LIFE09200604007 Tabish - Homi Bhabha National Institute

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Materials and Methods Method: Restriction digestion of the PCR product with appropriate restriction enzyme (Table 2) was done to analyse the genotype. 10 μl of PCR product out of 25 μl reaction mixture was incubated with restriction enzyme at a concentration of 2 U/10 μl and 1/10 th volume of appropriate buffer in the final volume of 20 μl at 37 °C overnight. Restriction digestion products were analysed on 2% agarose gel electrophoresis described in section 8.2 along with 100 bp DNA ladder. 3.8.5 SNaPshot reaction: SNaPshot assay developed on the principle of single base extension of primer serves as a simple, sensitive, robust, time saving and specific approach to detect multiple known SNPs in a pooled PCR product containing more than one gene. A primer which anneals immediately adjacent to the SNP is extended by one base using a fluorescently labelled ddNTP. Unlike PCR-RFLP, in SNaPshot assay the presence of any restriction site for the detection of polymorphism is not mandatory. This method further offers a direct visualization of the genotype of the SNP present in the expected region on a DNA fragment by its novel four different fluorescently labeled ddNTPs which give a coloured peak when incorporated in the SNP region on a electropherogram after capillary electrophoresis. Thus, allowing SNP genotyping on a large scale and in a short time. Materials: 1. Amplified PCR product 2. PCR products 3. EXO-1 (10 U/μl) (USB) 4. SAP (1 U/μl) (USB) 5. SNaPshot ready reaction mix (provided in the kit) (Applied Biosystems) 6. Primers (Sigma-aldrich) 7. PCR buffer (10X) 8. Thermalcycler (Tetrad2 by BIO-RAD) 9. Distilled water 10. Genetic analyzer (Applied Biosystems- Avant 3100/3500) Method: First the desired gene was amplified by PCR using gene specific primers as described in section 8.3. SNaPshot assay was performed which consists of three steps 71
Materials and Methods viz. EXO-SAP purification, SNaPshot reaction and Post SNaPshot purification. For PCR product purification EXOI and SAP enzymes are required. EXO-I (Exonuclease-I) is an enzyme with 5' to 3' exonuclease activity which helps clearing off the short singlestranded primers. SAP (Shrimp alkaline phosphatase) is a hydrolase enzyme responsible for dephosphorylation of nucleotides thereby hampering their migration and preventing their competition with ddNTPs in the SNaPshot reaction. EXO-SAP purification: This step deals with the purifications of PCR products as it contains free, unused dNTPs, primer and primer-dimers. It is important to get rid of these unwanted materials to avoid non-specific addition of ddNTPs resulting in false positive peaks in the SNaPshot electropherogram and these may also result in quenching of the signals. Reaction mixture for EXO-SAP purification Component Vol. (μl) Final concentration EXO-I (1 U/μl) 0.1 0.01 U SAP (1 U/μl) 1 0.1 U PCR-buffer (10X) 1 1 D/W 1.9 - Product 6 - Total 10 - Thermal-cycler program for EXO-SAP purification Step Temperature (⁰C) Duration Enzyme activity 37 2 h Enzyme deactivation 72 15 min Reaction end 4 30 min SNaPshot reaction: In this step, single base extension of the primer takes place. Purified PCR product was added in a reaction mixture containing ready reaction mix (contains fluorescently labelled ddNTPs with a different label for each of the 4 ddNTPs, DNA polymerase and essential buffers) and SNaPshot primers. 72
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Genotype-Molecular Phenotype Correl
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CONTENTS Page No Synopsis 1 List of
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Synopsis
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Synopsis Genotype-Molecular Phenoty
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Synopsis Materials and Methods: 1.
