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
Affymetrix uses photolabile groups, how does this work
Use of a photlabile group between the nucleotides
When you shine a light on the labile group it removes the block and thus allows adding of the next nucleotide
This is repeated until we get a full sequence
What are some uses of Affymetrix?
Can be used for different organisms or different human genes
Different chips available for different genes etc
Commercial which avoids having to make your own arrays etc -> dont have to design your own probes etc
What is the surface of the array for Affymetrix
A silicon wafer
The oligonucleotides are synthesised onto this
What are the uses of microarrays by Affymetrix
Creation of arrays that can be used for:
- tilling arrays
- high resolution CGH
- SNP arrays
How do gene expression microarraays work?
Fluorescently labelled target sequences bind to a probe sequence
Fluorescence signal quanitified by laser scanner
Total strength of signal depends upon the amount of target sample
How can microarrays be used to investigate gene expression in cancer
Two cell cultures: one nomal and one cancer cel biopsy etc
Isolate mRNA from both cell types
Must convert the mRNA into cDNA using reverse transcriptase enzyme
Use an olligo dT primer to bind to any polya tails of sequences
Once you have your two cDNA pools you can fluorescently label it using fluorescently labelled nucleotides
Mix the two cell populations together in liuid.hybridisation buffer and incubae on surface of array at 60-65 degrees
Sequences (if present) will bind to their complementary probes on the array and fluorescence will occur
Red, green or yellow fluorescence
Why do we use an oligo dT primer when converting mRNA ito cDNA, what is a dT primer
An oligo dT primer is just a row of Ts which will prime reverse transcription
This only works for sequences with poly-A tails i.e. eukaryotic sequences as bacteria dont have poly-A tails on the mRNA
At what temperature do you hybridise your cDNA onto the array when investigating gene expression?
At 60-65 degrees
What are the different kinds of fluorescence youlll see with gene expression microarray and explain these results
Yellow = indicates equal binding i.e. gene is equally expressed in both cel populations
Grey = gene not expressed in either cancer or healthy
Green = gene present in normal cells only
Red = gene present in cancer cells only
Green could be a tumour suppressor i.e only present in healthy -> and not present in cancer
If only in cancer it could be an oncogene
What are the different ways of fluorescent detection in microarrays
Labek DNA with Cy3 dCTP or Cy5 dCTP nucleotides
Cy3 absorps light at 550nm and emits light at 570nm
- green light
Cy5 absorps light at 650nm and emits light at 670nm
- red light
These are generated separately but overlay toether
How do you prepare your sample when comparing normal sample to disease, how would you do this in practice
The two samples to be compared are grown/acquired
RNA, DNA or DNA/RNA bound to a protein
- isolate RNA separately using trisol in acid conditions -> aqueous phase = RNA
The purified RNA is analysed for quality (by capillary electrophoresis and quantity by using a nanodrop spectrometer
- good RNA should have an a260:a280 ratio above 1.7
- below this indicates potential protein contamination
How would you label your RNA for microarray analysis, when doing this in practice
Your mRNA undergoes reverse transcription into cDNA. al mRNAs will have a polyA tail, oligo dT used as a primer
Add your fluorescent label either in the Reverse transcription step or in an additional step after amplification if present
Add Cy3 or Cy5 using a polymerase - done overnight at 37 degrees -> one population with Cy3 and the other with Cy5
The labelled smaples are then mixed with a hybridisation solution
What are the different components of hybridisation solution
SDS
SSC
dextran sulfate
Blocking agent
Formamide
What is SDS
Sodium dodecyl sulfate
- a detergent and nionic surfactant
- it disrupts the noncovalent bonds in protein molecules
- very high sodium concentration
- means only high speciicity binding occurs
What is dextran sulphate
A very sticky viscous solution allowing for mixing
What is the blocking agent in hybridisation solutino
CDT1 -> part of the human genome which is very repetitive
It stops repetitive sequencs from binding
What is formamide in hybridisationb buffer?
This prevents non spcific binding
Maintains speecificity of binding
What do you do after mixing your sequences with hybridisation buffer?
