction the two ends of the padlock probes are ligated. The circularized padlock probe is used as a template for target primed RCA before hybridization of fluorescently labelled oligonucleotides.

18

Single cell gel electrophoresis

Single cell gel electrophoresis was first described in 1984. The method which is known as the “comet assay” is a method for measuring DNA dam-age and repair. The comet assay is a good test for gene toxicity which should be performed before animal testing when developing new drugs. The number of animals which are used for animal testing can thus be reduced.

In the procedure, cells are embedded in low-melting-point agarose and treated with radiation or a chemical. Cells are then lysed and exposed to an electric field. DNA with strand breaks will migrate through the gel creating what resembles a comet “tail”. Undamaged DNA will remain in the nucleus which looks like the “head” of the comet. DNA is stained with a fluorescent dye e.g. DAPI before microscopy.

Single- and double strand breaks in the DNA causes relaxation of the su-percoiling in the DNA. This relaxation causes the DNA to stretch out into the gel during electrophoresis and is the reason for appearance of DNA in the tail instead of the head, rather than whole fragments of DNA migrating through the gel. The range of damage which can be measured in the comet assay is between 0.06-3 per 109 Da65.

Comets can be run under either neutral or alkaline conditions. The alka-line conditions make DNA single stranded and has shown to resolve the tail more from the head than in neutral comets. After electrophoresis the alkaline comet is neutralized causing DNA in the head to re-anneal66.

Comet assays can detect double and single strand breaks but it can also be used for detecting other types of DNA damage. This is done by converting DNA damage into DNA breaks by using DNA repair enzymes67 or by using lesion specific endonucleases68.

The common scoring procedure of comets is based on measurements of the relationship between the amounts of DNA in the tail compared to DNA in the head and sometimes tail length. Manual scoring is sometimes used where comets are scored with a number from 0-4 depending on the damage level, where 0 is undamaged. Comets can also be scored with image analysis software with manual or automatic selection of comets69.

The comet assay is mostly used to study DNA damage and repair in total DNA but there has been studies of DNA damage and repair of specific DNA sequences within the comet. Spatial determination of telomeres, centromers and gene sequences have been done with FISH on comets of human lym-phocytes70. FISH has also been used for monitoring repair of specific genes

19

in comets71. Though Comet FISH can give some insight into the repair of specific gene sequences it has some disadvantages. Only quite large frag-ments can be detected by FISH. Therefore it is difficult to study specific genes; it is only possible to study the gene-region. Also there is a risk of unspecific signals when using FISH on comets since the agarose gel is sensi-tive to stringent conditions and high temperatures.

20

Viruses

Even though viruses are seen as a biological group they are genetically very diverse. Their genomes can be either DNA or RNA and either single or dou-ble stranded. Single stranded viruses can exist as double stranded during periods of replication. RNA viruses are called sense (+) if they have the same polarity as the mRNA and antisense (–) if they have the complemen-tary strand to the mRNA.

Viruses have only a few genes in their genomes and can thus not replicate on their own but require help from a host cell. The viruses’ genes code for proteins that enable the virus entering a host cell and for a nucleocapsid pro-tein which is used for building a capsid before the virus leaves the host cell for infecting other cells. In addition RNA viruses have a gene for a poly-merase to be able to express mRNA.

Viruses have been detected in situ using ISH72 and in situ PCR39. Since viruses often are present in multiple copies these techniques have been somewhat successful but the present techniques have limitations when it comes to strand distinction, multiplexing and discrimination between closely related strains. This limits the possibility of studying the replication process and transcription.

21

Present investigation

The following work describes applications of padlock probes and RCA in situ. In the first paper padlock probes and RCA were used to study DNA damage in the comet assay. In the second paper we developed a twelve-gel format for the comet assay which will increase throughput of the comet as-say and in the third paper we studied gene-specific repair in the comet assay using padlock probes and RCA. In the fourth paper padlock probes and RCA were used for detection of an RNA virus and its transcript in cells.

Paper I: Detection of Alu sequences and mtDNA in comets using padlock probes Aim of the study In Paper I we wanted to test if padlock probes could be used for detection of DNA in the comet assay. Earlier FISH has been used for detection of spe-cific DNA sequences in comets but there are several advantages of using padlock probes instead. Padlock probes can be used under gentle, low strin-gent conditions which would help to preserve the comet in the gel. Using padlock probes should also increase the speed of the assay and the specific-ity should be greatly improved. Padlock probes need much shorter recogni-tion sequences than FISH which should make it possible to detect single genes instead of gene regions as with FISH.

It was previously unknown how mtDNA behaves in the comet assay. To study the mtDNA we chose a padlock probe that has previously been used for genotyping of the MELAS mutation in mtDNA56. For detection of chro-mosomal DNA we choose to detect Alu repeats. Alu repeats are present at ~1,000,000 copies in the genome73 and many of them share a 26 bp core sequence74. We used a padlock probe designed to hybridize to this core se-quence in Alu repeats and let them represent the genomic DNA.

Methods We used HeLa cells to prepare DNA representing different stages of the comet assay and Human osteosarcoma cells 143B as positive control for in situ mtDNA genotyping with padlock probes and RCA. Cells were fixed in ethanol before applying padlock probes.

22

In the method for genotyping mtDNA the target strand was prepared for hybridization enzymatically56. Here we took the same approach. After treat-ment with pepsin in HCl, the DNA targets in the comets were made single stranded with a combination of restriction enzymes and exonucleases before hybridization and ligation with padlock probes. MtDNA was cut with MscI just as in the earlier paper and genomic DNA was cut with AluI which is known to cut Alu repeats.

We hybridized padlock probes on fresh cells in agarose, cells in agarose treated with lysis solution, cells in agarose treated with lysis solution and incubated in alkaline electrophoresis solution and cells electrophoresed after treatment with lysis solution and alkaline solution. The same experiments were performed with cells that were treated with H2O2 after embedding in agarose and before lysis.

After hybridization the target strand was used as a primer for RCA with the ligated padlock probes as template. RCPs were detected by hybridization of fluorescently labelled oligonucleotides and were studied with fluores-cence microscopy.

