Assignment Task:
Introduction
In this report two oncogenes were explored, T-Cell Acute Lymphocytic Leukemia (TALL) a protein coding gene, associated with cancer causing diseases such as Lymphoblastic Leukemia and Lymphocytic Leukemia and the Cadherin gene (CDH1), associated with lung epithelial. In this experiment genetic variation is explored through gene expression and DNA methylation of these two genes to analyse if it is an effect of mutation. From this it was hypothesized that differences in the expression of TAL1 or CDH1 is due to differences in DNA methylation and/or variation in genetic sequence.
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Get Help Now!It was found that CDH1 and TAL1 was more expressed in cancer-causing cells relative to non-cancer causing, in which sample two for both oncogenes expressed higher levels of the genes CDH1 and TAL1. Where there was a difference in gene expression there was a difference in DNA methylation from enhancers, in which MSRE4 in CDH1 and MSRE5 in TAL1 showed signs of DNA demethylation from both sample two. Although the results differed for the nanopore sequencing between oncogenes, where a 12bp insertion was discovered where there were high peaks of histone acetylation for MSRE5 in TAL1 sample two. CDH1 showed no insertions or deletions, however there were high levels of histone acetylation in MSRE4, where altering haplotypes was also discovered between CDH1 samples.
This report concluded that gene expression is correlated with insertion mutations in regard to TAL1, and perhaps the TALL samples are biologically different as only changes were discovered between samples and not from regions of the genes. The results from CDH1 conclude that high levels of gene expression is associated with low DNA demethylation and changes in gene expression are correlated with different combinations of alleles, respective to a vitro study.
Methods
Gene Expression of TAL1 and CDH1
Firstly, cDNA was synthesised from RNA, a total volume of 20 microliters was made up from 4 ul of RNA, (final concentration 5ng/ul), 4ul TransAmp buffer, 1 ul reverse transcriptase and remaining water. A quantitative polymerase chain reaction (qPCR) was prepared using ABI 7500 FAST to analyse the expression of TAL1 and CDH1, relative to NFYB used as a reference gene. SensiFAST SYBR green, forward and reverse primers (0.2uM) were the components used to make up a total volume of 10ul, including 2ul of cDNA and water to make up the final volume. After the completion of polymerase activation, denaturation and collection of fluorescence, the data was normalised respective to NFYB where an average relative fold difference was calculated.
MSRE (methylation-sensitive Restriction Endonuclease) assay – DNA methylation
Firstly, three reactions were set up containing genomic DNA (gDNA) digested with Hpall or Mspl and undigested no enzyme as a control. The following components made up a final volume of 15ul; 4ul (100ng) of gDNA, 1ul of enzyme (Mspl or Hpall), 1.5ul of restriction endonuclease buffer and 8.5ul water to make up final volume. The undigested treatment contained 9.5ul of water. DNA was digested for one hour at 37 degrees Celsius, and heat inactivation for fifteen minutes at 95 degrees Celsius.
Quantitative PCR (QPCR) was set up to amplify two specific regions of CDH1 and TAL1 gene using different primers (MSRE3 and MSRE4 for CDH1 and MSRE4 and MSRE5 for TAL1). gDNA samples were diluted to a concentration of 5ng/ul, 2ul was added to 5ul of SensiFAST SYBR Green, 1ul (0.2uM) of each forward and reverse primer and 1ul of water.
Nanopore DNA Sequencing ligation-mediated PCR library
PCR is used to isolate and amplify specific sequences within CDH1 (1610bp) and TAL1 (2032) genes, first the DNA was amplified in a master mix with the following components; 3ul of gDNA (1.5ng/ul), 25ul of traq mix, 5ul (0.5uM) forward and reverse primer mix and 12ul water to make up a final volume of 50ul.
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