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MM12 - Disease diagnosis molecular biology methods 2 (methods for large…
MM12 - Disease diagnosis molecular biology methods 2 (methods for large scare variation)
G-banding karyotyping
determines no. + banding pattern of chromosomes
detects aneuploidy, large translocations, inversions, very large CNVs (>10 million bases)
carried out on any tissue type
amniotic fluid for foetal karyotype
blood for germline mutations
bone marrow for somatic mutations
1) cells stimulated to divide in culture
2) arrest cells in metaphase + burst them open using hypotonic solution
3) put cells on slide + stain them with giemsa
4) examine under microscope
visible are euchromatin (light, not condensed, gene rich) + heterochromatin (dark, condensed, gene poor)
Fluorescent in-situ hybridisation (FISH)
visualises specific DNA regions + determines their location
higher resolution than karyotyping
can detect smaller insertions/deletions
1) fluorescently label probe (designed to hybridise with target sequence)
2) denature probe (now ss)
3) arrest target DNA in metaphase + hybridise with probe
4) fluorescence microscopy
can detect gene fusions, unbalanced translocations, aneuploidy, gain/loss of chromosomal region
clinical uses
cancer detection (compare tumour sample to blood sample)
find causes of birth defects or metal retardation
e.g. micro deletion syndromes (< 5 million bases, e.g. prader-willi or Angelman)
limitations
can only detect what probe is designed for, ie.e you must know what you're looking for
won't detect submicroscopic changes or balanced translocations
labour-intensive
Array-based Comparative Genomic Hybridisation (CGH)
detects aneuploidy, CNVs + imbalanced translocations
genome wide, high resolution, can detect submicroscopic changes
rapid, cheap, less labour-intensive
1) isolate test genomic DNA
2) fluorescently label test DNA + control DNA (2 different colours)
3) hybridise to microarray (slide with probes) + post-hybridisation washing
control + test DNA compete to hybridise with probes
4) data analysis
excess test DNA - amplification
excess control DNA - deletion
uses: cancer (deletions of tumour suppressor genes, amplifications of oncogenes), unexplained developmental delay, multiple congenital abnormalities
limitations
can't detect balanced translocations
hard to distinguish between normal CNVs + phenotype-causing ones