LIFE09200604007 Tabish - Homi Bhabha National Institute

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Materials and Methods SMAD6 AREG HMGA2 F-TCGCGACGAGTACAAGCCACTG R-GCTGGCGTCTGAGAATTCACCC F-CTGTCGCTCTTGATACTCGGC R-GGTCCAATCCAGCAGCATAATG F-GCCAACGTTCGATTTCTACCTCAGC R-AACCTGGGACTGTGAAGGGATTACA 109 bp 49 bp 217 bp Table 3: PCR programs for different genes Gene of interest Denaturation Annealing Elongation GSTM1 94 ⁰C 1 min 55 ⁰C 1 min 72 ⁰C 1 min GSTT1 94 ⁰C 1 min 66 ⁰C 1 min 72 ⁰C 1 min GSTP1 94 ⁰C 1 min 52 ⁰C 1 min 72 ⁰C 1 min MPO 94 ⁰C 30 sec 60 ⁰C 30 sec 72 ⁰C 30 sec SULT1A1 95 ⁰C 1 min 62 ⁰C 30 sec 72 ⁰C 45 sec Fas 95 ⁰C 30 sec 61 ⁰C 30 sec 72 ⁰C 45 sec CyclinD1 95 ⁰C 30 sec 56 ⁰C 30 sec 72 ⁰C 1 min XRCC1-exon6 94 ⁰C 30 sec 57 ⁰C 45 sec 72 ⁰C 45 sec XRCC1-exon9 94 ⁰C 30 sec 53 ⁰C 1 min 72 ⁰C 30 sec XRCC1-exon10 94 ⁰C 45 sec 59 ⁰C 1 min 72 ⁰C 30 sec XRCC3-exon7 94 ⁰C 30 sec 60 ⁰C 1 min 72 ⁰C 30 sec hOGG1 95 ⁰C 30 sec 60 ⁰C 1 min 72 ⁰C 30 sec XPD 94 ⁰C 30 sec 64 ⁰C 45 sec 72 ⁰C 1 min p21 94 ⁰C 30 sec 55 ⁰C 30 sec 72 ⁰C 30 sec p53 94 ⁰C 1 min 55 ⁰C 1 min 72 ⁰C 1 min NAT2-1 94 ⁰C 30 sec 57 ⁰C 30 sec 72 ⁰C 30 sec NAT2-2 94 ⁰C 30 sec 55 ⁰C 30 sec 72 ⁰C 30 sec NAT2-3 94 ⁰C 30 sec 54 ⁰C 30 sec 72 ⁰C 30 sec BRCA2-1 94 ⁰C 30 sec 58 ⁰C 30 sec 72 ⁰C 30 sec BRCA2-2 94 ⁰C 30 sec 54 ⁰C 30 sec 72 ⁰C 30 sec GPX 94 ⁰C 30 sec 60 ⁰C 30 sec 72 ⁰C 30 sec mEH 94 ⁰C 30 sec 55 ⁰C 30 sec 72 ⁰C 30 sec β-actin 94 ⁰C 1 min 55 ⁰C 1 min 72 ⁰C 1 min 30 sec For all PCR reactions initial denaturation was done at 95 °C/10 min, final elongation was done at 72 ⁰C/ 10 min and number of cycles ranged between 30-35. 77
Materials and Methods Table 4: SNaPshot primers Gene Mutation SNaPshot Primer ( 5’-3’) Size Wildtype* Variant* NAT2-1 NAT2-2 NAT2-3 BRCA2-1 BRCA2-2 GPX mEH T→C G→A G→A G→A A→C G→A T→C (36)-X- TTCACCTTCTCCTGCAG GTGACCA (6)-X- AAAAAATATACTTATTT ACGCTTGAACCTC (47)-X- GCCCAAACCTGGTGATG (27)-X- GGTCTTCTGTTTTGCAG ACTTATTTACCAA (19)-X- AAATGATACTGATCCAT TAGATTCAAATGTAGCA (19)-X- GGCGCCCTAGGCACAG CTG (16)-X- TGGAAGAAGCAGGTGG AGATTCTCAACAGA 60 T C 36 G A 64 G A 57 G A 53 A C 38 G A 46 T C X is the number of uncomplimentary bases added at the 5‟ end for tailing the primers. These bases are added to the primers so that there is difference in lengths of different fragments on the SNaPshot electropherogram during multiplex reactions. *Indicates the nucleotide added by extension of the primer and the colour of the corresponding peak in the wild type and variant DNA sample. 78
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Genotype-Molecular Phenotype Correl
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CONTENTS Page No Synopsis 1 List of
Page 9 and 10:
Synopsis
Page 11 and 12:
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
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List of Figures Fig. 28: Percent re
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Chapter 1 Introduction
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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 and 84: Materials and Methods 7. Filter ste
Page 85 and 86: Materials and Methods viz. EXO-SAP
Page 88 and 89: Materials and Methods Table 2: List
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
Page 134 and 135: Results Fig. 37a: Representative el
Page 136 and 137: Results Table 13: Consolidated geno
Page 138 and 139: 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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