After this you add your sequence to the array and incubate it to allow binding
This is done in a hybridisation oven for 24-48 hours at 60-65 degrees -> constantly rataing and mixing the solution
Then wash away all the liquid and dried
Then put into a scaner hich will exite the samples wih a laser -
A detector measures the emission of light to give you all the different wavelengths of coloured spots etc
After that the raw data is analysed
Give an example of a new microarray available or diagnosis of cancer, what are the pros and cons
MammaPrint dx 70-gene expression array
Developed by Netherlands Cancer Institute
Prognostic and predictive diagnostic test for early stage breast cancer patients
Looks at 70 different genes
One single test costs about 3000 euro but it does give the patient an early diagnosis and could impact their prognostic outcome i.e. whether they will respond well to aggressive chemotherapy or not
How does the MammaPrint ddx 70-gene expression array work
Looks at the expression profile of 70 genes used to categorically calssify the patient as being at either high or low risk of breast cancer recurrenc
Helps physicians determine whether or not a patient will benefit from chemotherapy
Any signals below the threshold = poor gene expression -> breast cancer more likely to spread below this line -> will benefit from chemo
Can determine if cancer will metastasise
-> looks at underexpression and overexpression of certain genes -> if certain genes present this could be good or bad if some genes missing this coujld be good or bad dependeing on if in cancer or not etc
How does the MammaPrint ddx 70-gene expression array work
Looks at the expression profile of 70 genes used to categorically calssify the patient as being at either high or low risk of breast cancer recurrenc
Helps physicians determine whether or not a patient will benefit from chemotherapy
Any signals below the threshold = poor gene expression -> breast cancer more likely to spread below this line -> will benefit from chemo
Can determine if cancer will metastasise
-> looks at underexpression and overexpression of certain genes -> if certain genes present this could be good or bad if some genes missing this coujld be good or bad dependeing on if in cancer or not etc
How does the MammaPrint ddx 70-gene expression array work
Looks at the expression profile of 70 genes used to categorically calssify the patient as being at either high or low risk of breast cancer recurrenc
Helps physicians determine whether or not a patient will benefit from chemotherapy
Any signals below the threshold = poor gene expression -> breast cancer more likely to spread below this line -> will benefit from chemo
Can determine if cancer will metastasise
-> looks at underexpression and overexpression of certain genes -> if certain genes present this could be good or bad if some genes missing this coujld be good or bad dependeing on if in cancer or not etc
Other than gene expression in cancer what is another application of microarrays?
Deletion, duplications and disease diagnosis
How common are visible deletions in humans
Cytogenetically visible deletions occur in 1 in 7,000 llive births
Give some examples of deletion disorders
Cri du cha “cry of the cat” syndrome
Prader-Willi syndrome
Smithe Magenis syndrome
Williams Beuren syndrome
What kind of micrarrays are used to detec deletions and duplications?
Array cgh
Talk about cri du chat syndrome
Disinctive cry affected infants make sue to malformations of the laryns
Affects 1 in every 50,000 live births
Caused by a deletion on the short arm of Chr 5
Also characterised by mental retardation as well as other physical defects
Talk about prader willi syndrom, how does it occur, what does it do
Caused by a deletion on the long arm of Ch15
Other chromosomal abnormalities can also lead to the syndrome
Genetic region involved subject to genomic imprinting - phenotype differs depending upon which parent deletion is inherited from
Both disorders are characterised by mental retardation and a number of physical defects
How did we used to detect prader wili
Used to use chromosomal bandin
Used to have to visibly detect the deletions in chromosomes by looking down the mic
Array CHG replaced this
Give an example o a duplication disease
Charcot-Marie-Tooth disease typ1
17p12 affected
What kind of diseases are smith-magenis syndrome and willimas beuren syndrome?
Theyre both serious neurodevelopmental disorders
What is Charcot-Marie Tooth Disease Type 1?
A group of inherited conditions that damage the peripheral nerves - type 1 is not as serious
Named after the three scientist that found it
Grouped according to what kind of duplication is present
Progressive loss of muscle tissue and touch sensation across various parts of the body
Hammer toes and very high foot arches
Duplication of a large region ont he short arm of Ch17 that includes PMP22 gene
An extra copy of this gene causes damage to the myelin nerves resulting in lose of sensation etc
What is Williams-Beuren Syndrome
A developmental disorder that affects many parts of the body - serious
Causes mild to moderate intellectual disabbility with particular problems with visual spatia tasks
Problems with teeth, heart problems and period of high blood calcium are common
Caused by a deletion of about 27 genes from the long arm of one of the two chromosome 7s - not sure which gene is causative yet
Tend to be social individuals with a characteristic look and mild to moderate intellectual disbility
Calcaemia causes problems with the heart meaning they dont tend to live very long
What deletion is responsible for prader wili and angelman syndromes
Deletion of chromosome 15q11-13
Give two examples of hot spots for deletions
Chromosome 15 and 22
What deletion is reponsible for DiGeorge/Velocardiofacial syndrome
Deletion of 22q11.21-11.23
What duplications are we concerned with in chromosome 15
Smaller inv duplications in 15 are not associated with phenotypic abnormalities
Largere inv duplicatons are associated with an abnormal phenotype
What is duplications in chr22 associated with?
