Identification And Comparative Assessment of the SNP rs281865419 of Gene TNF ALPHA Involved In Arthritis

Author Name: * Sheela, Annamala Bhavani
Author Email: annamalabhavani143@gmail.com

Abstract

Arthritis, one of the primitive but yet unsolved problem affecting majority of the population, has been considered in the current study for the identification of the SNP involved. The SNP rs281865419 has been considered in the work.  The blood samples from a total of 10 individuals were collected, of which 8 were affected with arthritis and two were normal used as controls. All the samples were subjected for blood DNA extraction and were purified with ethanol treatment and preserved in TE buffer. The quality of the DNA was tested by Agarose Gel electrophoresis. In order to sequence the TNF alpha gene (involved in arthritis), the sequence was retrieved from the databases and primer was designed using online tool Primer 3. Using the primer the product was amplified and the amplicons were sent for sequencing. The 10 sequences thus obtained were compared for the presence of SNP using Insilco analysis. The work concludes that from a total of 8 diseased samples 1 was showing the SNP under study. This indicates that there is only a limited percentage of mutation affecting the onset of the disease. Also, all the normal individuals were free from the SNP. However the final conclusion can be made with the increase in the number of the samples.

Keywords

Isolation, Extraction, Preservation, Electrophoresis, Polymerase chain reaction, Amplification, Sequencing and Alignment

Introduction

Arthritis is an autoimmune disorder which causes inflammation of one or more joints in the body. Normal wear and tear, inflammation, injury can cause inflammation of joints. TNF-α plays a dominant role in rheumatoid synovitis (Table 1). In cultures of synovial cells from patients with RA, blocking TNF-α with antibodies significantly reduced the production of IL-1, IL-6, IL-8, and GM-CSF.4 Hence, the blockade of TNF-α may have a more global effect on inflammation than the blockade of other cytokines present in high concentration in synovial fluids, such as IL-1.The gene coding for human TNF-α is located on the short arm of chromosome 6 within the major histocompatibility gene complex. A 1.7kb TNF mess Arthritis, one of the primitive but yet unsolved problem affecting majority of the population, has been considered in the current for the identification of the SNP involved. The SNP rs267600955 has been considered in the work.  The blood samples from a total of 10 individuals were collected, of which 8 were affected with arthritis and two were normal used as controls. All the samples were subjected for blood DNA extraction and were purified with ethanol treatment and preserved in TE buffer. The quality of the DNA was tested by Agarose Gel electrophoresis. In order to sequence the TNF alpha gene (involved in arthritis), the sequence was retrieved from the databases and primer was designed using online tool Primer 3. Using the primer the product was amplified and the amplicons were sent for sequencing. The 10 sequences thus obtained were compared for the presence of SNP using Insilco analysis. The work concludes that from a total of 8 diseased samples 2 were showing the SNP under study. This indicates that there is only a limited percentage of mutation affecting the onset of the disease. However all the normal individuals were free from the SNP. However the final conclusion can be made with the increase in the number of the samples.

Conclusion

The current work aimed to analyze the importance of SNP rs281865419 of the gene tumour necrosis factor in causing Arthritis. For this 8 Arthritic blood samples were collected and the genomic DNA was extracted. The DNA was purified and run on agarose gel electrophoresis. After the extraction the gene sequence was retrieved from NCBI data base and the SNP under study was identified. The specific gene region containing the SNP was selected for primer designing as the amplification limit is 150 to 200bp only. The Primer was designed for the selected gene region using Primer 3 tool. The primer was then used for PCR Amplification and the amplicons were sent for sequencing. The sequences thus obtained were further analyzed for the presence of the SNP using CLUSTAL W tool. The multiple sequence analysis result showed that out of the 10 samples analyzed (2 normal and 8 diseased) only 1 were showing the SNP and the other 9 samples were normal. This indicates that only 1SS0% of the diseased individuals were reported with the SNP. However this can be concluded only with the increase in the sample size. This study can be used as a support to increase the sample size and extend the work

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