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DiscussionIt may seem like unnecessary work to perform a scanning method since it must becomplemented with a diagnostic method anyway. Why not just go for the diagnostic methodand get all results in one analysis? The answer is that different problems require differentsolutions. If the mutation is already described and we know what we are looking for, thediagnostic approach may be used. The BRAF, KRAS and PIK3CA genes have knownmutation hotspots, in which a diagnostic method like direct sequencing is highly suitable. Forgenes with large coding regions and no known hotspots (exemplified by NF1) a scanningmethod is quite useful. Direct sequencing of the 61 NF1 exons in 24 carcinomas wouldrequire a minimum of ~1500 sequences with a 100% success rate, meaning the final numberwould be higher. In paper III we pre-screened all 61 exons in 24 samples for NF1 mutationsusing denaturing high performance liquid chromatography (DHPLC) and only those with anabnormal elution profile were subjected to direct sequencing. The employment of a scanningapproach saved us from performing a large amount of unnecessary sequencing, resulting in amore time and cost effective approach.Denaturing high performance liquid chromatographyDHPLC was first described in 1995[145] and today several DHPLC-systems arecommercially available, such as the MultiMax LH 750 (Rainin Instrument, Woborn, MA,USA) and the WAVE systems (Transgenomic, Crewe, UK). The principle for detection ofmutations is temperature-based separation of homoduplex and heteroduplex moleculesunder partial denaturing conditions[146]. DHPLC detects both single base-pair mismatchesand indels of single and multiple bases with success, but are unsuccessful at detecting largegenomic rearrangements.In paper III the WAVE system was used for the DHPLC analysis. Separation of DNAmolecules is achieved by means of a mobile phase of hydro-organic eluent containingtriethylammonium acetate (TEAA) and acetonitrile, and a solid phase consisting of a columnwith hydrophobic beads. During the mobile phase the system is first flushed with TEAA,causing the beads to be “coated” with positively charged triethylammonium ion (TEA). TEAis an amphiphilic ion, meaning it has both hydrophobic and hydrophilic ends. This makesthe negatively charged DNA molecule able to bind to the positive end of TEA, linking the48
DiscussionDNA fragments to the column. In order to elute the fragments, increasing concentrations ofacetonitrile is flushed through the column. As the acetonitrile concentration increases, thebridging capabilities of the TEA ions decrease and the DNA fragments are released.Heteroduplexes, with mismatched base pairs, are eluted first followed by the homoduplexesdue to differences in melting temperature. Finally, the fragments pass through an UVdetector which detects the absorbance over time at 260nm[146] *** .The sensitivity of DHPLC is by large determined by temperature[147]. Included with theWAVE equipment is the Navigator TM software which based on fragment length and basecomposition predicts the optimal separation temperature, which is when 75% of thefragment is double stranded. If the temperature is too low, neither the homoduplex norheteroduplex fragments will be denatured, and will be eluted at the same time. Too hightemperature causes all fragments to be fully denatured before acetonitrile is added, againeluting the fragments at the same time. Only at optimal temperatures the heteroduplexes willdenature slightly before the homoduplexes, making mutation detection possible. Due tointra-fragment variations in base composition, PCR products longer than 200 bases usuallycontain two or more melting domains which mean that the product must be analyzed atseveral temperatures, reducing the throughput of the method. Other factors important forsensitivity are the resolution of the homoduplex and heteroduplex species as well as thepurity of the PCR product. With a good study design the sensitivity and specificity ofDHPLC is considered to be higher than 96%[146].In addition to DHPLC, a handful of sensitive mutation scanning methods exist, includingSSCP (Single-Strand Conformational Polymorphism)[148], TTGE (temporal temperaturegradient gel electrophoresis)[149], CSGE (Conformation Sensitive Gel Electrophoresis)[150]and DGGE (Denaturing Gradient Gel Electrophoresis)[151]. However, most, if not all ofthese, are clearly unsuitable when analyzing large samples series as these are gel basedmethods. In this setting a high throughput method would be more suitable, such asconformation-sensitive capillary electrophoresis (CSCE), MALDI-TOF based methods[152],and of course the new generation of ultra high-throughput sequencing systems. AlthoughDHLPC has an inferior throughput compared to these, with an estimated sensitivity higher*** Transgenomic web pages – http://www.transgenomic.com49
Page 1 and 2: Novel genetic and epigenetic altera
Page 3 and 4: TABLE OF CONTENTSACKNOWLEDGEMENTS .
