Souvik Ghatak

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4-6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

Coextraction and PCR Based Analysis of Nucleic Acids From Formalin-Fixed Paraffin-Embedded Specimens

Retrospective studies of archived human specimens, with known clinical follow‐up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin‐fixed paraffin embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. Our optimized co‐extraction approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies because of the high importance and less availability of clinical FFPE specimen.

Protocol for Optimal Quality and Quantity Pollen DNA Isolation from Honey Samples

The present study illustrates an optimized sample preparation method for an efficient DNA isolation from low quantities of honey samples. A conventional PCR-based method was validated, which potentially enables characterization of plant species from as low as 3 ml bee-honey samples. In the present study, an anionic detergent was used to lyse the hard outer pollen shell, and DTT was used for isolation of thiolated DNA, as it might facilitate protein digestion and assists in releasing the DNA into solution, as well as reduce cross-links between DNA and other biomolecules. Optimization of both the quantity of honey sample and time duration for DNA isolation was done during development of this method. With the use of this method, chloroplast DNA was successfully PCR amplified and sequenced from honey DNA samples.

Xenobiotic Pathway Gene Polymorphisms Associated with Gastric Cancer in High Risk Mizo-Mongoloid Population, Northeast India

The aim of this study was to evaluate the risk of gastric cancer associated with individual or combined glutathione S‐transferases (GSTs) polymorphism and their interaction with environmental factors.

Polymorphism in mtDNA control region of Mizo-Mongloid Breast Cancer samples as revealed by PCR-RFLP analysis.

Abstract Mutations in mitochondrial D-loop region of DNA (mtDNA) may serve as a potential sensor for cellular DNA damage and marker for cancer development. We investigated the restriction fragment length polymorphism (RFLP) pattern of the D-loop region in the blood samples of breast cancer patients among Mizoram population. Significant differences were observed among breast cancer and healthy blood samples in the RFLP pattern using AluI, HaeIII and RsaI enzymes. Polymorphic information content (PIC – 0.258), band informativeness (∑Ib – 3.283) and marker index (MI – 0.006) were highest in the case of RsaI enzyme. Our data suggest that the RsaI polymorphic site in the mitochondrial control region is an informative marker for breast cancer development in Mizo population.

Novel APC gene mutations associated with protein alteration in diffuse type gastric cancer

BackgroundThe role of adenomatous polyposis coli (APC) gene in mitosis might be critical for regulation of genomic stability and chromosome segregation. APC gene mutations have been associated to have a role in colon cancer and since gastric and colon tumors share some common genetic lesions, it is relevant to investigate the role of APC tumor suppressor gene in gastric cancer.MethodsWe investigated for somatic mutations in the Exons 14 and 15 of APC gene from 40 diffuse type gastric cancersamples. Rabbit polyclonal anti-APC antibody was used, which detects the wild-type APC protein and was recommended for detection of the respective protein in human tissues. Cell cycle analysis was done from tumor and adjacent normal tissue.ResultsAPC immunoreactivity showed positive expression of the protein in stages I, II, III and negative expression in Stages III and IV. Two novel deleterious variations (g.127576C > A, g.127583C > T) in exon 14 sequence were found to generate stop codon (Y622* and Q625*)in the tumor samples. Due to the generation of stop codon, the APC protein might be truncated and all the regulatory features could be lost which has led to the down-regulation of protein expression. Our results indicate that aneuploidy might occurdue to the codon 622 and 625 APC-driven gastric tumorigenesis, in agreement with our cell cycle analysis. The APC gene function in mitosis and chromosomal stability might be lost and G1 might be arrested with high quantity of DNA in the S phase. Six missense somatic mutations in tumor samples were detected in exon 15 A-B, twoof which showed pathological and disease causing effects based on SIFT, Polyphen2 and SNPs & GO score and were not previously reported in the literature or the public mutation databases.ConclusionThe two novel pathological somatic mutations (g.127576C > A, g.127583C > T) in exon 14 might be altering the protein expression leading to development of gastric cancer in the study population. Our study showed that mutations in the APC gene alter the protein expression and cell cycle regulation in diffuse type gastric adenocarcinoma.

Mutation Profiling in Mitochondrial D-Loop Associated with Stomach Cancer and Tobacco Consumers

Within North East India, the people of Mizoram are mongoloid in origin and Cancer is a major disease condition among this tribal populace. A peculiar habit of consuming ‘‘tuibur’’ (tobacco smoke–infused aqueous solution) has been practiced in Mizoram. Blood and oral swab samples were collected from stomach cancer patients, tuibur consumers and healthy people. DNA was extracted followed by PCR amplification of the D-loop region of mtDNA. Restriction enzyme digestion of 1050 bp of the hyper variable control region of mtDNA was performed in order to gain an insight into the phylogenetic relationship of populace of Mizoram besides the genetic variations among tuibur consumers. The phylogram based on restriction enzyme analysis (AluI, HaeIII, MspI and KpnI) of the D-loop region subsumed within same mtDNA haplogroups and the markers resulted in a similar clustering of the population. The polymorphic samples were sequenced, analyzed and compared with the MITOMAP database. In the hypervariable control region, 292A>AA and 316C>CC are novel microsatellites instability as they have been reported for the first time as it has not been found in the mitochondrial database. 292A>AA position of MT-HV II region is a locus for the mtTF1 binding site Y. Due to the microsatellite instability of this position, the binding of mtTF1 may be altered. 316C>CC is present at the conserved sequence block II in MT-HV II region of human mitochondrial control region. The present study revealed a variety of mtDNA D-loop region mutations and polymorphisms among tuibur consumer besides stomach cancer patients of Mizoram, some of which might be involved in the development of carcinogenesis in stomach for the tuibur consumer.

Lifestyle chemical carcinogens associated with mutations in cell cycle regulatory genes increases the susceptibility to gastric cancer risk

In the present study, we correlated the various lifestyle habits and their associated mutations in cell cycle (P21 and MDM2) and DNA damage repair (MLH1) genes to investigate their role in gastric cancer (GC). Multifactor dimensionality reduction (MDR) analysis revealed the two-factor model of oral snuff and smoked meat as the significant model for GC risk. The interaction analysis between identified mutations and the significant demographic factors predicted that oral snuff is significantly associated with P21 3′UTR mutations. A total of five mutations in P21 gene, including three novel mutations in intron 2 (36651738G > A, 36651804A > T, 36651825G > T), were identified. In MLH1 gene, two variants were identified viz. one in exon 8 (37053568A > G; 219I > V) and a novel 37088831C > G in intron 16. Flow cytometric analysis predicted DNA aneuploidy in 07 (17.5%) and diploidy in 33 (82.5%) tumor samples. The G2/M phase was significantly arrested in aneuploid gastric tumor samples whereas high S-phase fraction was observed in all the gastric tumor samples. This study demonstrated that environmental chemical carcinogens along with alteration in cell cycle regulatory (P21) and mismatch repair (MLH1) genes may be stimulating the susceptibility of GC by altering the DNA content level abnormally in tumors in the Mizo ethic population.