IDENTIFICATION AND INSILICO ANALYSIS OF PATIENTS AFFECTED WITH GASTRO INTESTINAL INFECTIONS

Project Title

IDENTIFICATION AND INSILICO ANALYSIS OF PATIENTS AFFECTED WITH GASTRO INTESTINAL INFECTIONS

Project Area of Specialization Biomedical EngineeringProject Summary

Cancer is a disease in which some of the body’s cells grow uncontrollably and spread to other parts of the body. Cancers form solid tumors, but some cancers such as blood infected cancers, leukemia’s, generally do not develop tumors. Gastrointestinal Tract Cancer (GIT) is one of the most common types of cancer and the chief cause of morbidity and mortality globally. The TP53 consists of 20kb of DNA with 11 exons which on translation gives a 3.0 kb of mRNA (1179bp). This mRNA generates 53 kDa proteins. The TP53 is stimulated to accumulate in the nucleus in response to DNA damage by post-translational changes such as phosphorylation and acetylation. The current study was done to identify and perform in-silico analysis of variants in the TP53 gene in patients affected with gastrointestinal tract cancer from Balochistan. The pathogenic variants/mutations in TP53 were identified by targeted sequencing of the TP53 gene in 05 patients and 01 control. The 11 exons were optimized using a gradient thermocycler, the amplified product was purified and sequenced using Sanger sequencing. The sequences were analyzed for the 11 exons of the TP53 gene of the 6 individuals. The identified variants were c. Ser?phe and c.Glu ?His; In silico analysis, of the variant Ser?phe & Glu ?His identified in the TP53 gene, was performed by SWISS-MODEL. These variants could be pathogenic for causing any disorder. The results suggest that mutations in exon 4, 6, 7 & 8 of the TP53 gene probably could be the cause of developing GIT cancer. In silico analysis shows that the mutations that occur in the above-mentioned exons do not affect the phenotypic expression of the TP53 protein.

Project Objectives

To identify the varients in TP53 gene likely to cause GIT cancer

Sequencing of gene to find out causative mutation in gene

In silico analysis of varients of TP53 gene

Project Implementation Method

Development of a Questionnaire

An informed consent documented in a structured questionnaire to record all the necessary information about the patient regarding the disease, like demographic location, socioeconomic status, family and personal history, age, sex, life style of the patient and other related information.

4.2   Identification and enrolment of patients with GIT cancer

50 patients will be enrolled in this study that have been diagnosed positive with GIT cancer. Detail history will be obtained from the patients to record the presence or absence of any other disease/ abnormality. Biopsy reports and other related laboratory diagnosis if done will be collected from the patients.

4.3   Blood collection

3-5 ml venous blood will be collected from the patients and this collected blood will be dispensed into a collecting tube containing EDTA to avoid clotting of the blood. After collection the blood samples will be stored at -20 degree Celsius.

4.5    DNA Extraction

DNA will be extracted from the blood samples following a standardized protocol already published.

4.6    DNA Estimation

The final extracted DNA will be run in gel to check its quality and then amplified by PCR using the synthesized primers of Tp53 gene.

4.7    Sequencing of Gene Tp53

The amplified DNA will be sequenced to check any possible mutations for p53 gene by genetic sequencer.

4.8   In silico analysis

Once the DNA is sequenced and variant is identified in silico analysis will be performed to study the interaction of TP53 and MDM2 proteins.

Benefits of the Project

In this study, the prevailing risk factors of GIT cancer will be investigated in the population of Balochistan. The study will also be helpful to understand the pattern of disease and its prevalence in the region. As the disease prevalence is varying on the bases of geographical status, race, ethnicity, age, gender, socioeconomic status and lifestyle. The study will be helpful to investigate that which of the ethnic group and age group is more affected and what are the possible risk factors for the particular ethnic and age group and on parallel basis, variants rate in P53 gene in patients from Balochistan will be investigated.

Technical Details of Final Deliverable

For the successful completion of the research work, the work plan is distributed into the following tasks;

Years	Activates	Locations	Methodology/ Procedure
3 moths	Collection of Blood Samples	Field Work	5ml Blood Samples will be collected in EDTA Containing Tubes
	Genomic DNA Extraction from Whole Blood	Molecular Biology Laboratory BUITEMS	Extraction by Inorganic method and by kit method (blood washing) & (DNA extraction)
6 months	Amplification of Primers by Polymerase Chain Reaction and Gel Electrophoresis	Molecular Biology Laboratory BUITEMS	PCR Using Labelled Primers
	Sequencing of Amplified Product	China	Analyzing Data Through Different Software

Years

3 moths

Genomic DNA Extraction from Whole Blood

   

6 months

Sequencing of Amplified Product

Final Deliverable of the Project Hardware SystemCore Industry HealthOther Industries Education Core Technology OthersOther TechnologiesSustainable Development Goals Good Health and Well-Being for People, Quality EducationRequired Resources

Elapsed time in (days or weeks or month or quarter) since start of the project	Milestone	Deliverable
Month 1	Enrollment of patients	yes
Month 2	Collection of Blood Samples	yes
Month 3	Genomic DNA Extraction from Whole Blood	yes
Month 4	Primer designing	yes
Month 5	Amplification of Primers by Polymerase Chain Reaction and Gel Electrophoresis	yes
Month 6	Sequencing of Amplified Product	yes