Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
1998
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Freiburg (Breisgau), Univ., Diss., 2000 (Nur beschränkt für den Austausch) |
| Beschreibung: | 91 Bl. Ill., graph. Darst. : 30 cm |
Internformat
MARC
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| 100 | 1 | |a Feldmann, Kathrin Alexandra |d 1973- |e Verfasser |0 (DE-588)122725271 |4 aut | |
| 245 | 1 | 0 | |a Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients |c vorgelegt von Kathrin Alexandra Feldmann |
| 264 | 1 | |c 1998 | |
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Datensatz im Suchindex
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ABBREVIATIONS
4
1 INTRODUCTION 5
1.1 COLORECTAL CANCER 5
1.1.1 CLINICAL PROBLEM 5
1.1.2 MOLECULAR CHANGES IN THE DEVELOPMENT OF COLORECTAL CANCER 5
1.1.3 STAGING 7
1.1.4 MANAGEMENT 7
1.2 P53
8
1.2.1 ORGANISATION AND STRUCTURE OF THE P53 GENE 8
1.2.2 ORGANISATION AND STRUCTURE OF THE P53 PROTEIN 8
1.2.3 NORMAL P53 FUNCTION AT A CELL CYCLE CHECKPOINT 10
1.2.4 THE ROLE OF P53 IN TUMOUR DEVELOPMENT/CLINICAL IMPLICATIONS 12
1.3 CIRCULATING TUMOUR CELLS 13
1.3.1 PROCESS OF METASTATIC DEVELOPMENT AND THEIR CLINICAL IMPLICATIONS
13
1.3.2 HETEROGENEITY OF METASTASIS/CLONAL DIVERGENCE 14
1.3.3 MOLECULAR DETECTION OF CIRCULATING TUMOUR CELLS 14
1.3.4 AIMS OF THIS STUDY 15
2 MATERIALS AND METHODS 16
2.1 PATIENTS 16
2.2 SAMPLE COLLECTION 16
2.2.1 TUMOUR BIOPSIES 16
2.2.2 BLOOD SAMPLING 16
2.3 CELL LINES 17
2.4 FLOW CHART 17
2.5 SEPARATION OF BLOOD COMPONENTS BY USING AN ERYTHROCYTE LYSIS BUFFER
19
2.6 ISOLATION OF CIRCULATING TUMOUR CELLS 19
2.6.1 IMMUNOMAGNETIC SEPARATION OF CIRCULATING TUMOUR CELLS 20
2.6.1.1 CHARACTERISTICS OF DYNABEADS 20
2.6.1.2 CHARACTERISTICS OF THE HUMAN EPITHELIAL ANTIGEN BER-EP4 21
2.6.2 THE USE OF IMMUNOBEADS IN BIOMAGNETIC SEPARATION 21
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/960931864
2
2.6.2.1 IMMUNOMAGNETIC BEADS AND TUMOUR CELL BINDING: PROTOCOLS 22
2.7 RNA EXTRACTION 23
2.7.1 TOTAL RNA EXTRACTION FOR TISSUE AND BLOOD SAMPLES 24
2.7.2 A DETAILED PROTOCOL OF THE RNA ISOLATION PROCEDURE INCLUDES THE
FOLLOWING STEPS: 24
2.8 SPECTROPHOTOMETRIC DETERMINATION OF THE AMOUNT OF RNA 25
2.8.1 CONTROL OF THE RNA QUALITY ' 26
2.9 COMPLEMENTARY DNA SYNTHESIS (CDNA) 26
2.9.1 CDNA PRIMING 26
2.9.2 PROTOCOL OF CDNA SYNTHESIS 27
2.10 POLYMERASE CHAIN REACTION (PCR) 29
2.10.1 PCR PARAMETERS
30
2.10.2 PCR PROFILE: TIMES AND TEMPERATURES
33
2.11 ELECTROPHORESIS
36
2.11.1 PROTOCOL OF PROCEDURE FOR MAKING AND RUNNING AN AGAROSE GEL FOR
AMPLIFIED CDNA SAMPLES:
37
2.11.2 DETERMINATION OF THE SIZES OF DNA FRAGMENTS FROM 100-2000BP
(PHARMACIA BIOTECH) ON AGAROSE GELS
37
2.12 SINGLE STRAND CONFORMATION POLYMORPHISM (SSCP) ANALYSIS 38
2.12.1 SAMPLE PREPARATION FOR SSCP ANALYSIS
39
2.12.2 ANALYSING THE RESULTS
YY
39
2.13 AUTOMATED SEQUENCING 41
2.14 PURIFICATION OF PCR-AMPLIFIED TEMPLATE DNA 42
2.14.1 PCR PURIFICATION PROTOCOL
43
2.14.2 STATISTICS
43
2.15 APPENDIX
44
3 RESULTS
47
3.1 METHOD DEVELOPMENT
47
3.1.1 SEARCH FOR P53 MUTANTS IN TUMOUR TISSUES
47
3.1.1.1 MAGNESIUM CONCENTRATION
47
3.1.1.2 RNA QUALITY CONTROL AFTER EXTRACTION FROM TISSUE SAMPLES
47
3.1.1.3 GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) 48
3