Cat eye syndrome whereby patients eyes will fluorescene in the dark
What are the four ways of detecting chromosomal rearragnements, how do they compare against each other
Karyotyping:
- large chromosomal rearangements
- low resolution down to 5mB
Fish;
resolution down to 100kb
- for sub-microscopic genomic imbalances
Microrrays (BAC and oligonucleotides):
- for both microscopic and sub microscopic detection of copy number changes for the whole genome in a single assay
High resolution microarrays (oligo CGH arrays):
- most efficient means of idenitfying genomic imbalances
- approaching base pair reslution - high resolution
What does array CGH stand for?
Comparative genomic hybridisation
What are the steps in array CGH
1-3: patient and control DNA are labelled with fluorescent dyes and applied to the microarray
4: patient and control DNA competes to attach, or hybridise to the microarray
5: the microarray scanner measures the fluorescent signals
6: computer software analyses the data and generates a plot
Explain in your own words how array CGH workd
Patient DNA sample e.g someone with WBS and a control DNA sample without the condition
Isolate DNA from both
Label patient with Sy3 (green) and control with Sy5 (red)
This allows for comparison between normal and patient
Co-hybridise the labelled patient and control
After 48 hours wash all off the array and look for fluorescence
Determine if deletion or duplication are present
Yellow = equal red and green binding = equal hybridisation = same copy number in patient and control
Red = more hybridisation of control DNA = gene lost in patient = affected patient
Green = duplication or dosage gain in patient compared to control DNA
How do we detect Williams-Beuren Syndrome?
Array CGH to detect the deletion of 10.9MB (large chunk)
- if trained in karyotyping you could detect the deletion this way
- array CGH is the first line for the diagnosis of neurodevelopmental conditions such as WBS
The deletion includes more than 60 genes
- dont know which exact one is causative of the disease
The deletion includes the WBS region at chromosome region 7q11 plus extra genes
- region in Chr7 is a hotspot for deletions and duplications
What can array CGH detect
DNA copy number variations (CNVs) across the genome
CNVs present in healthy control populations as well as individuals with disease
Comparison of cases to controls frequently required to understand contribution of CNVs to disease
Array CGH can be used for the diagngosis of what conditions?
Intellectual disabiity/developmental delay (ID/DD)
Autusm spectrum disorders (ASDs)
Multiple Congenital Anomalies (MCA)
Array CGH can be used for the diagngosis of what conditions?
Intellectual disabiity/developmental delay (ID/DD)
Autusm spectrum disorders (ASDs)
Multiple Congenital Anomalies (MCA)
Talk about array CGH for autism
Range of different mutations present hence a ‘spectrum’
Dont generally know which mutations are causative as many dont go for testing
But generally those with autism have deletions in certain genes
What are the two main companies providing array CGH
Agilent or illumina
What are some high resolution array CGH platforms
Agilent technologies
Affymetrix
Illumina
NimbleGen
Talk about high resolution array CGH, how do you select the right resolution
Large spacing e.g. 43kb away between probes means much lower resolution
1 million, 2.1kb away from each other will give a much higher resolution but this comes at a cost as there are many more probes etc
This means you might only be able to run a few patient samples
It depends on the condition your investigating, e.g. for WBS you could use a cheaper array as its a very large deletion of many genes, 60 genes thuhs plenty of room for probes
However if a small deletion you will need high resolution probes very close together to target such small sequences
What are the two CNV databases
dbGaP
Decipher
What is dbGaP?
Database of Genotype and Phenotyp0e at NCBI
Database for CNV and phenotype data generated from clinical laboratories
What is DECIPHER
A database of chromosomal imbalance and phenotyp0e in humans using Ensembl Resources