Results We got strong signals from the padlock probe targeting mtDNA in fresh cells. The signals were located around the cell. After one hour incubation in lysis solution fewer signals were seen. They were also more spread in the gel. After alkaline incubation the signals were even more dispersed and after alkaline electrophoresis the association with the comet was totally lost. H2O2 did not affect the distribution pattern. The negative controls where T4 DNA ligase was excluded from the ligation reaction were negative and the 143B cells had strong signals from mtDNA.

Since the diffusion of mtDNA happened already during lysis we wanted to study the dynamics of the diffusion of mtDNA. After embedding 143B cells in low-melting-point agarose we added lysis solution. After adding lysis solution we studied the diffusion of the mtDNA for one hour. We could see diffusion of mtDNA already after 2.5 min and most diffusion after the longest incubation time which was one hour.

We detected Alu repeats in both neutral and alkaline treated comets from H2O2 treated HeLa cells. The Alu signals remained located in both the head and the tail of the comet throughout the procedure. Controls without ligase contained no signals from the Alu specific padlock probe.

Discussion In this paper we showed that DNA can be detected in the comet assay using padlock probes and target primed RCA, after enzymatic target preparation. As expected there were many signals from the Alu padlock probe. The sig-

23

nals were located in the head and tail of the comets and stayed associated with the comet throughout the procedure.

We also showed that after lysis the mtDNA disperses away into the gel, eventually showing no association with the DAPI staining of the comet. The reason for the nuclear DNA to stay associated while the mtDNA diffuses away could be because the DNA in the nucleus is attached to the nuclear matrix while the mtDNA is free in the cytoplasm. The mtDNA is also much shorter than the average nuclear DNA segment and it is also circular and would thus diffuse more easily through the gel. Another reason for mtDNA to diffuse is the fact that mtDNA is located in the cytoplasm and would therefore not be associated with the nucleus even before electrophoresis.

Specific DNA sequences have been detected earlier with FISH but our approach with padlock probes and RCA has several advantages. Padlock probes can be used under gentle, low stringent conditions, they are specific, the assay is faster and it is possible to detect shorter sequences, which means that single genes are a possible target.

Paper II: Twelve-gel slide format optimised for comet assay and fluorescent in situ hybridisation Aim of the study Comet assays are conventionally performed with two 20 mm square gels on a microscope slide. Incubations with chemicals, enzymes or cells extracts are performed under a cover slip or by putting the whole slide in a Coplin jar. This limits the number of samples that can be run at one time since there are limits on how many slides that can be handled at one time point and also how many slides that fit into an electrophoresis tank.

In paper II we wanted to increase throughput of the comet assay by intro-ducing a new twelve- gel approach and comparing it to the conventional comet assay. By keeping the twelve gels in holes of a silicone gasket which is under pressure, the gels will be isolated. This would enable the use of dif-ferent reagents on all twelve gels.

Methods Agarose with suspended HeLa cells or human lymphocytes was dropped on microscope slides and left to dry. For the two-gel slides drops were put un-der cover slips but for the 12-gel slides drops were left un-covered. Some cells were treated with H2O2 or with Ro and visible light to induce 8-oxoGua. Some cells were exposed to Ro plus light and formami-dopyrimidine DNA glycosylase (FPG) which detects 8-oxoGua as well as other oxidised purines75.

24

A silicone gasket with twelve holes was moulded in a custom made mould. During incubations the silicone gasket was pressed onto the micro-scope slide with steel pin in a custom designed chamber.

Incubations with or without colour and increasing and decreasing concen-trations of H2O2 on opposite rows on the slide were performed in holes in the silicone gasket to check for leakage between the holes.

After comet assays we also applied FISH with whole genome DNA la-belled with biotin to some slides. We also performed rolling circle amplifica-tion of a padlock probe targeting Alu repeats in the holes of the silicone gas-ket.

Results With the twelve-gel approach instead of the conventional two-gel approach throughput of the comet assay was increased.

We performed experiments to check for leakage between the wells in the silicone gasket. Experiments with colour showed no leakage into neighbour-ing wells. Neither did the experiment with increasing or decreasing concen-trations of H2O2. Wells treated with no H2O2 showed no signs of damage even though there was high concentration of H2O2 in the next well.

Dose response curves after treatment with H2O2 were identical when using the twelve-gel format compared to the traditional two-gel format. The results after treatment with Ro plus light and FPG showed no difference between the 2 or 12 gel format.

FISH with total genomic DNA gave a strong staining of the whole comet and RCA gave strong signals when performed in the silicone gasket.

Discussion The conventional comet assay is performed with two agarose gels on a mi-croscope slide. This is a limiting factor for the throughput of the assay. In this paper twelve gels were put on the same slide in the comet assay and reactions were carried out in the wells of a silicone gasket. The twelve gel approach showed no signs of leakage and performed identical to the 2-gel format in dose response assays but increases throughput and reduces the number of cells and the reagents needed for an experiment. It also makes it possible to treat all twelve gels with different reagents since all gels are physically separated and there is no leakage between the holes in the silicone gasket.

RCA on comet assays were previously performed in a circle by a PAPpen (Dako). There was then a risk of leakage and evaporation which is prevented when incubating in the silicone gasket. There is also a defined area for the incubation making reaction conditions more consistent.

25

Paper III: Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification Aim of the study In paper III we wanted to study certain DNA repair genes in the comet assay. Certain transcribed sequences have been shown to have specific repair early after DNA damage76, 77. Repair genes have been hypothesized to be repaired early after DNA damage. If this is the case, after leaving cells to repair after DNA damage, the repair genes should be seen in the head of the comet in-stead of the tail to a higher degree than total DNA compared to cells with no or shorter time to repair. The genes we have chosen for this project are 8-oxoguanine-DNA glycosylase-1 (OGG1), the xeroderma pigmentosum group D (XPD), and the hypoxanthine-guanine phosphoribosyltransferase (HPRT). OGG1 and XPD are involved in DNA repair78, 79 and HPRT is a housekeeping gene80. We compared the repair of the three specific genes both with total DNA repair and with repair of Alu repeats.

Earlier specific DNA sequences have been studied in situ with comet FISH70. An advantage of using padlock probes compared to FISH for detec-tion of the genes is that much smaller regions can be detected and also the agarose gel does not have to be exposed to as high salt concentrations as in FISH.

