LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
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Synopsis<br />
8.3 Restriction fragment length polymorphism (RFLP): Restriction digestion of the<br />
PCR product with appropriate restriction enzyme was done to analyse the genotype.<br />
PCR product from gene specific PCR reactions was incubated with specific restriction<br />
enzyme with appropriate buffer. Restriction digestion products were analysed by<br />
agarose gel electrophoresis along with DNA ladder.<br />
8.4 SNaPshot reaction: SNaPshot genotyping is based on the dideoxy single-base<br />
extension of an unlabeled oligonucleotide primer which binds to a complementary<br />
template in the presence of fluorescently labelled ddNTPs and Taq DNA polymerase.<br />
The polymerase extends the primer by just one nucleotide, adding a single ddNTP to its<br />
3‟ end. First the desired gene was amplified by PCR using gene specific primers.<br />
SNaPshot assay was performed according to manufacturer‟s protocol which consists of<br />
three steps: (1) EXO-SAP purification (remove unused dNTPs, primer and primerdimers),<br />
(2) SNaPshot reaction (single base extension of the primer) and (3) Post<br />
SNaPshot purification (remove unused ddNTPs from SNaPshot reaction. The processed<br />
samples were then sequenced in Genetic analyzer for interpretation of SNaPshot<br />
results.<br />
8.5 Genotype Score: Genotyping of 22 candidate single nucleotide polymorphisms<br />
(SNPs) in 17 candidate genes involved in DNA repair, apoptosis, cell cycle regulation<br />
and carcinogen metabolism was done and a Genotype Score (G Score) was created<br />
from the number of variant alleles. A value of 2, 1 and 0 was allotted to homozygous<br />
variant, heterozygous and homozygous wild type alleles respectively, and a<br />
consolidated G Score was calculated taking values of all genes together.<br />
9. Microarray: Whole genome expression profiling was done for 5 MPN and 5 Control<br />
cell lines after 5 Gy γ-radiation exposure and 24 h time point using Toronto 27 K slides<br />
(University Health Network Microarray Centre, Toronto). RNA was isolated using<br />
TRIzol protocol and was used for first strand cDNA synthesis using Superscript II<br />
reverse transcriptase for each slide. Indirect labelling of test and reference cDNA<br />
samples was done using Cy5 and Cy3 dyes (Amersham), respectively. Labelled<br />
samples were added to microarray slides and were kept in hybridization chambers at 42<br />
o C in water bath overnight. After hybridization slides were sequentially washed using<br />
reducing concentrations of saline sodium-citrate (SSC) buffer containing SDS. The<br />
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