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Synopsis loading control. PCR produ
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Synopsis 7.1 Percent cell death aft
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Synopsis slides were then dried and
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Synopsis range 41.77% - 90.95% at 5
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Synopsis 5. Genotyping of genes: Ge
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Synopsis G2/M arrest and delaying e
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Synopsis may have an important bear
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Synopsis References: 1. Bobba, R.;
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Abbreviations MOPS : 3-(N-morpholin
Page 33 and 34: List of Figures Fig. 28: Percent re
Page 35 and 36: Chapter 1 Introduction
Page 37 and 38: Introduction In previous studies fr
Page 39 and 40: Introduction important carcinogenes
Page 41 and 42: Review of Literature The yet undeci
Page 43 and 44: Review of Literature Fig. 2: Incide
Page 45 and 46: Review of Literature sustained angi
Page 47 and 48: Review of Literature 2.6 Genotype p
Page 49 and 50: Review of Literature 2.7.2 Biologic
Page 51 and 52: Review of Literature Table 1: Steps
Page 53 and 54: Review of Literature future analysi
Page 55 and 56: Review of Literature Inefficient ce
Page 57 and 58: Review of Literature XRCC1 (X-ray r
Page 59 and 60: Review of Literature 2.9.2 Gene inv
Page 61 and 62: Review of Literature MPO gene polym
Page 63 and 64: Review of Literature important role
Page 65 and 66: Materials and Methods Source of che
Page 67 and 68: Materials and Methods Method: EBV-t
Page 69 and 70: Materials and Methods Immunophenoty
Page 71 and 72: Materials and Methods with 500 μl
Page 73 and 74: Materials and Methods cDNA it can b
Page 75 and 76: Materials and Methods Cobalt-60 iso
Page 77 and 78: Materials and Methods specific bind
Page 79 and 80: Materials and Methods h half of the
Page 81 and 82: Materials and Methods phenol and th
Page 83: Materials and Methods 7. Filter ste
Page 88 and 89: Materials and Methods Table 2: List
Page 90 and 91: Materials and Methods SMAD6 AREG HM
Page 92 and 93: Materials and Methods 3.9 Effect of
Page 94 and 95: Materials and Methods fresh eppendo
Page 96 and 97: Materials and Methods Power SYBR Gr
Page 98 and 99: Results Objective 1 To generate EBV
Page 100 and 101: Results Fig. 8: Frequency of second
Page 102 and 103: Results Table 6: Demographics of he
Page 104 and 105: Results Fig. 10: Cell population do
Page 106 and 107: Results Fig. 12: Cell surface marke
Page 108 and 109: Results 4.1.5 Expression of ATM gen
Page 110 and 111: Results Objective 2 To compare the
Page 112 and 113: Results Fig. 17: Standardization of
Page 114 and 115: Results Fig. 19: Standardisation of
Page 116 and 117: Results The cells were incubated fu
Page 118 and 119: Results 4.2.3.1 Percent G2 delay af
Page 120 and 121: Results 4.2.3.2 Effect of BPDE expo
Page 122 and 123: Results Fig. 29: Comparison of perc
Page 124 and 125: Results Fig. 31: Percent cell death
Page 126 and 127: Results normalization with baseline
Page 128 and 129: Results 4.2.5.2 Real time PCR valid
Page 130 and 131: Results homozygous wild-type, heter
Page 132 and 133: Results Fig. 36b: Electrophoresis o
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Results Fig. 37a: Representative el
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Results Table 13: Consolidated geno
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Results LCL Genes NAT2(1) NAT2(2) N
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Results Fig. 38: Distribution of Ge
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Results Fig. 39: Various combinatio
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Results Table 14: Genotype-phenotyp
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Discussion Introduction Squamous ce
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Discussion stimulated lymphocytes w
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Discussion H2AX is currently the mo
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Discussion or G2/M phase arrest. As
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Discussion differential expression
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Discussion DNA repair genes and MPN
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Chapter 6 Summary and Conclusions
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Summary and Conclusions Statistical
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Bibliography 1. Dikshit, R.; Gupta,
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Bibliography 43. Lei, D.; Sturgis,
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Bibliography 76. Fry, R. C.; Svenss
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Bibliography 115. Kote-Jarai, Z.; M
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Bibliography 149. Hashibe, M.; Bren
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Bibliography 183. Bergamaschi, D.;
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Bibliography 216. Bondy, M. L.; Wan
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Indian J Med Res 135, June 2012, pp
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822 INDIAN J MED RES, JUNE 2012 mar
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824 INDIAN J MED RES, JUNE 2012 (h)
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826 INDIAN J MED RES, JUNE 2012 Tab
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828 INDIAN J MED RES, JUNE 2012 lik
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CASE REPORT Eben L. Rosenthal, MD,
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FIGURE 2. Chromosomal breakage anal
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FA along with ataxia telangiectasia
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LIFE09200604007 Tabish - Homi Bhabha National Institute

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