Methods We designed six padlock probes against each of these three genes in order to increase the sensitivity of the assay. All six padlock probes targeting one gene have one common tag sequence which makes it possible to detect all six padlock probes simultaneously, but they also have individual tag se-quences which should make it possible to see where in the comet the differ-ent parts of the same gene are located.

Human lymphocytes were prepared from blood samples and the cells were embedded in agarose. Slides with cells were exposed to hydrogen per-oxide and then left to repair in medium for up to 55 minutes before electro-phoresis.

Padlock probes targeting one of the three genes and a padlock probe tar-geting Alu repeats were hybridized to the DNA in the comet. After ligation, RCA and hybridization of detection oligonucleotides we counted the number of signals from the gene specific padlock probes in 50 comets. In the comets with gene specific signals we also counted the number of signals from the Alu targeting padlock probe using the Blobfinder software57.

26

Results We tested our padlocks on comets and discovered that we got too many sig-nals from the XPD padlock probes. After investigating the probes we could exclude one of the probes that seemed to target a repetitive sequence. After this most signals disappeared but there were still too many. We concluded that is was necessary to use a thermo stable ligase to improve specificity. For all the following experiments T4 DNA ligase was replaced with Ampligase.

To be able to hybridize padlock probes the target DNA has to be single stranded and to do RCA the target sequence has to have a nearby 3’-end. It is not known to which extent the DNA in the comet tail is single- or double stranded. Furthermore, the absolute number of breaks in the DNA is not known. So to provide some insights on the nature of the DNA in the comet tails and to optimize the detection reaction we performed a number of ex-periments to probe the structure of the comet tail DNA. Therefore we tried two approaches for making sure DNA was single stranded and had a nearby break. We tried restriction digestion and exonucleasis but this causes loss of RCPs. We also tried site specific cleavage with MutY and EndoIV but this did not improve the assay compared to no cleavage. Therefore there was no cutting performed in the repair experiments.

To study DNA repair we treated cells with H2O2. Cells were left to repair for up to 55 minutes before electrophoresis. XPD, OGG1 or HPRT were then detected with padlock probes together with a padlock probe detecting Alu repeats. Total DNA damage was also measured with SYBR Gold. After two minutes repair there were more RCP from all genes as well as Alu re-peats. As comets were left to repair longer there were fewer signals from the padlock probes. The three genes had different rate of repair. HPRT de-creased fastest and OGG1 decreased slowest. All genes and Alu repeats re-paired fast comparing to total genomic DNA. Another thing that was noted was that the number of signals after damage was highest from HPRT and lowest from OGG1.

Discussion Single-copy genes have previously not been detected in situ with padlock probes and RCA. The reason for this is probably because the nuclear matrix is tightly packed and pre-treatments that make it possible to access the nu-clear DNA in cells and tissue causes DNA loss. In the comet assay the genes are available in the gel and at the same time attached to the nuclear matrix preventing DNA loss. That makes it possible to detect single genes within comet preparations.

In the previous two papers we detected Alu repeats and mtDNA using T4 DNA ligase which is an efficient enzyme which has the advantage of having optimal temperature at 37˚C. This is preferable since there will be little ef-

27

fect on the morphology of the investigated cell or tissue. Though this has been satisfactory when detecting more abundant sequences, when detecting single copy genes there are higher demands on specificity than when geno-typing mtDNA or studying repeated sequences. A single unspecific signal per cell will give misleading results. We therefore replaced T4 DNA ligase with Ampligase, which has a higher optimal temperature which increases hybridization specificity. The gel appeared unaffected even though ligation was performed at 55˚C.

The nature of the DNA in the comet tail is to some extent unknown. Therefore different approaches of cutting and making DNA single stranded were tested. Cutting DNA in the comet assay did not increase the number of RCPs. This could be because the DNA in the tail of the comet assay already is single stranded and has many breaks. When cutting DNA with restriction enzymes the signals were lost. This could be due to the double stranded DNA in the head is cut. The DNA in the tail would then loose attachment to the nucleus which could cause the DNA to diffuse out in the gel.

There are three forces that put a gene in the tail of a comet or in the head, accessibility which is associated with transcription, damage of the gene and repair of the gene. Since HPRT is a house-keeping gene it is probably tran-scribed before damage. It would therefore be available and quickly give RCP in the tail of the comet. OGG1 on the other hand is probably not transcribed until the cell is left to repair. It would therefore give fewer RCPs. The three genes repaired at different rates but all three repaired faster than total ge-nomic DNA. Alu repeats were also repaired quicker than total genomic DNA. The density of Alu repeats is strongly associated with gene density81. This could indicate that genes in general are repaired early after damage.

The RCPs from all genes increase in number after two minutes repair compared to no repair. This could be because of increased single strand breaks during repair since single strand breaks is an intermediate step in base excision repair82. The alkaline electrophoresis could also give strand breaks if a base is missing during DNA repair.

The majority of the signals were seen in the tail of the comet. The head is more tightly packed and thus less available and also DNA in the head is to a greater extent double stranded. When excluding cutting of the DNA we would not expect to have signals in the head but only in the tail. Counting the total number of signals will therefore give an indication of the level of damage whereas earlier studies of DNA repair of specific genes have made comparisons between DNA in the head and in the tail.

Our approach makes it possible to study damage and repair of specific genes and even parts of genes within comet preparations. This has not been possible before since studies with FISH require long target sequences and the result will be detection of a gene region rather than the gene.

28

Paper IV: Simultaneous detection of Crimean Congo Hemorrhagic fever virus viral RNA and complementary RNA in situ by padlock probes and rolling circle amplification combined with viral protein visualization Aim of the study The Crimean Congo hemorrhagic fever virus (CCHFV) belongs to the genus Nairovirus and the family Bunyaviridae. CCHFV causes fever, prostration and severe hemorrhages in humans with a high mortality rate but infected animals are usually asymptomatic83. CCHFV is tick borne and mainly trans-mits through bites from Hyalomma ticks84. The distribution of the virus fol-lows the distribution of the Hyalomma ticks in Asia, Africa and southeast Europe85.

