Download Microarrays = Gene Chips

Microarrays
= Gene Chips
What is a Microarray (Gene chip)?
• A microarray is an orderly arrangement of short
pieces of single stranded (unpaired) DNA in a grid
on a suitable surface - often a glass slide
• A specialized robot places the spots at precise
intervals (20 to 100 mm diameter)
• Each spot is a single sequence (gene)
• Each spot contains millions of copies of that single
sequence
– A bacterium’s entire genetic make-up can be
contained on a single gene chip
• A computer keeps track of which piece of DNA is
contained in each spot
• Tens of thousands of spots on a single slide
How does it work?
• Single stranded DNA likes to form a double
stranded molecule by matching with a
complementary strand
• One of the basic driving properties of DNA
• Microarrays or gene chips take advantage of this
– DNA:DNA or DNA:RNA
• Based on the ability of single strands of DNA to
hybridise (stick) to complementary (matching)
sequences
• Example, a scientist might wish to figure out which
bacteria are present in a soil sample
Process: DNA:DNA
1. Buy a gene chip with genes representative of bacterial
species (dictionary of barcodes)
2. Extract DNA from the soil sample
3. Use PCR to amplify genes that identify different
bacteria (barcodes)
Extract
DNA
4. Label the PCR products with a fluorescent dye
PCR
5. Wash the labelled PCR products over the gene chip and
allow complementary strands to stick together
6. Wash off any unstuck PCR products
7. Use a laser to detect the fluorescent dyes and create a
visual image of the pattern of the dyes
8. If the PCR product has stuck on it will glow
9. The computer can then say which of the bacterial species
the PCR products have stuck to and this indicates which
species are present in the sample
+
Label
Process: DNA:RNA
• Question – what genes are active in the “on
state”
• Looking at all the genes expressed in a cell,
some are specific but some are general (eg
those involved in respiration)
OFF
• Extract mRNA from two cells – one in off
state and one in on state
• Label one green and one red
• Wash over a gene chip that has a selection
of genes on it
• If the gene matches it will bind to the chip
• Excite with two lasers – one for the green
dye and one for the red dye – computer
overlays the image
Stick to
microarray
ON
• Green = only in off
state
• Red= only in on
state
• Yellow = equal in
both states
www.brunel.ac.uk/.../researchgroups/mf/mp
'GreeneChip' - New diagnostic
tool that rapidly and accurately
identifies multiple pathogens
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The Greenechip System
Diagnosis of infectious diseases, including those caused by viruses, bacteria,
fungi, or parasites
Can be used on a wide variety of samples, including tissue, blood, urine, and
stool
Can get an answer in 6 hours
The chip has almost 30,000 pieces of genetic material taken from thousands of
different viruses, bacteria, fungi and parasites
– represent all recognized 1,710 vertebrate viral species and 135 bacterial,
73 fungal, and 63 parasite genera.
For each family or genus at least 3 separate genomic target regions are used
The test was used on a sample from a healthcare worker who had died of fever
and liver failure during the Marburg outbreak in Angola. Previous tests for
hemorrhagic fevers had been negative. The GreeneChip result pointed to
Plasmodium falciparum malaria ( www.cdc.gov/EID/13/1/06-0837.htm).
Plant health diagnostics on a microchip
In Europe:
• A wide range of organisms can attack plants, from viruses to
insects
• Those that are serious pests and diseases are quarantined under
the EU Plant Health Directive (almost 300 pathogens)
• This legislation controls the passage of plant material between
countries
• To implement the legislation, Member States and Acceding
countries must be able to diagnose all the pests and pathogens,
some of which are obscure
• Currently, diagnosis requires a wide range of expertise, from
traditional taxonomy to classic microbiology and molecular
biology
• The Diag Chip project, with a consortium of plant disease
scientists from Germany, France, Spain and the UK aimed to
create a gene chip for this detection – currently under
construction
Example
•
•
Previously no means to identify
new cancer classes (class
discovery) or for assigning
tumours to known classes (class
prediction)
Important for targeting
treatment
1999 Science 286:531-537
• The 50 genes
most highly
associated with
the ALL-AML
class distinction
• row = a gene
• column = a
patient
• Expression
levels are
normalised
using the mean
• > mean = red
• < mean = blue