dc.contributor.author
Borodina, Tatiana
dc.date.accessioned
2018-06-07T19:29:18Z
dc.date.available
2005-11-07T00:00:00.649Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/6140
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-10339
dc.description
1\. TITLE PAGE, TABLE OF CONTENTS, ABBREVIATIONS 6
2\. INTRODUCTION 7
2.1. SINGLE NUCLEOTIDE POLYMORPHISM - DEFINITION AND APPLICATIONS 7
2.1.1. What are SNPs? 7
2.1.2. SNP maps 8
2.1.3. Technology development for SNP detection 9
2.2. CURRENT STATE IN THE FIELD OF SNP DETECTION 9
2.2.1. Hybridization-based methods 10
2.2.1.1. Microarrays 10
2.2.1.2. DASH 12
2.2.1.3. Homogenous assays 14
2.2.2. Enzymatic methods 17
2.2.2.1. SNP discrimination by polymerase 17
2.2.2.1.1. Allele-specific PCR 17
2.2.2.1.2. Primer extension 19
2.2.2.1.2.1. Primer extension on microarrays 19
2.2.2.1.2.2. Homogenous primer extension assays 21
2.2.2.1.2.3. Primer extension with MALDI-TOF detection 22
2.2.2.1.2.4. Pyrosequencing 23
2.2.2.2. SNP discrimination by ligase 25
2.2.2.3. Invasive cleavage 29
2.2.2.4. Site-specific cleavage 31
2.2.3. Chemical ligation 31
3\. MATERIALS AND METHODS 34
3.1. MATERIALS 34
3.1.1. Oligonucleotides 35
3.1.2. DNA samples 35
3.1.3. SNP loci 35
3.2. METHODS 35
3.2.1. Universal oligonucleotides 35
3.2.2. Design of locus-specific oligonucleotides 36
3.2.3. Preparation of detector oligonucleotides 37
3.2.4. Ligation detection reaction (LDR) - TaqMan SNP detection 38
3.2.4.1. One tube - one locus procedure 38
3.2.4.2. One tube - many loci procedure 39
3.2.4.3. Determination of minimal concentrations of DOs 39
3.2.4.4. Influence of PEG on the hybridization of DOs 39
3.2.5. Hot start Taq polymerase preparation 40
3.2.5.1. Purification of Taq polymerase. 40
3.2.5.2. Determination of Taq polymerase activity 41
3.2.5.3. Preparation of hot start Taq polymerase 42
3.2.5.4. Estimation of the efficiency of formaldehyde inactivation 42
3.2.6. Pfu DNA Ligase preparation 43
3.2.6.1. Purification of Pfu DNA ligase. 43
3.2.6.2. Estimation of Pfu DNA Ligase activity 44
3.2.7. T4 DNA Ligase preparation 44
3.2.8. Genomic DNA purification 44
3.2.8.1. Plant Genomic DNA purification using CTAB buffer 44
3.2.8.21. Plant Genomic DNA purification using homemade silica spin-columns.
45
3.2.8.3. Genomic DNA purification from saliva. 46
4\. RESULTS AND DISCUSSION 48
4.1. BACKGROUND 48
4.2. LDR-TAQMAN SNP DETECTION METHOD 50
4.2.1. The structure of detector oligonucleotides 51
4.2.2. The "one tube - one locus" procedure (Figure 13) 53
4.2.3. The "one tube - many loci" procedure (Figure 14) 55
4.2.4. Discussion of the LDR-TaqMan protocol 58
4.3. LDR-TAQMAN GENOTYPING KITS 64
4.3.1. Preparation of enzymes and Detector oligonucleotides 64
4.3.1.1. Homemade Pfu DNA ligase 65
4.3.1.2. Homemade hot start Taq DNA polymerase 65
4.3.1.3. Plastic consumables 66
4.3.1.4. Plant and human genomic DNA isolation using homemade spin columns
66
4.3.1.5. Ligation-based synthesis of Detector Oligonucleotides (DOs) 66
4.3.2. Arabidopsis thaliana SNP kit 69
4.3.3. Human SNP kit 75
5\. SUMMARY 79
5.1. ZUSAMMENFASSUNG 80
6\. REFERENCES 81
7\. SUPPLEMENTS 98
dc.description.abstract
The LDR-TaqMan SNP genotyping method was elaborated and patented [Borodina et
al., 2004; EP03019521]. The method is based on the ligation detection reaction
(LDR), which is performed directly on genomic DNA. During the LDR, the
biallelic state of an SNP locus is converted into the bimarker state of
ligated detector oligonucleotides. The state of the markers is then determined
by the 5'-nuclease assay (TaqMan) with universal fluorescent probes. The
technology is sensitive, has high discrimination power, needs no locus-
specific optimization and is applicable both for individual and multiplex SNP
analyses.