CCHFV is a negative strand single stranded RNA virus which consists of three RNA segments that are called small (S), medium (M) and large (L). The S segment codes for the nucleocapsid protein (NP), M codes for two glycoproteins, Gn and Gc, and L codes for the RNA polymerase. CCHFV enters host cells through endocytosis and replicates in the cytoplasm fol-lowed by assembly in the Golgi84. Little is known about this virus, mainly because the study of it has been limited since it is dangerous to work with, requiring laboratories with bio safety level 4. It has also been difficult to generate an animal model although a mouse model was described recently86. Therefore, in this project we aimed to develop a method to study this virus and single stranded RNA viruses in general. Our approach was to reverse transcribe virus RNA (vRNA) and complementary RNA (cRNA) into cDNA and detect it with padlock probes and target primed RCA. We also wanted to see intra-cellular localization of vRNA and cRNA and the NP which can be detected with immunostaining.

Methods We designed padlock probes for target sequences in the S segment of the vRNA and cRNA. Vero cells were infected with CCHFV and left for six or 24 hours. After fixation of the cells the vRNA and cRNA were reverse tran-scribed into cDNA using LNA primers. After degradation of the RNA with RNaseH the padlock probes were hybridized to the cDNA and ligated. The target molecule was then cleaved site specifically with MutY and EndoIV to produce a starting point for RCA. The padlock probes were then used as templates for RCA. The RCPs were detected by hybridization of fluores-cently labelled oligonucleotides.

We compared cells after six or 24 hours post infection with uninfected controls. Technical controls such as ligation without ligase, reverse tran-

29

scription without primer or without reverse transcriptase were also included to study the specificity of the assay. The infection of CCHFV was confirmed by immunostaining for the NP.

Experiments were performed in a bio safety level 4 laboratory and slides were required to be fixed in formamide for 48 hours before being taken out from the laboratory. The slides were studied with fluorescence microscopy and image analysis was performed with the Blobfinder software57.

Results We reverse transcribed vRNA into cDNA and detected the cDNA with pad-lock probes and RCA. To make sure the assay was specific we added a mock infected control. We also added technical controls for the vRNA such as no ligase in the ligation reaction and no reverse transcriptase or no primer in the reverse transcription reaction. The percentage of cells with RCPs from the vRNA was counted with the Blobfinder software. We were able to detect the vRNA in the infected cells. The mock infected cells and the controls without ligase or reverse transcriptase had few positive cells but the control without primer in the reverse transcriptase step had more positive cells.

We then compared the percentage of cells with vRNA or cRNA signals in cells with immunostaining for the NP after different times after infection. The percentage of positive cells was higher 24 hours post infection for both vRNA and cRNA compared to 6 hours post fixation. Both cells where the NP was detected and cells where the NP couldn’t be detected had signals from the vRNA and the mRNA. Cells with NP staining had a higher per-centage of cells with RCPs than cells without NP staining. The vRNA showed no intra-cellular co-localization with the NP. The cRNA on the other hand showed some co-localization with the NP. When counting the number of RCPs in individual cells we could see that cells with immunostaining for the NP had more signals from both vRNA and cRNA than cells without staining from the NP.

vRNA and cRNA were also detected simultaneously with padlock probes and RCA. The number of signals from vRNA and cRNA were not as high when detected simultaneously as they were when detected separately. The signals from the vRNA and cRNA showed no co-localization.

Simultaneous detection of vRNA and cRNA was also made together with staining for the NP. Here it was confirmed that cRNA and NP co-localizes in certain areas of the cytoplasm whereas the vRNA is more spread throughout the cytoplasm.

30

Discussion We were able to detect vRNA in infected cells. To show that our method is specific we performed a number of controls. The mock infected slide had very few cells containing signals. This shows that the padlock probes are specific, not giving signals from other nucleic acids present in the cells. Omitting ligase from the ligation reaction also resulted in very few signals. Since unligated padlock probes cannot be amplified with RCA, the visible signals should be a result of auto fluorescence from the cells or slides. When excluding reverse transcriptase in the reverse transcriptase step we expected no signals. Signals would mean that padlock probes were ligating against RNA as well as cDNA and the method would thus not be strand specific. Without the reverse transcriptase there were as expected very few signals which means that with this method we can distinguish between the two complementary strands. In the control where the primer was omitted from the reverse transcriptase there were some signals. There has however been a report about self-priming by reverse transcriptase87.

vRNA and cRNA were detected in cells six and 24 hours post infection. The percentage of infected cells were higher in the 24 hours slide which could be because there is an ongoing infection where there are more virus per cell, which results in that we are able to detect cells with viruses to a higher degree with the method, but also because there has been a second round of infection. There were signals both in cells with immunostaining and in cells without immunostaining. Cells without immunostaining could be at an earlier stage of infection where the NP is not expressed yet. When count-ing the number of signals per cell it was clear that cells with immunostaining for NP have more signals from the padlock probes.

Here we developed a method for detection of both strands of CCHFV, but this method can be used for single stranded RNA viruses in general. With this method we were able to distinguish between the two opposite strands of RNA which is otherwise difficult.

By using padlock probes for detection of single stranded RNA viruses in situ we can detect single virus molecules. This should make it possible to see clear signals even early after infection. Using ligases for detection it should also be possible to distinguish between closely related strains.

LNA primers made the reverse transcription more efficient but also re-sults in a localized detection since the part of the RNA that is hybridized to the LNA primer is not degraded by RNaseH88 and the RCPs are therefore still linked to the target RNA.

This whole project was performed in a P4 lab and slides had to be fixed for 48 hours before microscopy. The RCPs survived 48 hours in formamide without loosing fluorescence or structure. Also, all reagents except enzymes had to be mixed before entering the P4 lab. This shows that this is a robust technique.

31

Future perspectives

Padlock probes and RCA has a potential to be used for a number of applica-tions. Earlier it has been used for genotyping of mtDNA and mRNA and detection of bacteria. Here we have used if for detection of genomic and mtDNA in the comet assay as well as an RNA virus and its cRNA in cul-tured cells.

In this thesis, detection of a single stranded RNA virus is presented but padlock probes and RCA can also be used for DNA viruses. We have used it for detection of Porcine circovirus type 2 (PCV2) in cells and tissues.

