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7/29/2019 FISH Analysis of Chromosomes and DNA Recombination
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FISH analysis of chromosomes:
Fluorescent IN SITU hybridization
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metaphase spread chromosomes stained with
DAPI, a fluorescing stain that specifically binds
double stranded DNA
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green = probe for end of chromosome 4
Expose DAPI-stained metaphase
chromosomes to fluorescent probes
red = control probe for centromere of
the X chromosome & another probe forend of chromosome X
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DiGeorge syndrome/CATCH22microdeletion on chromosome 22
birth defect that affects the immune system
absence of or underdevelopment of the thymus and
parathyroid glands
facial features include low-set ears, wide-set eyes, small
jaw, and bowing up of upper lip
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FISH tests: DiGeorge syndrome
Expose DAPI-stained
chromosomes to mixture
of fluorescent probes
green = control probe for
chromosome 22
red = probe for DiGeorge
region on long arm of
chromosome 22
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FISH tests: Painting chromosomes
Expose chromosomes to fluorescent probes that highlight
entire chromosomes
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FISH tests: Painting chromosomes
Expose chromosomes to fluorescent probes that highlight
chromosomes 13, 18, 21, X, and Y
nuclei fromthe same
fetus
green = chromosome 13
red = chromosome 21
aqua = chromosome 18
green = X chromosome
red= Y chromosome
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DNA Recombination
General recombination
Site specific recombination
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General DNA Recombination
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Heteroduplex joint
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General Recombination
Two homologous DNA molecules cross over
The site of exchange can occur anywhere
A strand of one DNA molecule has become
base-paired to a strand of the second DNA
to create heteroduplex joint
No nucleotide sequences are altered
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The procedure of general recombination
DNA synapsis: base pairs form between
complementary strands from the two DNA molecules
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DNA Hybridization
The initial step for DNArecombination
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RecA protein-mediated DNA synapsis
Rec A has multiple DNA binding sites, hence can hold a single strand
and a double helix together
Rec A is also a DNA-dependent ATPase
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DNA Branch Migration
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Holiday Junction for DNA recombination
Exchange of the first single strand between two different DNA double helices is slow and
difficult, then intermediate state Holiday Junction, then complete exchange
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Holiday Junction for DNA
recombinationand its resolution
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Summary for General
RecombinationGeneral recombination allows large fraction of
genetic information to move from one
chromosome to another.
General recombination requires the breakage ofdouble helices, beginning with a single strand
breakage.
General recombination is facilitated by Rec A inbacteria and its homologs in eucaryotes.
Holiday junction is the intermediate state of
general recombination
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Site-specific recombinationMoves specialized nucleotide sequence (mobile
genetic elements) between non-homologous sites
within a genome.
Transpositional site-specific recombination
Conservative site-specific recombinatinon
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Transpositional site-specific
recombinationModest target site selectivity and insert mobile
genetic elements into many sites
Transposase enzyme cuts out mobile genetic
elements and insert them into specific sites.
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Three of the many types of mobile genetic elements found in bacteria
Transposase gene: encoding enzymes for DNA breakage and joining
Red segments: DNA sequences as recognition sites for enzymes
Yellow segments: antibiotic genes
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Cut and Paste Transposition
DNA-only
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The structure of the central intermediate formed by transposase (integrase)
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Replicative Transposition
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Retrovirus-based Transposition
Retroviral-like retrotransposition
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Reverse Transcriptase
RNA
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Non-retroviral retrotransposition
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Conservative Site Specific Recombination
Integration vs. inversion
Notice the arrows of directions
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