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MM13 - Disease diagnosis molecular biology methods 3 (mRNA array…
MM13 - Disease diagnosis molecular biology methods 3
Array hybridisation
analyses gene expression patterns in diseased tissues
application: gene expression in cancerous tumours may help...
diagnosis of tumour-type
predict risk of metastasis
develop effective treatment strategies
analyses genetic differences in germline DNA
mRNA array hybridisation
detects presence of different RNA types (qualitative) + quantifies their levels
detects gene expression only (not germline/somatic variation)
transcriptome analysis
tells us gene expression, hence cellular physiology
NB in mechanisms of disease development
1) isolate test + control RNA
2) use reverse transcriptase (from retrovirus) to convert RNA to copy (c) DNA (complementary)
3) add mRNA degrading enzymes mRNA-cDNA to form ss cDNA
4) fluorescently label test + control cDNA
5) add DNA microarray - competitive binding of test + control cDNA
6) data analysis (look @ colour)
Genotype DNA array hybridisation
detects SNPs + mutations
used to identify carriers + identify genetic risk variants for complex disease
method = mostly illumina
1) design genotyping array with oligonucleotides (stop 1 base before SNP target) to target SNPs of interest
oligonucleotides designed for specific mutation (not any)
2) DNA fragmentation + added to array with fluorescent tags
3) fragments hybridise to array
4) colour analysis
can't detect somatic changes, germline only
Next Generation Sequencing
more efficient that Sanger (cheaper + quicker)
best possible resolution - small + large scale variations detectable
COMPLETE ascertainment of germline + somatic variation (not just 1 SNP)
sequences complete genomes/exomes
coding regions only = cheaper
won't identify non-coding variations
many methods, mostly illumina sequencing by synthesis (SBS)
1) prepare sequencing library: fragment DNA + add adaptor sequences (known oligo sequences, can hybridise to sequencing flow cell by complementation)
2) cluster generation: bridge amplification (strand bends over and attaches to a 2nd oligo forming a bridge. A polymerase synthesises the reverse strand. The 2 strands release and straighten) of same read forms flow cell (necessary to achieve required signal)
3) fluorescent labelling + hybridisation - each fluorescent signal (reversible terminators) captured by camera
4) Align to reference + analyses points of variation
types...
targeted
more depth + sensitivity
for cancer genes
used in molecular diagnostic labs
RNA-sequencing
quantifies gene expression
detects fusion transcripts - cancer