Detection of PCV2 in situ PCV2 is a single stranded circular DNA virus89, 90 but it is double stranded in its replicative form. It has been discussed if it exists in both polarities. The replication is unknown but it is thought to replicate with rolling circle repli-cation91. Two padlock probes targeting both strands of the virus genome were designed so that they were in a conserved region and thus would target 21 known Swedish isolates. The two probes target regions close to each other but not overlapping sequences since this would cause hybridization between the two probes. We studied these viruses in tissue and cells at dif-ferent stages after infection. During the course of infection the number of viruses increased.

The PCV2 genome predominantly exists as single stranded but in the rep-licative form it can be double stranded. Therefore the DNA was first made single stranded with restriction digestion and exonucleasis and to make sure that there was an end for RCA DNA was also site specifically cleaved with MutY and EndoIV by introducing an AG mismatch in the padlock probe.

The padlock probes were applied to experimentally infected tissue, natu-rally infected tissue and uninfected tissue as well as cultured cells that had been infected for different time. PCV2 viruses were found in cells and tis-sues. There was a domination of the virus strand over the replicative strand. Viruses appeared in the cytoplasm of the cells and in high numbers in certain nuclei. Around these nuclei the replicative strand could often be found indi-cating that the virus replicates in these cells. The number of virus also in-creased over time which was confirmed with PCR. We also detected PCV2 in tissue sections from lymph nodes from pig, but the method could be used

32

to study infection of DNA viruses in cells and tissues in general. Even though most of the PCV2 viruses were found in the cytoplasm there seem to be a replication centre in the nucleus of a few cells where there are lots of PCV2 viruses and also the replicative strand is visible. This is in agreement with immunostaining of the nucleocapsid protein which is visible in certain cell nuclei. Padlock probes and RCA were also applied on cells which were stained with Mitotracker92. There has been a discussion whether PCV2 repli-cates or is localised in the mitochondrion93. We could however not see any indication of co-localization between the mitochondria and either the ge-nomic strand or the replicative strand.

Figure 3.Detection of PCV2 with padlock probes and rolling circle amplification in a) experimentally infected tissue, b) naturally infected tissue and c) uninfected tis-sue. Rolling circle products detecting the genomic strand is seen as green dots and the replicative strand are seen as red dots. Nuclei are counter stained with DAPI.

Conclusions Padlock probes and RCA have several advantages, such as high specificity due to the requirement of ligation to produce a signal and high signal to noise because of the signal amplification with RCA. Padlock probes and RCA have other advantages such as strand specificity, no need for expensive apparatuses, relatively quick, low labour and a possibility to multiplex. Here a maximum of two targets were used at a time but there is a possibility to use more target sequences. The limitation for multiplexing lies in the number of fluorophores that can be spectrally separated. Using combinations of fluoro-phores will increase the number of possible targets. This should make them useful in a range of applications such as loss-of heterozygosity, mono-allelic expression, copy number variations and distinction between closely related species in viral or bacterial infections.

Efforts have been made to detect single copy genes in cultured cells and tissues. However, detection of single copy genes requires a very low back-ground and also it is difficult reaching a balance between fixation and per-meabilisation making DNA available without causing DNA loss. Detection

33

of single copy nuclear targets is therefore difficult. Our efforts have resulted in detection efficiencies of ~5% but for most applications higher detection efficiencies are necessary.

Padlock probes and RCA are successfully used for fresh frozen tissue. Formalin fixed paraffin embedded tissues have proven more difficult. Exist-ing bio banks contain mostly formalin fixed paraffin embedded tissue. It will be a challenge adapting padlock probes and RCA for these tissues.

The work presented in this thesis will enable study of gene specific repair within comet preparations and make it possible to study viral infections in situ. In the comet assay the short target sequence of the padlock probes and the high signal-to-noise of RCA make it possible to detect specific genes instead of as earlier gene regions. When using padlock probes and RCA for detection of viruses it is possible to discriminate even between closely re-lated species. Another advantage when detecting viruses is that strand spe-cific detection is possible which has otherwise been difficult.

34

Acknowledgements

The work presented in this thesis was performed at the Department of genet-ics and pathology at Uppsala University. I would like to thank the people who contributed. First of all I would like to thank my supervisor Mats Nils-son for giving me this opportunity and for always being positive and enthu-siastic. I would also like to thank my co-supervisor Ulf Landegren for creat-ing such an open and creative scientific environment. Many thanks as well to my fellow in situ padlockers: Chatarina Larsson for taking care of me when I was a new student, Ida Grundberg for colour coding in the lab, helping me practise for my oral presentation in Cyprus and for GR8 times in New York, as well as our newer co-workers Marco Mignardi and Rongqin Ke. Thanks also to Lore for being a great inspiration and for being able to discuss practi-cal in situ issues.

Many thanks to my collaborators Sergey Shaposhnikov and Andrew Collins at Oslo University as well as the rest of the COMICS people. You are a happy life-loving bunch. Thanks also to my collaborators at SMI Ce-cilia Andersson and Ali Mirazimi and my collaborators at SVA Mikael Berg and Anne-Lie Blomström. Special thanks to Cecilia for writing manuscript under pressure.

I would also like to thank Carolina Wählby for help with image analysis, Lena Lindbom for helping me with experiments and for organizing ”girls-nights”. Thanks also to Paco for showing up at the microscope in just five minutes every time I need help and to Johan Oelrich for giving CPR to my computer time and time again.

Thanks to my project students I have had over the years: Johannes Bergström, Johan Heldin and Christina Classon. Special thanks to Christina for all her work on the nuclear DNA detection, for baking cookies and for still going out buying lunch with me every day.

My office room mates have been many over the years and I would like to thank Anders Alderborn for always being in a cheerful mood keeping a nice atmosphere in the office. I survived a lot of Monday mornings thanks to you. Thanks also to Agata Zieba for funny and intense chats in the office, Maria Hammond for importing goodies to me from the US and the rest of my office room mates Kerstin Henriksson, Mats Gullberg, Sigrun Gustafsdottir, Carolina Rydin, Elin Ekberg, Erik, Tony, you have all been great office neighbours. Many thanks to my former office room mate Katerina Pardali for showing me the real Greece and for great office gossip as well as serious talks.

