Gene Genome & Human Health Research

What need to be included in the report?

Introduction:

Hypothesis: The hypothesis of the experiments is that any differences in the expression of TAL1 or CDH1 between different lung epithelial leukaemia cell lines is due to differences in DNA methylation and/or variation in genetic sequence

Aim: three assays in this report gene expression , DNA methylation and DNA sequencing will allow test an increase in gene expression is associated with decreased DNA methylation and if there is changes can be correlated with variation in DNA sequence

  topics to be covered briefly :

1.function of gDNA ( T-Cell Acute Lymphocytic Leukemia (TALL)  , Cadherin gene (CDH1), associated with lung epithelial Lepi: lung epithelial)

2. restriction enzymes (Hap II ,NSP I and when no enzyme digestion )
3. mutation
4. gene regulation (promoter , enhancer )
5. primer, dNTP,ddNTP

(this should be concise and probably no more than 400 words). The introduction places the experiments in context, drawing on published studies. The rationale for the experiments is described in the hypothesis which is followed by the aims of the experiments. The introduction concludes with a summary of key findings and outcomes of the experiments.( total of 4 different genomic DNA (gDNA) and RNA samples 1. Lepi_1 2. Lepi_2 3. TALL_1 4. TALL_2)

Methods. A very brief summary of key methods (MSRE assay, cDNA synthesis and quantitative real-time PCR, ligation-mediated PCR for preparing Nanopore sequencing libraries)

1. Gene expression :  keywords of what to write in this section and interpret the table below

• Aim : convert of total RNA to cDNA , qPCR for cDNA synthesis
• Set up of qPCR use ddCT comparative :
• test gene are TALL1,2 and CDH1 ,2 , and normlizer or endogenous gene is NFYB then run the reation in the ABI 7500 fact PCR machine
• use SYBR green to calculate gene expression
• high PCR cycle number = low CT
• Two RNA samples, 25ng/µl, from either TALL or Lepi cells  2. SensiFAST cDNA Synthesis Kit

DNA Methylation : use MSRE (methylation-sensitive Restriction Endonuclease) assay

• Many promoters contain concentration of 5’-CG-3’ repetitive sequence  called CPG island
• Enzymes recognize at 2nd G of the sequence
• Mention about methylation and histone acetylation and the relationship with  transcription activation as well as restriction enzymes (HapII , NspI and when there is no enzyme )
• High methylation  =low CT value =high pcr reaction , low methylation =high CT value =low PCR reaction
• Relationship between CT values and restriction enzyme : HAPII methylated=low CT ,no enzyme control = low CT . MSPI cut = high CT , HAPII not methylated =high CT
• Location of MSRE4 , 5 qPCR for TALL 1,2 (upstream ) and compare it to non-TALL in terms of histone acetylation
• Location of MSRE3,4 qPCR for LEPI (large introns) and compare it with skin epithelial , breast epithelial and lung epithelial in terms of histone acetylation. Gene Genome & Human Health Research Report

Nanopore DNA Sequencing ligation-mediated PCR library

• Mention about adaptor DNA that needs ligation
• Regions around or in TALL1,2 and CDH1,2 sequencing
• High acetylation in Gdna TALL  DNA maybe due to mutation compare it to non-TALL

The concentration of DNA IS 0.1 mg/Ml

1. DNA End Repair: blunting and phosphorylation of DNA for subsequent use in bluntend ligation 2. Ligation: blunt-end ligation of adaptors to the DNA amplicon

Note: if there is ligation means + result

If there is no ligation mean – result

Results: Figures have figure legends that are placed under each figure. The figure legend does not interpret the figure, but provides information that allows the reader to understand the content of the figure. A figure legend has a brief descriptive title. Tables do not have legends but are identified by a number and a title that is placed above the table. The figures and tables are accompanied by a text that provides a narrative that links the results into a cohesive story. The narrative identifies important findings, but does not interpret the significance of the results.

( pic of agrose DNA gel added in result section )

1. What are the sizes of the CDH1 and TAL1 amplicons?
2. Why did we ligate the sequencing adaptor to the CDH1 and TAL1 amplicons?
3. Why are there no PCR products in lanes 3 and 6 of the agarose gel? Because number 3 and 6 are controls

Gene Genome & Human Health Research Report

The Nanopore DNA sequencing output has the following parts: 1. The location of the amplicon on the human genome 2. Information on coverage 3. Sequencing results of forward and reverse DNA strands 4. Comparison to the human genome reference sequence 5. Identification of sites that differ from the human reference genome

1. Which sample shows the highest expression of TAL1: TALL1 or TALL2? TALL2
2. What would be your prediction for DNA methylation in the sample showing the highest level of TAL1 expression? TAL1 is the highest level of expression at TALL2 equal highest level of histone acetylation which means lowest  level of DNA methylation
3. Based on the Nanopore DNA sequencing result, would you expect to see a change in DNA methylation at: a. MSRE5 only b. MSRE4 only c. Both MSRE4 and MSRE5
4. Was there a difference in CDH1 gene expression between LEPI1 sand LEPI2?
5. If there was a difference, which sample showed the highest expression of CDH1?
6. What were the results of your MSRE assay? Was there a difference between MSRE3 and MSRE4 within LEPI1 and/or within LEPI2; was there a difference between MSRE3 or MSRE4 between LEPI1 and LEPI2?
7. Did MSRE3 or MSRE4 map to peaks of histone acetylation?
8. Are you able to correlate gene expression with DNA methylation and localization to a peak of histone acetylation?
9. There were no rare mutations discovered in the part of the CDH1 gene that we sequenced. However, there was a difference between the two samples in terms of the haplotypes across this region?
10. How might different haplotypes be associated with differences in gene expression?

Note : all questions above (TALL&LEPI) required to be answered in discussion below

Discussion: The discussion will begin with a summary of key findings and will then discuss significance of the results in the context of the published literature

Concultion :

Resources:

1. The results generated in the practical classes form the basis of this report.
2. You will be required to present your data as scientifically accurate graphs, figures and tables and include these results in the report.
3. You will accurately describe the content of the graphs, figures and tables and interpret the significance of the results.
4. You must include appropriate references to support your arguments.
5. References are peer-reviewed research and review articles that are available through PubMed ( https://pubmed. ncbi.nlm.nih.gov/)
6. It is NOT PERMITTED to cite lectures from this unit as a reference (marks will be deducted)

Important notes :

• Length A 1500 word report (figure legends and references are not included in the word count). There is no minimum word count, but reports in excess of 1700 words will be penalized.
• 5-10 references needed for this report , avoid using websites , only from articles

Goals of the report

need to understand the relationship between genetic variation and our genome – this requires mapping of common and rare genetic variants to specific locations of the genome, and to functionally understand the consequences of genetic variation

consider the genome as the sum of genes that encode RNA (and in most cases protein), as well as the genetic switches (regulatory DNA) that turns genes on and off

epigenetic landscape – the chemical modification of DNA and proteins (histones) that are bound to DNA that package our genome into domains of active and repressed gene expression. Gene Genome & Human Health Research Report