WK 3 Flashcards
Fluorescence in situ hybridization - FISH
Denature DNA sample, add probe, lower temperature, allow hybridization
Observe areas of hybridization → areas of DNA sequence you are looking for
If you know gene X is in chromosome 7 but FISH shows it in chromosome 1 then there is a translocation
Array-comparative genomic hybridization (array-CGH)
Used for the detection of chromosomal abnormalities that change the copy number of a given sequence
Test (patient) and reference DNA samples are labeled with different colored fluorophores (red = pt DNA, green = reference), fragmented, and made single-stranded. They are mixed unequal genomic amounts and allowed to hybridize to the microarray. Each spot on the array contains a large number of identical single-stranded DNA molecules from a known genomic location. Corresponding fragments of the differently colored test and reference DNA compete to hybridize to the molecules on the array. The average color of a spot on the array after hybridization is a measure of the relative amounts of the corresponding fragments in the test and reference samples. Results: red > green = pt has a duplication, red = green (shown as yellow) = pt has normal copy number, green > red = pt has a deletion.
Array-CGH uses a tiling array – an array chip spotted with genomic sequences that together represent the entire genome; the higher the density of the array, the smaller the genetic region each spot represents and thus the higher resolution the assay can provide.
cannot test
Inverses
balanced translocation
Etc
anything where there is no gain or loss of DNA
whole-genome sequencing
Many millions of sequence reads are analyzed and aligned to the reference genome to obtain statistically significant support for a diagnosis.
Genome sequencing is
highest resolution
can detect micro and macro mutations
VERY EXPENSIVE → always a barrier to using this
SNP
single nucleotide polymorphism
Indels
insertions and deletions
STR
short tandem repeat
CNV
copy number variation
Polymorphism
Variant present in the population at > 1% frequency
Microsatellites
repeat of short sequences
Microscopic Structural variation:
Large changes observable under the microscope
>3 Mb in size
Aneuploidies, large rearrangements, heteromorphisms, fragile sites
Detection methods: banding, FISH, SKY
Sub-microscopic Structural variation:
Too small to be observed under the microscope
Much more frequent than microscopic
~1 kb - 3 Mb in size
CNVs
Detection methods: array-CGH, (long-read) sequencing
Small-scale Structural variation:
SNPs, short repetitive elements, small rearrangements (<1 kb)
Very frequent
Detection methods: molecular biology methods, conventional sequencing
Conventional sequencing = genome sequencing
Changes in chromosome structure are also called chromosome rearrangements. Includes
deletions, duplications, inversions and translocations.
Two major causes of changes to chromosome structure are
DNA breakage and rejoining, and crossing over between repetitive DNA segments. → between non homologous chromosomes.
BREAKAGE AND REJOINING
both DNA strands must break at two different locations, followed by a rejoining of the broken ends to produce a new chromosome rearrangement
the chromosomal breakage can be artificially induced using ionizing radiation
how are chromosome rearrangements produced by breakage:
Each chromosome = single dsDNA
Two or more dsDNA breaks (DSBs) occur
DSBs are potentially lethal unless repaired
DSB Repair systems of the cell repairs DSBs
If two ends of same break are rejoined, original DNA order restored
If two ends of different breaks are joined, we get a chromosomal rearrangement