35

The rest of the people in the lab: Irene, thanks for making excellent ice-cream and for making me run “Tjejmilen” I would never have done that if it wasn’t for you, Carla for sharing my interests whether it comes to yoga, salsa or skiing, Ola for teaching me the necessities in life: how to crawl and what a padlock probe is, Rachel for being my lab-bench neighbour, for fun party nights and for short exercise sessions followed by long sessions in the sauna. Tim, my partner in crime, thanks for refilling my paper tissues and for being a reliable teaching partner. Kalle thanks for great company in the lab for many years and for having the answer to almost any question, Gucci for being sweet and energetic and always making me run an extra ten minutes, Jenny for sharing mingling experiences and late night talks in Helsinki, Hen-rik for entertainment in the lab and for showing me Zotero, Magnus for checking the surveillance cameras, Masood for practical jokes, Spyros for sorting out all my troubles with ELN, Yuki for making excellent sushi at group gatherings, Carl-Magnus for re-filling the chocolate supply, Ulla for sorting out all administrative issues, David for fresh input in discussions, Jonas and Olle for good comments during group meetings. Thanks to Mi-kaela, Johan Vänelid, Elin Falk, and Malin for nice talks during lunches and coffee breaks and to the new people Ling, Andries, Lotte, Wu Di, Anne-Lie, Pier thanks for contributing to making such a nice place to work.

There are people outside that lab that I would like to thank as well. Marie thanks for taking me through my undergraduate years in Uppsala, for “light lunches” and for being my party planner, Marie and Karin thanks for being amazing colleagues during my time at BMC and for encouraging me to do a PhD. Ida E thanks for exercising me in the forest, even though I slow you down.

Most of all I would like to thank my family. Mamma and Pappa thanks for always supporting whatever I do, Lena thanks for always being a nice older sister dragging me along and for entertaining me in Uppsala. Lisa thanks for showing me fun things and updating me on “daily” matters. Mat-tias my brother in law thanks for telling me the harsh truth and for introduc-ing me to doodle jump. Ida and Rasmus thanks for always showing me something new you can to do.

Finally my Mats at home… Thank you for not being a scientist, for show-ing me “things” and for our little family.

36

References

1. Frazer, K.A. et al. A second generation human haplotype map of over 3.1 mil-lion SNPs. Nature 449, 851-861 (2007).

2. Sebat, J. et al. Large-scale copy number polymorphism in the human genome. Science 305, 525-528 (2004).

3. Venter, J.C. et al. The sequence of the human genome. Science 291, 1304-1351 (2001).

4. Pruitt, K.D., Tatusova, T. & Maglott, D.R. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35, D61-D65 (2007).

5. Gall, J.G. & Pardue, M.L. Formation and detection of RNA-DNA hybrid mole-cules in cytological preparations. Proc. Natl. Acad. Sci. U.S.A 63, 378-383 (1969).

6. Bauman, J.G., Wiegant, J., Borst, P. & van Duijn, P. A new method for fluores-cence microscopical localization of specific DNA sequences by in situ hybridi-zation of fluorochromelabelled RNA. Exp. Cell Res 128, 485-490 (1980).

7. Hopman, A.H., Wiegant, J., Tesser, G.I. & Van Duijn, P. A non-radioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhy-dryl-hapten ligands. Nucleic Acids Res 14, 6471-6488 (1986).

8. Lichter, P., Cremer, T., Borden, J., Manuelidis, L. & Ward, D.C. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum. Genet 80, 224-234 (1988).

9. Schröck, E. et al. Multicolor spectral karyotyping of human chromosomes. Sci-ence 273, 494-497 (1996).

10. Kokalj-Vokac, N., Alemeida, A., Gerbault-Seureau, M., Malfoy, B. & Dutril-laux, B. Two-color FISH characterization of i(1q) and der(1;16) in human breast cancer cells. Genes Chromosomes Cancer 7, 8-14 (1993).

11. Tagarro, I., Fernández-Peralta, A.M. & González-Aguilera, J.J. Chromosomal localization of human satellites 2 and 3 by a FISH method using oligonucleo-tides as probes. Hum. Genet 93, 383-388 (1994).

12. Bayani, J. & Squire, J.A. Fluorescence in situ Hybridization (FISH). Curr Pro-toc Cell Biol Chapter 22, Unit 22.4 (2004).

13. Kelly, R.B., Cozzarelli, N.R., Deutscher, M.P., Lehman, I.R. & Kornberg, A. Enzymatic synthesis of deoxyribonucleic acid. XXXII. Replication of duplex deoxyribonucleic acid by polymerase at a single strand break. J. Biol. Chem 245, 39-45 (1970).

14. Gebeyehu, G., Rao, P.Y., SooChan, P., Simms, D.A. & Klevan, L. Novel bioti-nylated nucleotide--analogs for labeling and colorimetric detection of DNA. Nucleic Acids Res 15, 4513-4534 (1987).

15. Feinberg, A.P. & Vogelstein, B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal. Biochem 137, 266-267 (1984).

37

16. Landegent, J.E., Jansen in de Wal, N., Dirks, R.W., Baao, F. & van der Ploeg, M. Use of whole cosmid cloned genomic sequences for chromosomal localiza-tion by non-radioactive in situ hybridization. Hum. Genet 77, 366-370 (1987).

17. Arvey, A. et al. Minimizing off-target signals in RNA fluorescent in situ hy-bridization. Nucleic Acids Res 38, e115 (2010).

18. Sugimoto, N. et al. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11211-11216 (1995).

19. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585-590 (1998).

20. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877-879 (2008).

21. Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. & Litt, G.J. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoas-says. J. Immunol. Methods 125, 279-285 (1989).

22. Kerstens, H.M., Poddighe, P.J. & Hanselaar, A.G. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem 43, 347-352 (1995).

23. Raap, A.K. et al. Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum. Mol. Genet 4, 529-534 (1995).

24. Cao W, Connolly J, Zagala M, Beard C, Hirsch A, Ku L, Kolberg J, Teramoto Y A sensitive, rapid and non-isotopic in situ bDNA assay for detection of hnRNP A2 mRNA. Proc Am Assoc Cancer Res 89th Annu Meet (1998).

25. Antao VP, Player AN, Kolberg JA In situ hybridization using the bDNA tech-nology. In Patterson BK, ed. Techniques in Quantification and Localization of Gene Expression. Boston, Birkhauser Press 81-83 (2000).