Page 5 and 6: ACKNOWLEDGEMENTSThe present work ha
Page 7 and 8: Prefacetechnology[3]. This new tech
Page 10 and 11: SummaryThe subgroup of carcinomas w
Page 12 and 13: Introduction“Epigenetic inheritan
Page 14 and 15: Introductionamino acid change it is
Page 16 and 17: Introductionmethylation during embr
Page 18 and 19: IntroductionDNA is most of the time
Page 20 and 21: IntroductionFigure 5. DNA methylati
Page 22 and 23: IntroductionFigure 6. Incidence rat
Page 24 and 25: IntroductionFigure 8. Tumor staging
Page 26 and 27: Introductioninasmuch as 80% of colo
Page 28 and 29: IntroductionInstabilities involved
Page 30 and 31: Introductionthere seems to be a fid
Page 32 and 33: Introductionsevere alterations are
Page 34 and 35: Introductionpopulation-wide screeni
Page 36 and 37: IntroductionFigure 12. Present and
Page 38 and 39: RESULTS IN BRIEFPaper Ia. “DNA hy
Page 40 and 41: Results in Briefinstability, and se
Page 42 and 43: Results in BriefUnivariate survival
Page 44 and 45: Discussionseveral factors, and full
Page 46 and 47: Discussionlow threshold, we increas
Page 50 and 51: Discussionthan 96% DHPLC do not sta
Page 52 and 53: DiscussionFigure 13. Mutation detec
Page 54 and 55: DiscussionClinical impact of molecu
Page 56 and 57: Discussionmarkers with a very high
Page 58 and 59: Discussionchromosomes in metaphase[
Page 60 and 61: DiscussionThese examples underline
Page 62 and 63: Discussiongenes. One is based on mu
Page 64 and 65: CONCLUSIONSWe have identified novel
Page 66 and 67: Future PerspectivesMolecular risk a
Page 68 and 69: REFERENCES1. Breasted J (1930) The
Page 70 and 71: References29. Deng G, Chen A, Pong
Page 72 and 73: References57. Al-Sukhni W, Aronson
Page 74 and 75: References84. Kunkel TA (1993) Nucl
Page 76 and 77: ReferencesLeggett B, Levine J, Kim
Page 78 and 79: References133. Lind GE, Thorstensen
Page 80 and 81: References156. Meling GI, Lothe RA,
Page 82 and 83: ReferencesT, Song X, Day RH, Sledzi
Page 84 and 85: References196. Honda S, Haruta M, S
Page 86 and 87: ORIGINAL ARTICLESAPPENDIXAppendix I
Page 89 and 90: GASTROENTEROLOGY 2007;132:1631-1639
Page 91: Paper IbGuro E Lind, Terje Ahlquist
Page 94 and 95: Journal of Translational Medicine 2
Page 96 and 97: Journal of Translational Medicine 2
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Journal of Translational Medicine 2
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Paper IITerje Ahlquist, Guro E Lind
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BackgroundMost cases of colorectal
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ADAMTS1 CDKN2A CRABP1 HOXA9 MAL MGM
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pseudogene, leading to a high rate
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strands. Proc Natl Acad Sci U S A 1
Page 116 and 117:
concomitant absence of transcript a
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Volume 10 Number 7 July 2008 pp. 68
Page 121 and 122:
682 RAS Signaling in Colorectal Car
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Table W2. Detailed Somatic Events o
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Identification of RCC2 as a prognos
Page 133 and 134:
INTRODUCTIONMicrosatellite instabil
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unselected series of primary tumors
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specificity, i.e. that they only am
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On the assumption that DNA repair a
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In order to ensure that gene mutati
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Figure 2. Mutation frequency differ
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and TAF1B (0.50), ACVR2A and ASTE1
Page 147 and 148:
Multivariate analysesA multivariate
Page 149 and 150:
When comparing our findings of muta
Page 151 and 152:
The test series included a low numb
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entering M-phase remains to be seen
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12. Duval A, Reperant M, Hamelin R
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34. Martineau-Thuillier S, Andreass
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AppendicesAppendix I:List of abbrev
Page 163 and 164:
Critical Reviews TM in Oncogenesis,
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TARGET GENES OF MSI COLORECTAL CANC
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