To transfer the technology to scientific partners, the following additional
tasks were solved:
* a method for cost-effective synthesis of long (60-120nt) oligonucleotides with block structure was developed and patented [Borodina et al., 2003; PCT/EP 2004/003921];
* a procedure for DNA isolation from plant material and saliva on home-made silica spin-columns was established [Borodina et al., 2003a];
* enzymes required for the method (thermostable Pfu DNA ligase and hot start Taq DNA polymerase) were cloned and expressed.
The LDR-TaqMan method was applied for SNP genotyping of human and Arabidopsis
thaliana DNA samples:
* a kit for genotyping of 9 clinically important human SNPs was prepared and used for determination of allele frequencies of these SNPs in two East European populations (Ukrainians and Belorussians, 216 DNA samples) [Kozhekbaeva et al., 2004];
* a 138-loci genotyping kit for Arabidopsis thaliana accessions Columbia (Col-0) and C24 was prepared and tested in the blind genotyping of 10 plants.
A comparison of the LDR-TaqMan genotyping results with those obtained by
conventional methods (sequencing, RFLP, SNaPshot, Amplifluor [Rickert et al.,
2004]) demonstrated the efficiency and reliability of the LDR-TaqMan
procedure.
Ready-to-use genotyping kits (including enzymes, buffers and sets of detector
oligonucleotides) were transferred to collaborators in Golm (Germany) and
Moscow (Russia).
de
dc.description.abstract
Im Rahmen der Arbeit wurde die LDR-TaqMan SNP-Genotypisierungsmethode
entwickelt und patentiert [Borodina et al., 2004; EP03019521]. Die Methode
basiert auf der direkt mit genomischer DNA durchgefuhrten
Ligationsdetektionsreaktion (LDR). Abhangig vom allelischen Zustand des SNP
Locus wird eine korrespondierende Markersequenz in das Ligationsprodukt
integriert. Die Markersequenz wird im folgenden 5'-Exonukleaseassay (TaqMan)
durch universelle Sonden identifiziert. Die Methode ist sensitiv, hat ein
hohes Diskriminierungsvermogen, bedarf keiner lokusspezifischen Optimierung
und ist in Single- als auch Multiplexanalysen anwendbar.
Um die Technologie unseren wissenschaftlichen Partnern zur Verfugung zu
stellen, wurden folgende Aufgaben gelost:
* die kosteneffiziente Synthese langer (60 - 120nt) Oligonukleotide mit Blockstruktur [Borodina et al., 2003; PCT/EP 2004/003921];
* Entwicklung einer effektiven Methode zur DNA Isolation aus pflanzlichem Material und Speichel in Silica basierten Saulen [Borodina et al., 2003a];
* Klonierung und Expression von fur die Methode benotigten Enzymen (thermostabile Pfu DNA Ligase und HotStart-DNA Polymerase).
Die LDR-TaqMan Methode wurde zur SNP-Genotypisierung von humanen und
Arabidopsis thaliana Proben verwendet:
* ein Genotypisierungskit fur 9 klinisch bedeutenden humanen SNPs wurde angefertigt und zur Bestimmung der Allelfhaufigkeit dieser SNPs in zwei Osteuropaischen Populationen verwendet (Ukrainer und Wei?russen - 216 DNA Proben) [Kozhekbaeva et al., 2004];
* ein 138-Loci Genotypisierungskit fur Arabidopsis thaliana der Okotypen Columbia (Col-0) und C24 wurde angefertigt und in einem Genotypisierungsversuch mit 10 Pflanzen getestet.
Der Vergleich der Genotypisierungsergebnisse mit den Ergebnissen von
komerziellen Produkten (Sequenzierung, PDRF, SnaPshot, Amplifluor [Rickert et
al., 2004]) bestatigte die Effizienz und Zuverlassigkeit der LDR-TaqMan
Methode. Daraufhin wurden gebrauchsfertige Genotypisierungskits (Enzyme,
Puffer und lokusspezifische Oligonukleotide) Partnern in Golm (Deutschland)
und Moskau (Russland) zur Verfugung gestellt.
de
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
ligation detection
dc.subject
genotyping kits
dc.subject.ddc
500 Naturwissenschaften und Mathematik::570 Biowissenschaften; Biologie::570 Biowissenschaften; Biologie
dc.title
Development of the Ligation Detection Reaction (LDR)-TaqMan Assay
dc.contributor.firstReferee
Prof. Dr. Hans Lehrach
dc.contributor.furtherReferee
Prof. Dr. Volker Erdmann
dc.date.accepted
2005-10-12
dc.date.embargoEnd
2005-11-24
dc.identifier.urn
urn:nbn:de:kobv:188-2005002980
dc.title.subtitle
a Novel SNP-genotyping Method
dc.title.translated
Entwicklung des Ligationsdetektionsreaction (LDR)-TaqMan Assay
de
dc.title.translatedsubtitle
eine neue SNP-Genotypisierungsmethode
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000001825
refubium.mycore.transfer
http://www.diss.fu-berlin.de/2005/298/
refubium.mycore.derivateId
FUDISS_derivate_000000001825
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access
