ENVB 437 Lecture Notes - Lecture 19: 18S Ribosomal Rna, Oxidative Stress, Breastfeeding
4 Jun 2018
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Eukaryotic cells and viruses – lecture 19 (March 21st, 2018)
Slide 1 – DNA Microarrays – analysis of whole cell gene expression
- Average cell expresses about 10,000 genes
- How to study?
o Northern blot – but can
▪ only study 1 or a few genes at a time
▪ time consuming and difficult to study gene interactions
- DNA chips allow simultaneous monitoring of many genes
Slide 2 – Central dogma and methods
- Traditional detection;
o Southern blot – the DNA duplicates
▪ PCR
o Northern blot – the RNA synthesis
▪ RT-PCR
o Western blot → the protein synthesis
Slide 3 – Development of genomic/proteomic technology
- Tools have been developed to map entire genomes, measure smaller
amounts of mRNA and protein, and simultaneously measure many
more mRNAs and proteins
find more resources at oneclass.com
find more resources at oneclass.com
Slide 4 – the beginnings of microarray technology
- Schena et al., 1995
- Science
- “Quantitative monitoring of gene expression patterns with a complementary DNA microarray”
- Lockhart et al., 1996
- Nature Biotechnology
- “Expression monitoring by hybridisation to high-density oligonucleotide arrays”
o looking at the expression of Mt
o march, 2018:>85,000 papers (PubMed)
- Microarrays can be put on chips and use them to screen for diseases and conditions
Slide 5 – Microarray hybrodization
- Watson-Crick base pairing of complementary DNA sequences.
- Specifically spot a gene sequence that we get with all of our genome sequences
o And we can know the location of all those genes by printing then in a specific location
- By measuring the ratio of the intensities, you can get a measurement of the transcription of this in the entire cell
- Microarrays have 1000’s of spots, each representing a piece of one gene, immobilized on a glass slide.
- The intensity (or intensity ratio) of each spot indicates the amount of labeled cDNA hybridized, thus, representing
the starting mRNA transcript abundance.
- → VIDEO
Slide 6 – Uses and appliccations
- Extremely reproducible so if two different labs use these, results should be the same
find more resources at oneclass.com
find more resources at oneclass.com
Slide 7 – Two main types of arrays
- Spotted cDNA arrays
o cDNA clones or PCR products (>100 nt)
o “spotting” is variable
o Inexpensive (about $100)
- Oligonucleotide arrays
o Photolithography allows ssDNA probes of 25-60 nt to be built in situ
▪ Affymetrix (25 nt oligos, redundant probes)
▪ Agilent (60 nt oligos)
▪ Amersham (30 nt oligos)
o Extremely reproducible mass of target
o Expensive (about $1000)
- After amplification and purification can be printed on a slide with a printer
- You can modify the printer to print on a slide
o Using this technology, you can print easily 10k different probes on a slide
- Each base that is added, is added to a photoprotection group as well
o You can then de-protect different areas
- You apply a new mask and repeat the process over and over again and repeat the process for all the genes
Slide 8 – Microarray preparation
- cDNA clones (probes)
- Spotted microarrays
- cDNA amplified by PCR (500-2,000bp)
- Or synthesize oligonucleotides
- >10,000 probes per glass slide
Slide 9 – Array preparation (2) – Oligonucleotide
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Eukaryotic cells and viruses lecture 19 (march 21st, 2018) Slide 1 dna microarrays analysis of whole cell gene expression. How to study: northern blot but can, only study 1 or a few genes at a time time consuming and difficult to study gene interactions. Dna chips allow simultaneous monitoring of many genes. Traditional detection: southern blot the dna duplicates, northern blot the rna synthesis, pcr, rt-pcr, western blot the protein synthesis. Tools have been developed to map entire genomes, measure smaller amounts of mrna and protein, and simultaneously measure many more mrnas and proteins. Slide 4 the beginnings of microarray technology. Quantitative monitoring of gene expression patterns with a complementary dna microarray . Expression monitoring by hybridisation to high-density oligonucleotide arrays looking at the expression of mt: march, 2018:>85,000 papers (pubmed) Microarrays can be put on chips and use them to screen for diseases and conditions. Watson-crick base pairing of complementary dna sequences.
