Samantha M. Hurndon Week 11: Week 11: DNA Microarray Journal Club

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#**PCR products were designed to represent all ORFs in H pylori NCTC26695 aand J99.  
#**PCR products were designed to represent all ORFs in H pylori NCTC26695 aand J99.  
#**Micro arrays were constructed by robotic spotting of PCR products on UltraGaps amino-silane-coated glass slides and post-print-processed
#**Micro arrays were constructed by robotic spotting of PCR products on UltraGaps amino-silane-coated glass slides and post-print-processed
 +
#*CGH
 +
#**Strain CCUG17874 was used to study global transcription patterns of different flagellar mutants (the genome of this strain as not been sequenced).
 +
#**The macro diversity of this strain was investigated using CGH.
 +
#**Array hybridiztions for the wt. and the mutant were performed in duplicate.
 +
#**Quantile normalization was performed
 +
#**The output files give three lists: divergent genes, uncertain genes and present genes.
 +
#*Confirmatory analysis of selected CGH data by PCR
 +
#**under standard conditions, confirmed selected results obtained from CGH.
 +
#**Seven genes were selected from CGH data
 +
#**Primer pair sequences were designed using the Primer3 online software.
 +
#*Type II Microarray analysis
 +
#*Quantitative analysis of transcription by real-time PCR
 +
#*Reverse transcription and amplification of intergenic regions by PCR
 +
#*Bioinformatics analysis
 +
#Results

Revision as of 19:05, 9 November 2011

Definitions

  1. Adenocarcinoma is a cancer of an epithelium that originates in the glandular tissue.
  2. MALT lymphoma: development of cancer in the B Cells. Rare amoung people under the age of 50 and affects men much more than women. Signs and symptoms: Swelling in the neck , groin and armpit due to enlargement of lymph nodes. Loss of unger and body fatigue, high fever and weight loss. http://maltlymphoma.net/
  3. B cells: Help the immune system fight against the invasion of harmful microorganisms into the body. http://maltlymphoma.net/
  4. Ablation: Removal or excision. Ablation is usually carried out surgically. For example, surgical removal of the thyroid gland (a total thyroidectomy) is ablation of the thyroid. http://www.medterms.com/script/main/art.asp?articlekey=2089

Outline

  1. Abstract
    • Helicobacter pylori is a motile Gram-negative bacterium. This bacterium persists in the human gastric mucosa
    • FliK is the gene that controls hook length during flagellar assembly.
    • Microarry analysis was done on fliK-null mutant, this revealed that under the control of the sigma 54 factor RpoN there is an incase in transcription of genes.
    • Sigma 45 fact is responsible for transcription on the class II flagellar genes (flgE and flgB).
    • Flik is involved in three processes:
      • hook-length control
      • export substrate specificity
      • control of RpoN transcriptional activity
  2. Introduction
    • Helicobacter pylori is an infection that causes gastrointestinal disorders (peptic and duodenal ulcers). It is also a predisposing factor of gastric adenocarcinoma and B-cell MALT lymphoma.
    • This is seen often in developing countries due to their living conditions and lack of health care.
    • A key feature of H. pylori is motility, which is required for colonization and persistence. The motility in this bacteria is due to flagella.
    • three RNA polymerase sigma factors (80, 54, 28) control the transcription of H. pulori flagellar genes.
    • There are three classes of regulation
      • Class one: Sigma 80 modulates the transcription of the early flagullar genes.
      • Class two: RpoN deals with the regulation of the middle flagellar structural genes. RpoN is regulated FlgR/FlgS activation system and HP0958
        • Class two regulation encodes hook-length-control proteins.
      • Class three: Transcription of these genes is controlled by FliA (sigma 28) and an anti sigma factor (FlgM).
    • It was observed that the initial annotation of H.pylori genome sequences did not identify all flagellar genes expected by comparison with the Salmonella/Escherichia coli paradigm.
    • Flik in Salmonella controls hook length. In h pylori fliK gene impairs motility. The cells, in both H. pylori and Salmonella, harbor polyhook structures
    • it is suggested that the FliK protein is required to turn off the RpoN regulation during flagullar assembly.
    • In their study they used pan-H.pylori array. The array was based on the genomes of strains NCTC26695 and J99.
  3. Methods
    • Used a pan-H. pylori array based on the genomes of strains NCTC26695 and J99
    • They preformed array comparative genomic hybridization to identify specific chromosomal regions that were missing in CCUG17874
    • Global transcipt analysis of a CCUG17847 mutant lacking the fliK gene was performed inorder to find out more infomation on the role of FliK in flagellar biogenesis in H. pylori.
    • Bacterial strains, media and growth conditions
      • The mutants used in this study were cultured on Columbia agar base plates containing the appropriate antibiotic
    • Extraction of genomic DNA frp, H. pylori
      • Genomic DNA was isolated using the DNeasy tissue kid. Then was quantified using Nanodrop ND 1000 spectrophotometer.
    • RNA isolation from H. pylori
      • RNA was isolated from 20 liquid cultures, the cells were harvested and centrifuged for 15 secs.
      • Cell plates were then resuspended in bacteria RNA protected reagent.
      • total RNA samples DNase-treated
    • H. pylori microarray design and construction
      • PCR products were designed to represent all ORFs in H pylori NCTC26695 aand J99.
      • Micro arrays were constructed by robotic spotting of PCR products on UltraGaps amino-silane-coated glass slides and post-print-processed
    • CGH
      • Strain CCUG17874 was used to study global transcription patterns of different flagellar mutants (the genome of this strain as not been sequenced).
      • The macro diversity of this strain was investigated using CGH.
      • Array hybridiztions for the wt. and the mutant were performed in duplicate.
      • Quantile normalization was performed
      • The output files give three lists: divergent genes, uncertain genes and present genes.
    • Confirmatory analysis of selected CGH data by PCR
      • under standard conditions, confirmed selected results obtained from CGH.
      • Seven genes were selected from CGH data
      • Primer pair sequences were designed using the Primer3 online software.
    • Type II Microarray analysis
    • Quantitative analysis of transcription by real-time PCR
    • Reverse transcription and amplification of intergenic regions by PCR
    • Bioinformatics analysis
  4. Results
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