3.1.1.4 RT- PCR AMPLIFICATION OF P53 TOTAL IN TUMOUR TISSUES 49
3.1.1.5 DETECTION OF SPECIFIC EXONS IN TISSUES SAMPLES 51
3.1.1
.6
SINGLE STRAND CONFORMATION POLYMORPHISM (S SCP) 51
3.1.1.7 DIRECT SEQUENCING 53
3.1.2 BLOOD SAMPLES 55
3.1.2.1 BLOOD PROCESSING DEVELOPMENT 56
3.1.2.2 RT-PCR AMPLIFICATION OF P53 FOLLOWING BEAD SELECTION OF TUMOUR
CELLS FROM BLOOD 57
3.1.2.3 1-STEP OR 2-STEP AS TEMPLATE FOR SPECIFIC EXONS 58
3.2 ANALYSIS OF PATIENT SAMPLES 60
4 DISCUSSION 67
4.1 DISCUSSION OF METHODS 67
4.1.1 MULTIPLE BLOOD SAMPLING 67
4.1.2 IMMUNOMAGNETIC SEPARATION OF BLOOD COMPONENTS 68
4.1.3 REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) 69
4.1.4 2-STEP PCR 70
4.1.5 SINGLE STRAND CONFORMATION POLYMORPHISM (SSCP) 70
4.1.6 VARIABILITY OF PCR AMPLIFICATION PRODUCTS UNDER DIFFERENT
CONDITIONS 71
4.2 DISCUSSION OF RESULTS 72
4.2.1 SITE OF MUTATION 73
4.2.1.1 SPECIFIC P53 MUTATIONS WITH DOMINANT-NEGATIVE AFFECTS 74
4.2.2 HETEROGENEITY IN SUBPOPULATIONS 75
4.2.3 P53 POLYMORPHISMS 75
4.2.4 RELEVANCE OF SPECIFIC P53 MUTATIONS TO PROGNOSIS 76
4.2.5 FUTURE PROSPECTS 77
5 SUMMARY 78
6 REFERENCES 79
ACKNOWLEDGEMENT 90
91
CURRICULUM VITAE |
| any_adam_object | 1 |
| author | Feldmann, Kathrin Alexandra 1973- |
| author_GND | (DE-588)122725271 |
| author_facet | Feldmann, Kathrin Alexandra 1973- |
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| bvnumber | BV013667145 |
| ctrlnum | (OCoLC)48734644 (DE-599)BVBBV013667145 |
| discipline | Medizin |
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| indexdate | 2024-10-09T18:14:43Z |
| institution | BVB |
| language | English |
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| physical | 91 Bl. Ill., graph. Darst. : 30 cm |
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| spelling | Feldmann, Kathrin Alexandra 1973- Verfasser (DE-588)122725271 aut Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients vorgelegt von Kathrin Alexandra Feldmann 1998 91 Bl. Ill., graph. Darst. : 30 cm txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2000 (Nur beschränkt für den Austausch) Hochschulschrift gtt (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009338674&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Feldmann, Kathrin Alexandra 1973- Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients Hochschulschrift gtt |
| subject_GND | (DE-588)4113937-9 |
| title | Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients |
| title_auth | Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients |
| title_exact_search | Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients |
| title_full | Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients vorgelegt von Kathrin Alexandra Feldmann |
| title_fullStr | Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients vorgelegt von Kathrin Alexandra Feldmann |
| title_full_unstemmed | Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients vorgelegt von Kathrin Alexandra Feldmann |
| title_short | Circulating tumour cell p53 mutations identified in blood from colorectal cancer patients |
| title_sort | circulating tumour cell p53 mutations identified in blood from colorectal cancer patients |
| topic | Hochschulschrift gtt |
| topic_facet | Hochschulschrift |
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