Shane 5: Microarrays Flashcards
What are the two methods of DNA microarray analysis
Gene Expression Arrays
Comparative Genomic Hybridisation (array CGH)
What are DNA microarrays
A collection of microscopic DNA spots attached to a solid surface
Each DNA spot contains picomoles (10^12 moles) of a specific DNA sequence (probes or oligos)
Each DNA probe occupies a particular “spot” on the chip
Fluorescently labelled target sequences bind to probes and generate a signal - this cn be quanitified
What can a DNA microarray also be called?
A DNA chip
What kind of solid surfaces are commonly used in microarrays?
Glass slides
How are the DNA spots on microarrays formed?
Robots deposit the DNA on to the slides
Companies will synthesis short oligonucleotides on the surface of the array or you can use PCR products
How is the fluorescence from microarrays read
Any yellow spot means equal fluorescence
Green is positive
red is negative
What is the historical background behind microarrays
Southern blotting developed in 1975 by Edwin Southern
The concept of DNA microarrays began in the mid 1980s
Pin based robotic system was developed by Lehrach’s group in 1990
‘Quantitative Monitoring of Gene Expression Patterns with a complementary DNA microarray” Patrick Brown, Mark Schena and colleagues in Science
What is the principle behind microarrays?
Single-stranded DNA probes specific for sequence A
Mixure of single-stranded mRNA molecules in the cell
Mix the two together
A* can only bind to its complementary sequenct A
What exactly are we looking for with microarrays
Looking for DNA fragments in a gnome sequence
In microarrays, what do the complementary sequences bind to
Probes
Who was the first to use microarrays and how
Patrick Brown was the first to use microarrays on plants
How do DNA microarrays work?
Core principle behind microarrays is hibridisation
Samples are labelled using fluorescent dyes
Usually two samples are hybidised to chip
Complementary nucleic acid sequences pair via Hydrogen bonds
Washing off of non-specific binding sequences
How are nucleic acids hybridised for microarray analysis
A duplex of complementary base-pairing strands is denatured using salts and heats
Complementary strands are then hybridised to these single strands through labelled probes
What are the two main types of DNA microarrays?
Spotted DNA arrays
Oligonucleotide arrays
What are the two types of oligonucleotide arrays?
Gene Chips (affymetrix)
Inkjey microarrays (agilent)
What are spotted DNA arrays?
The first method used - developed by Patrick brown in stanford
PCR products or long oligos from known genes (-100nt -1kb) spotted on glass, plastic or nylonn support
Bacterial artificial chromosome (BAC) clone inserts (150-350kp) array CGH
Customisable and off the shelf
What gene did Patrick Brown use
Arabidopsis - a plant genome
Made PCR products of these and spotted them on to solid surface
What are BACs?
Longer sequences of DNA
You can use these clonse inserts for human DNA microarrays
How are spotted DNA arrays customisable?
You can make your own PCR product and spot whatever you want
What company utilsies gene chips
Affymetrix
What are gene chips?
Several 20-25 mers/gene on silica substrate
Tiling arrays for array CGH
Enabled by photolithogrophy from the computer industry
You can buy different genes in the human genome
Probes are quite small
Used to look for smal deletions and duplications
High resolution of these mutations
Who uses Ink-Jet microarrays?
Agilent
What are ink-jet microarrays
Large number of 25-60mers printed directly on glass
Four cartridges A, C, G and T
Flexible, rapid
Can build up probe sequence for any gene of interest
What is the principle behind spotted DNA arrays
Probe DNA molecules spotted onto a slide by a robotic printer - directly apply DNA to slide
What are the pros and cons of spotted DNA arrays?
Can be done inexpensivley and in house
Can achieve greater probe length than oligo arrays
- inexpensive as once you have your PCR products you have an unlimited amount of product to spot on
Disadvantages are speed, spotting distance and number of probes deposited
Also requires a glass slide coated in poly-L-lysine to allow binding of DNA onto slide
How is DNA spotted onto glass in spotted DNA analysis
A robot with tiny pins goes into the 96 well plate containing the PCR producrs
They take up a very small amount of DNA and deposit this onto the surface of the array
What must glass be coated in spotted microarrays, why
poly-L-lyine
To allow binding of DNA onto the slide
What should spotted DNA arrys be used for
For analysis that doesnt require a high number of probes
What kind of DNA is needed for spotted microarrays, how is this proven
Single stranded, reverse or foward
Proven using double stranded, forawrd single and reverse single strands -> double wont fluorescence as much as single, but reverse will fluorescence just as much as forward
In spotted microarrays what needs to be added to primes
An amino linker must be added ont the end of a primer
Only one strand will have the amino linked i.e. not the reverse
Why do you need to add the amino linker in spotted microarrays?
This is crucial for the binding of PCR products/DNa onto the surface of the array
Glass array is coatd in poly-L-lysins which will bind to this amino linker in the squence
Why do you need to add the amino linker in spotted microarrays?
This is crucial for the binding of PCR products/DNa onto the surface of the array
Glass array is coatd in poly-L-lysins which will bind to this amino linker in the squence
What do we do after complementary sequence and probe has bound to arrray in spotted microarray
We dont want the probe - so we heat it up and wash it away
The linker will keep th complementary strand attached thus were only left with our sequence
Explain in your own way how spotted microarray works?
(need to confirm with chat gpt)
Fluorescently labelled probes designed to bind complementary strand of interest
Complementary sequence/PCR product etc binds to probe
Amino linker binds to complementary strand
Amino linker:Probe binds to Poly-L-lysine on array
Heating removes the probe to just leave the complementary strand
What is the principle behind the affymetric method of oligonucleotide microarrays
Uses photolithogrophay to place probes on to glass or silicon surface
Affymetrix uses photolabile groups between nucleotides (break in response to light and allows the sequence of probes to be determined)
Millions of probes can be fixed using this fabrication technique
Creation of arays that can be used for tilling arrays, high resolution CGH and SNP arrays