26. Player, A.N., Shen, L., Kenny, D., Antao, V.P. & Kolberg, J.A. Single-copy Gene Detection Using Branched DNA (bDNA) In Situ Hybridization. J. Histo-chem. Cytochem. 49, 603-612 (2001).

27. Jensen, K.K., Orum, H., Nielsen, P.E. & Norden, B. Kinetics for Hybridization of Peptide Nucleic Acids (PNA) with DNA and RNA Studied with the BIAcore Technique†. Biochemistry 36, 5072-5077 (1997).

28. Nielsen, P.E., Egholm, M., Berg, R.H. & Buchardt, O. Sequence-selective rec-ognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500 (1991).

29. Orum, H. et al. Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection. BioTechniques 19, 472-480 (1995).

30. Lomakin, A. & Frank-Kamenetskii, M.D. A theoretical analysis of specificity of nucleic acid interactions with oligonucleotides and peptide nucleic acids (PNAs). J. Mol. Biol 276, 57-70 (1998).

31. Lansdorp, P.M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet 5, 685-691 (1996).

32. Chen, C., Hong, Y.K., Ontiveros, S.D., Egholm, M. & Strauss, W.M. Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH. Mamm. Genome 10, 13-18 (1999).

33. Koshkin, A.A. et al. LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetra-hedron 54, 3607-3630 (1998).

34. Singh, S.K., Koshkin, A.A., Wengel, J. & Nielsen, P. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem. Commun. 455-456 (1998).

38

35. Silahtaroglu, A., Pfundheller, H., Koshkin, A., Tommerup, N. & Kauppinen, S. LNA-modified oligonucleotides are highly efficient as FISH probes. Cytogenet. Genome Res 107, 32-37 (2004).

36. Darnell, D.K., Stanislaw, S., Kaur, S. & Antin, P.B. Whole mount in situ hy-bridization detection of mRNAs using short LNA containing DNA oligonucleo-tide probes. RNA 16, 632-637 (2010).

37. Mullis, K. et al. Specific enzymatic amplification of DNA in vitro: the poly-merase chain reaction. 1986. Biotechnology 24, 17-27 (1992).

38. Lawyer, F.C. et al. High-level expression, purification, and enzymatic charac-terization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity. PCR Methods Appl 2, 275-287 (1993).

39. Haase, A.T., Retzel, E.F. & Staskus, K.A. Amplification and detection of lenti-viral DNA inside cells. Proc. Natl. Acad. Sci. U.S.A 87, 4971-4975 (1990).

40. Nuovo, G.J., Gorgone, G.A., MacConnell, P., Margiotta, M. & Gorevic, P.D. In situ localization of PCR-amplified human and viral cDNAs. PCR Methods Appl 2, 117-123 (1992).

41. Pulimood, A.B., Peter, S., Rook, G.W.A. & Donoghue, H.D. In situ PCR for Mycobacterium tuberculosis in endoscopic mucosal biopsy specimens of intes-tinal tuberculosis and Crohn disease. Am. J. Clin. Pathol 129, 846-851 (2008).

42. Hishikawa, Y., An, S., Yamamoto-Fukuda, T., Shibata, Y. & Koji, T. Improve-ment of in situ PCR by optimization of PCR cycle number and proteinase k concentration: localization of x chromosome-linked phosphoglycerate kinase-1 gene in mouse reproductive organs. Acta Histochem Cytochem 42, 15-21 (2009).

43. Nuovo, G.J. In situ detection of precursor and mature microRNAs in paraffin embedded, formalin fixed tissues and cell preparations. Methods 44, 39-46 (2008).

44. Koch, J.E., Kølvraa, S., Petersen, K.B., Gregersen, N. & Bolund, L. Oligonu-cleotide-priming methods for the chromosome-specific labelling of alpha satel-lite DNA in situ. Chromosoma 98, 259-265 (1989).

45. Koch, J., Hindkjaer, J., Kølvraa, S. & Bolund, L. Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cytogenet. Cell Genet 71, 142-147 (1995).

46. Pellestor, F., Girardet, A., Lefort, G., Andréo, B. & Charlieu, J.P. Selection of chromosome-specific primers and their use in simple and double PRINS tech-niques for rapid in situ identification of human chromosomes. Cytogenet. Cell Genet 70, 138-142 (1995).

47. Hindkjaer, J. et al. Fast, sensitive multicolor detection of nucleic acids in situ by PRimed IN Situ labeling (PRINS). Cytogenet. Cell Genet 66, 152-154 (1994).

48. Landegren, U., Kaiser, R., Sanders, J. & Hood, L. A ligase-mediated gene de-tection technique. Science 241, 1077-1080 (1988).

49. Nilsson, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085-2088 (1994).

50. Nilsson, M. et al. Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21. Nat. Genet 16, 252-255 (1997).

51. Fire, A. & Xu, S.Q. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. U.S.A 92, 4641-4645 (1995).

52. Lizardi, P.M. et al. Mutation detection and single-molecule counting using iso-thermal rolling-circle amplification. Nat. Genet 19, 225-232 (1998).

39

53. Banér, J., Nilsson, M., Mendel-Hartvig, M. & Landegren, U. Signal amplifica-tion of padlock probes by rolling circle replication. Nucleic Acids Res 26, 5073-5078 (1998).

54. Blanco, L. & Salas, M. Characterization of a 3'----5' exonuclease activity in the phage phi 29-encoded DNA polymerase. Nucleic Acids Res 13, 1239-1249 (1985).

55. Howell, W.M., Grundberg, I., Faryna, M., Landegren, U. & Nilsson, M. Glyco-sylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets. Nucleic Acids Res (2010).doi:10.1093/nar/gkp1238

56. Larsson, C. et al. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat. Methods 1, 227-232 (2004).

57. Allalou, A. & Wählby, C. BlobFinder, a tool for fluorescence microscopy image cytometry. Comput Methods Programs Biomed 94, 58-65 (2009).

58. Wamsley, H.L. & Barbet, A.F. In situ detection of Anaplasma spp. by DNA target-primed rolling-circle amplification of a padlock probe and intracellular colocalization with immunofluorescently labeled host cell von Willebrand fac-tor. J. Clin. Microbiol 46, 2314-2319 (2008).

59. Lohmann, J.S., Stougaard, M. & Koch, J. Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis. BMC Mol. Biol 8, 103 (2007).

