Gene expression and DNA microarrays by ppc90937

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									    Gene expression and DNA
          microarrays
• Old methods.
• New methods based on genome sequence.
  – DNA Microarrays
• Reading assignment - handout
  – Chapter 4 - 195-232, 239-250, 255 MIAME
  – Box 4.1, 4.2 (Significance test only), 4.3, and
    4.4.
           Genome of the week
• E. coli O157:H7.
   – Comparison to the laboratory strain of E. coli.
• Causes haemorrhagic colitis
   – Initially identified in 1982 during an outbreak of severe
     bloody diarrhea.
   – Linked to contaminated ground beef from Michigan
   – Can be lethal
• 75,000 cases per year
• Major findings:
   – Comparison of E. coli O157:H7 with E. coli K-12
     (common lab strain) found that the O157:H7 genome is
     ~ 1Mb larger than K-12 and contains 1,387 genes
     specific for O157:H7.
   – Genomes share a 4.1 Mb backbone with species
     specific DNA interspersed throughout the genome
      • K-islands - specific to K-12 (0.53Mb)
      • O-islands - specific
   – Lateral transfer of DNA occurs much more frequently
     than previously thought. Especially high for
     enterobacteria.
      • O-island specific DNA encoded genes required for virulence
        and a large number of phage and phage associated genes.
• Five sequences of E. coli - 4 pathogens and 1 lab
  strain.
   – Pathogens are as different from each other as they are
     from the non-pathogenic lab strain.
             Gene expression
• What is gene expression?
• Methods for measuring a single gene.
   – Northern Blots
   – Reporter genes
   – Quantitative RT-PCR
• Operons, regulons, and stimulons.
• DNA microarrays.
   – Expression profiling
   – Identifying protein binding sites.
   – Comparing gene content of different strains.
      What is gene expression?
• The amount of RNA produced from a gene.
• Level of RNA produced from a gene is controlled
  by:
   – Transcription
   – Stability/Degradation
• Transcriptome - Expressed transcripts in a cell
  under defined experimental conditions.
   – mRNA(5-10% of total RNA).
   – rRNA, tRNA - make up most of total RNA
   – scRNA (protein secretion), tmRNA (rescue stalled
     ribosomes).
       Regulons and Stimulons
• Operon - group of genes co-expressed on a single
  transcript.
   – One location of the genome
• Regulon - genes that are regulated by a single
  transcription factor.
   – Genes and operons throughout the genome
• Stimulon - collection of genes that are regulated in
  response to environmental changes.
   – Can be multiple regulons affected at once.
• Regulatory network - alternative term for regulon.
  Analysis of gene expression at
      the single gene level.
• Northern Blots
  – Measure RNA levels by hybridization of a
    labeled probe to total RNA.
• Reporter Genes
  – Use of an enzyme to measure the amount of
    transcription from a promoter.
• Quantitative real-time RT-PCR.
• Brief review in book.
Quantitative real time RT-PCR
          Challenges Facing Genomics

                       Detailed
Depth of knowledge




                     analysis of             New tools in
                     single gene              genomics:
                                            microarrays and
                                              proteomics


                                               Genome
                                              sequencing



                              Breadth of knowledge
Assaying the regulation of 1000s
 of genes in a single experiment
• DNA microarrays
  – DNA molecules printed at high density used to
    determine the level of RNA or DNA in a
    sample.
  – Can be thought of a “reverse Northern blots”
• Other technologies (described in chapter 4).
  – SAGE
  – Microbeads
  DNA Microarrays -Introduction
• Spotted DNA arrays (glass slides)
  – Competitive binding of samples - internal
    control
  – Fluorescent detection - Cy3 and Cy5
  – Small sample sizes (10-30µl).
  – PCR or cDNA arrays - double stranded
  – Long oligonucleotide arrays - single stranded
     • Better specificity, cheaper, easier to work with.
• Short oligonucleotide arrays
  – ex. Affymetrix
• DNA spotted onto nylon membranes
  (macroarrays)
          Applications of DNA
              microarrays
• Expression profiling
   – Determining the relative levels of RNA in two or more
     samples.
• DNA/DNA hybridizations
   – Investigate gene content between different strains
   – Determine gene dosage
   – 16S arrays - microbial communities (being developed).
• Identification of protein binding sites
   – ChIP-Chip. Immunoprecipitation of protein/DNA
     complexes. Assaying those interactions with
     microarrays.
     Uses of DNA microarrays
• Detection of candidate genes
   – Expression profiling
   – DNA/DNA hybridizations
• Annotation of gene function
   – Expression - compendium approach
• Defining regulatory networks
   – Expression profiling
   – ChIP/chip experiments
• Molecular phenotyping
   – Expression profiling
   – DNA/DNA hybridizations
Microarray experimental overview
37C             25C

                          Grow cells



                          Isolate RNA
                          Make labeled cDNA




                          Mix and hybridize




                           Scan slide
                           Analyze data
   Hybridization: basic concept
The ability of two strands to hybridize is
dependent on their complementarity.
More complementarity=better hybridization
      Bacterial DNA microarrays
•   Small genome size
•   Fully sequenced genomes, well annotated
•   Ease of producing biological replicates
•   Genetics
  B. subtilis DNA microarrays
• PCR generated microarrays using custom primers
  (Sigma-Genosys).
• Each PCR product represents a single gene.
• 4074 genes of 4101 on the array.
• Printed on Corning CMT-GAPS slides.
• 4 E. coli controls, each represented 15-20 times on
  the array.
  How a DNA microarray works
• Comparing the genome content of two B.
  subtilis strains.
• The two strains differ only by the fact that
  JH642 is lysogenized with the
  bacteriophage SPb.
• JH642 vs PY79 genomic DNA
  hybridization.
  – PY79 does not contain SPb.
  – SPb spots will be red.
    JH642

    PY79



Array size = 16mm x16mm
Spot size = 150mM
        SPb genes   E. coli control
JH642


PY79

								
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