Microarrays Flashcards
What’s a SNP
Single nucleotide polymorphism. DNA variation occurring commonly within a population (>1%). Where a single nucleotide differs between members of a species
What’s the issue with SNP arrays
SNP are naturally occurring in genome and have an uneven distribution, with them occurring higher in non-coding regions.
If a SNP is in a non-coding region what can it affect
Splicing, transcription, mRNA degradation, sequence of non-coding RNA
Why have SNP-Oligo arrays been developed
To overcome the issue of uneven coverage of SNP distribution over genome
Give two examples of SNP Oligo platforms
Affymetrix: human SNP array 6.0, OGT: cytosure ISCA+SNP 4x180, Agilent: cytochipSNP 4x180
Describe affymetrix SNP array system briefly
906,000 SNPs 946,000 CNVs. Target genome digested by two restriction endonucleases, fragments are amplified and labelled. After hybridisation read outs are generated.
Oligo: strength of signal indicates copy number. SNP: 100%complemetary to 25mer will be bright yellow.
Describe Illumina high density human SNP (only) bead array
Bead based platform: set in microcells on a silicon array. Whole genome isothermal amplification, DNA fragmentation, hybridisation onto beads (1 SNP allele per bead/ multiple probes), wash non-specific binding/ non-hybridised DNA, extension step, fluorescence read outs.
Describe the two Illumina SNP extension methods briefly
Single nucleotide extension: the 50mer corresponds to the sequence adjacent to the SNP. At the extension step ddNTPs are added (G/C: Biotin labelled green) (A/T: dinitrophenyl labelled red) ddNTP will be incorporated that’s homologous to the SNP on the target DNA: colour will be detected.
Allele-specific primer extension: 50mer extends over the SNP site so the 3’end overlaps the SNP site. At extension add labelled dNTPs and sequence is synthesised, only perfect hybridisation between probe and target DNA will allow extension: read signal output
Why chose Oligo SNP array
Detect copy number change, higher resolution than SNPs, can detect LOH/UPD (Oligo can’t), alleles specific oligonucleotides
In oncology which genes are found mutated in UPD regions (detected by SMP arrays)
WT1, FLT3, CEBPA, RUNX1
Name some cancers that have know LOH/UPD regions
Lymphomas, MM, CRC, AML (~20%)
Name a constitutional disease where SNP arrays/analysis helps with treatment
Duchenne muscular dystrophy. Glucocorticoid corticosteroids therapy: 39 SNPs differentiate into 2 groups: high and low responders.
Name two types of Oligo array
Chips: Agilent: 25-85mer laser jet printed onto a glass slide; Affymetrix oligonucleotide synthesised onto a silica surface.
Beads: Illumina: 70mer Oligo with the last 25mer unique for sequence- 100,000 per bead
What are the advantages of Oligo arrays
Multiple pts per slide, good reproducibility, high resolution (50-200bp),
What are the disadvantages of Oligo arrays
Can have poor signal to background ratio due to small probe size, increased number probes required to make a call, CNVous often detected, can’t detect bal rearr, expensive, no positional info
What’s the advantage of SNP arrays
Detect LOH/UPD, high resolution, multiple pts per slide,
What’s are the disadvantage of SNP arrays
Can’t detect bal rearr, uneven coverage across genome, expensive, labour intensive, CNVous found, no positional info
What are the advantages of BAC arrays
Due to large size of probe- they’re very stable, less CNVous, good for mosaicism, has replicates so increases confidence when called, tolerates poor quality DNA.
What’s are the disadvantages of BAC arrays
Lower resolution than other arrays, miss small imbalances, expensive (2 pt per slide), no positional info, can’t detect bal rearr
What is an expression array
It allows the simultaneous investigation of 1000s genes as it compares the gene expression levels (quantitative) of RNA (cDNA).
What are advantages of expression arrays
Can compare the expression of a set of genes simultaneously in different tissue types, different stages of malignancy, normal vs tumour, etc. can labelled two sets of cDNA with red and green and run on same array to directly compare
What is a disadvantage of expression arrays
Stringent normalisation is required. Requires clustering algorithms to be able to analyse the data
What’s it important to think about the actual copy number of a call when analysing and array (not just if its deal or dup)
A deletion might be casual, but a duplication not. An amplification might have a phenotype, but a duplication not. A homozygous deletion might manifest whereas a hemizygous deletion doesn’t.
How would you class aCNV as benign
Common polymorphism (found in >1% of the population), documented in multiple peer reviewed publications or curated databases as a benign variant.
(Miller et al 2010: Inherited from a healthy parent, similar CNV present in a healthy relative, gene poor, devoid of know regulatory elements)
How would you class aCNV as uncertain, likely benign
Reported in a small number of normal variation databases but too low to be called common, no genes but region is large
How would you class aCNV as uncertain
Insufficient evidence for unequivocal determination of significance
How would you class aCNV as uncertain/likely pathogenic
Single case report with defined breakpoints and phenotype relevant to patient. A gene with compelling function that’s relevant to patient
How would you class aCNV as pathogenic
Documented as clinically significant in multiple peer reviewed journals. Known disease/syndrome region. Most cytogenetically visible alterations.
(Miller et al 2010: expanded cnv inherited from parent, cnv from affected parent/relative, CNV overlaps CNV in a DD/ID, ASD, MCA database, overlaps a know syndrome, overlaps a morbid OMIM gene, CNV is gene rich, it’s a homozygous deletion, it’s an amplification)
Name some databases used for CNV interpretation
DGV: database of genomic variation.
DECIPHER. ISCA. OMIM. Pubmed. NCBI gene reviews