60. Nilsson, M., Barbany, G., Antson, D.O., Gertow, K. & Landegren, U. Enhanced detection and distinction of RNA by enzymatic probe ligation. Nat. Biotechnol 18, 791-793 (2000).

61. Nilsson, M., Antson, D.O., Barbany, G. & Landegren, U. RNA-templated DNA ligation for transcript analysis. Nucleic Acids Res 29, 578-581 (2001).

62. Lagunavicius, A. et al. Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA. RNA 15, 765-771 (2009).

63. Stougaard, M., Lohmann, J.S., Zajac, M., Hamilton-Dutoit, S. & Koch, J. In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS. BMC Biotechnol 7, 69 (2007).

64. Larsson, C., Grundberg, I., Söderberg, O. & Nilsson, M. In situ detection and genotyping of individual mRNA molecules. Nat Methods (2010).doi:10.1038/nmeth.1448

65. Ostling, O. & Johanson, K.J. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun 123, 291-298 (1984).

66. Collins, A.R., Dobson, V.L., Dusinská, M., Kennedy, G. & Stĕtina, R. The comet assay: what can it really tell us? Mutat. Res 375, 183-193 (1997).

67. Gedik, C.M., Ewen, S.W. & Collins, A.R. Single-cell gel electrophoresis ap-plied to the analysis of UV-C damage and its repair in human cells. Int. J. Ra-diat. Biol 62, 313-320 (1992).

68. Collins, A.R., Duthie, S.J. & Dobson, V.L. Direct enzymic detection of endoge-nous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733-1735 (1993).

69. Collins, A.R. et al. The comet assay: topical issues. Mutagenesis 23, 143-151 (2008).

70. Santos, S.J., Singh, N.P. & Natarajan, A.T. Fluorescence in situ hybridization with comets. Exp. Cell Res 232, 407-411 (1997).

71. Horváthová, E., Dusinská, M., Shaposhnikov, S. & Collins, A.R. DNA damage and repair measured in different genomic regions using the comet assay with fluorescent in situ hybridization. Mutagenesis 19, 269-276 (2004).

40

72. Brahic, M. & Haase, A.T. Detection of viral sequences of low reiteration fre-quency by in situ hybridization. Proc. Natl. Acad. Sci. U.S.A 75, 6125-6129 (1978).

73. Lander, E.S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860-921 (2001).

74. Rüdiger, N.S., Gregersen, N. & Kielland-Brandt, M.C. One short well con-served region of Alu-sequences is involved in human gene rearrangements and has homology with prokaryotic chi. Nucleic Acids Res 23, 256-260 (1995).

75. Dusinská M., C.A. Detection of oxidised purines and UV-induced photopro-ducts in DNA of single cells, by inclusion of lesion-specific enzymes in the comet assay. Altern. Lab Anim. 405–411 (1996).

76. Bohr, V.A., Smith, C.A., Okumoto, D.S. & Hanawalt, P.C. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40, 359-369 (1985).

77. Mellon, I., Bohr, V.A., Smith, C.A. & Hanawalt, P.C. Preferential DNA repair of an active gene in human cells. Proc. Natl. Acad. Sci. U.S.A 83, 8878-8882 (1986).

78. Lu, R., Nash, H.M. & Verdine, G.L. A mammalian DNA repair enzyme that excises oxidatively damaged guanines maps to a locus frequently lost in lung cancer. Curr. Biol 7, 397-407 (1997).

79. Flejter, W.L., McDaniel, L.D., Askari, M., Friedberg, E.C. & Schultz, R.A. Characterization of a complex chromosomal rearrangement maps the locus for in vitro complementation of xeroderma pigmentosum group D to human chro-mosome band 19q13. Genes Chromosomes Cancer 5, 335-342 (1992).

80. Pernas-Alonso, R., Morelli, F., di Porzio, U. & Perrone-Capano, C. Multiplex semi-quantitative reverse transcriptase-polymerase chain reaction of low abun-dance neuronal mRNAs. Brain Res. Brain Res. Protoc 4, 395-406 (1999).

81. Grover, D., Mukerji, M., Bhatnagar, P., Kannan, K. & Brahmachari, S.K. Alu repeat analysis in the complete human genome: trends and variations with re-spect to genomic composition. Bioinformatics 20, 813-817 (2004).

82. Lindahl, T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair. Prog. Nucleic Acid Res. Mol. Biol 22, 135-192 (1979).

83. Ergönül, O. Crimean-Congo haemorrhagic fever. Lancet Infect Dis 6, 203-214 (2006).

84. Whitehouse, C.A. Crimean-Congo hemorrhagic fever. Antiviral Res 64, 145-160 (2004).

85. Hoogstraal, H. The epidemiology of tick-borne Crimean-Congo hemorrhagic fever in Asia, Europe, and Africa. J. Med. Entomol 15, 307-417 (1979).

86. Bereczky, S. et al. Crimean-Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice. J. Gen. Virol 91, 1473-1477 (2010).

87. Ståhlberg, A., Håkansson, J., Xian, X., Semb, H. & Kubista, M. Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem 50, 509-515 (2004).

88. Kurreck, J., Wyszko, E., Gillen, C. & Erdmann, V.A. Design of antisense oli-gonucleotides stabilized by locked nucleic acids. Nucleic Acids Res 30, 1911-1918 (2002).

89. Allan, G.M. et al. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J. Vet. Diagn. Invest 10, 3-10 (1998).

90. Meehan, B.M. et al. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J. Gen. Virol 79 ( Pt 9), 2171-2179 (1998).

41

91. Faurez, F., Dory, D., Grasland, B. & Jestin, A. Replication of porcine circovi-ruses. Virol. J 6, 60 (2009).

92. Poot, M. et al. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem 44, 1363-1372 (1996).

93. Rodríguez-Cariño, C., Sánchez-Chardi, A. & Segalés, J. Subcellular immunolo-calization of porcine circovirus type 2 (PCV2) in lymph nodes from pigs with post-weaning multisystemic wasting syndrome (PMWS). J. Comp. Pathol 142, 291-299 (2010).

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 575

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

Distribution: publications.uu.seurn:nbn:se:uu:diva-128446

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2010

application of padlock probe based nucleic acid analysis in situ331818/fulltext01.pdf ·...

Documents