methods of chromosome analysis - 123seminarsonly.com · 2. cell culturing – cells in different...
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Methods of chromosome Methods of chromosome analysisanalysis
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( He is 4 years old looks like 2. .)y o
Knows few words
Inadequate reaction
Abnormal face
Short hands
, Heart abnormalities kidneyabnormalities
Plurimapformative Plurimapformative syndromesyndrome
What is his karyotype?What is his karyotype?
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. 1 ( Collecting of nuclear cells just leucocytes )if blood is used
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2. Cell culturing – cells in different stages of cell cycle
Thermostat
37º C
72hours
Bloodcells
+ growth broth
Cells indifferen t stages of cellcycle
Proliferation
After 72hours
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3. Preparation of chromosomes for analysis
Cells indifferen t stages of cell
cycle
Hypotonicsolution
Cytostatic-
colchicine
Mitosisarresting
Cellsedimentatio
n
Chromosome dispersing
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Slide with fixed cellsSlide with fixed cells Slide with metaphase platesSlide with metaphase plates
PaintingPainting
4. Painting of chromosomes4. Painting of chromosomes
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5. Karyotype analysis5. Karyotype analysis
Printing ofpicture
Cutting individual
chromosomes
Preparation ofkaryotype
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47,XY,+21 –Down syndrom47,XY,+21 –Down syndrom
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Cytogenetic methods used for karyotype analysis:
To identify numeric aberrations – analysis of metaphase chromosomes uniformly painted.
To identify numeric or structural aberrations – analysis of metaphase or prometaphase chromosomes using Q, G, R banding.
For exact identification of chromosomes – analysis of metaphase chromosomes using T or C banding.
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Analysis of human karyotypeAnalysis of human karyotype
Metaphase chromosomes Prometapfase chromosomes Interphase chromosomes
Uniform painting, , , , G Q R C T banding, G R banding X and Y sex chromatin analysis Molecular citogenetical methods
, FISH mFISHSKYCGH
*** : - / / No of bands per haploid set 300 400 m 550 pm 850p
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Type of bandingType of banding Used dyeUsed dye
GG Giemsa Giemsa
QQ QuinacrinQuinacrin(fluorescent) (fluorescent)
RR ((reversrevers) ) Giemsa or Giemsa or fluorescentfluorescent
CC (centromere) (centromere) Giemsa or Giemsa or fluorescentfluorescent
TT (telomere) (telomere) Giemsa or Giemsa or fluorescentfluorescent
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Chromosome stainingChromosome staining
HomogenousHomogenous BandingBanding
GG QQ RR CC TT
Bands G+ = Q+ = R –Bands G+ = Q+ = R –
Bands G- = Q- = R +Bands G- = Q- = R +
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Cum se colorează diferit acelaşi crs?Cum se colorează diferit acelaşi crs?
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Uniform stainingUniform staining
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C bandingC banding
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Chromosomal bandingChromosomal banding
ChromosomeChromosome
BandBand
ArmArm
SubbandSubband
4p22.24p22.2
5q13.45q13.4
9p21.39p21.3
RegionRegion
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, , ( )( -46 XY del 1 p11)p34
1p35
1p22
1q23
1q41
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, , ( )( -46 XY del 1 p11)p34
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, , ( )( . - . )46 Y del X q12 1 q24 3
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Chromosome 4 Identification of X and 18 Identification of X, Y and 18
Identification of 18 of 21 Identification of 18 and 21
FISH FISH in chromosomal in chromosomal analysisanalysis
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46,XX46,XX 47,XX,+2147,XX,+21
47,XX,+2147,XX,+2147,XX,+2147,XX,+21
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SKYSKY
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mFISHmFISH
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KaryotypeKaryotype
,46 XX
,46 XY
, , +46 XX 9qh
, , -46 XY 16qh
, , ++46 XX 14s
, ,47 XX+21
, , -45 XY 12
,48 XXXY
,45 X
,69 XXX
,69 XXY, , -46 XX 1q
, , +46 XY 16p
, , (46 X r X)
, , (46 XY del 5p)
, , ( ,46 XX t 12 2)2
NormalNormal Normal Normal polymorphismpolymorphism
ss
AneuploidyAneuploidy PolyploidyPolyploidy Chromosomal Chromosomal abberationsabberations
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Abnormalphenotype
invtrob
Balanced structural aberrations
Fenotippatologic
– , ; , ; Trisomy 47 XXX 47 XXY,+ ; ,+ ; ,+ ; 47 21 47 13 47 18,+47 8
– ,Monosomy 45 X
Aneuploidies
Abnormalphenotype
– Chrs D and G variations in ph or s :
, , , , – Chrs 1 9 16 9 Y variations in qh
Polymorphisms
Normalphenotype
,46 XX,46 XY
Normal karyotype
Fenotippatologic
deldupri
Unbalanced structural aberrations
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??? Polymorphisms??? Polymorphisms
• p and q – contain coding and non-coding regions
• p+ or p-- abnormality • q+ or q- - abnormality• c longer or shorter – polymorphism• t longer or shorter – polymorphism• h+ or h- - polymorphism• s+ or s- - polymorphism
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Sex chromatin analysisSex chromatin analysis
X chromatin analysis = Barr body test – X chromatin analysis = Barr body test – identification of non-active X, in interphase nuclei of somatic cells
Y chromatin analysis = F body test – Y chromatin analysis = F body test – identification of 2/3 of qY identification of 2/3 of qY in interphase nuclei of somatic cells or spermsin interphase nuclei of somatic cells or sperms
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Normal karyotype
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- :Y chromosome 397 genes
• regulatory masculinisationgenes
•pseudogenes
•!!! q arm contains constitutive heterochromatin
X chromosome - : 1606 genes
• Structural somatic genes
• Regulatory feminisation;genes
• Structural feminisation; genes
• Structural masculinisationgenes
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!!! Important!!! Important
• X chromosome – is mandatory for ♀ and ♂:
• In 46,XX – only one X is active
• In 46,XY – are active both X and Y
• In 47,XXX - only one X is active
• In 47, XXY- are active only one X and one Y
• In 48, XXXY- are active only one X and one Y
• Y chromosome – is mandatory for ♂
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How to explain this?How to explain this?
•Sexual dimorphism - balancedSexual dimorphism - balanced• Primary sexual traits - differentPrimary sexual traits - different• Secondary sexual traits - differentSecondary sexual traits - different• Somatic traits - similarSomatic traits - similar
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45,X45,X 47,XXY47,XXY
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46,XX46,XX 46,XY46,XY
- Active Xeuchromatin
- Active Xeuchromatin
Y chromosome
– p arm active
- q arminactive
- Inactive Xheterochromatin
F body
∅ = , 0 25 μm
Barr body
∅ = 1μm
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Barr body – X sex chromatinBarr body – X sex chromatin
NrNrBarr Barr = X - 1= X - 1
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Barr body test
XXX X XXXX XXXXX
Y YY
F body test
48,XXXY48,XXXY 45,X45,X 50,XXXXYY50,XXXXYY 49,XXXXX49,XXXXX
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Barr body testBarr body test : X sexual chromatin
- , represents an inactive X facultative, , ; heterochromatin in somatic cells 46 XX
- represents a Barr body 1µ ;m in diameter- Barr body test is useful for identification of
: = No of X chromosomes in somatic cells No X No + .Barr 1
, – 46 XX 1 Barr body, – 46 XY 0, – 47 XXX 2, - 47 XXY 1, – 45 X 0, – 48 XXXX 3
!!! . ( ) - M Lyon 1961 explanation of Barr body
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Mary Lyon hypothesisMary Lyon hypothesis
After 16th day
!!! In 46,XX cells – after 16!!! In 46,XX cells – after 16thth day of development only one X remains active day of development only one X remains active
In 50% of cells – maternal X is active, in 50% - paternal XIn 50% of cells – maternal X is active, in 50% - paternal X
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,46 XX
, + 45 X active X paternalinactive
, + 45 X active X maternalinactive
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F body testF body test : Y sexual chromatin
- / , represents 2 3 of q arm of Y chromosome its a % constitutive heterochromatin in somatic cells or 50 of
( , ); sperms 23 Y only- ,represents fluorescent F body 0 25µ ;m in diameter- F body test is useful for prenatal identification of
: gender = No Y No F bodies
, – 46 XX 0, – 46 XY 1, – 47 XYY 2, - 47 XXY 1, – 48 XXYY 2, , ( ) – 46 X i Yp 0, , ( ) – ( ,46 X i Yq 1 0 5µ )m