137+FISH+slides

137+FISH+slides - O ld s t y le b a n d in g a llo w s id e...

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Unformatted text preview: O ld s t y le b a n d in g a llo w s id e n t ifi c a t io n o f e a c h c h r o m o s o m e b y b a n d s a n d s iz e Karyotyping 2007 Which chromosomes are there? Are they normal? Tools of the trade Jlmarsh, UCI, 2008 C h r o m o s o m e p a in tin g a n d b a n d in g a llo w s id e n tific a tio n o f e a c h c h r o m o s o m e Specific color pattern for each chromosome http://www.sciencemuseum.org.uk/exhibitions/genes/154.asp S p e c tra l k a ry o ty p in g ( S K Y ) a te c h n iq u e th a t a llo w s s c ie n tis ts to v is u a liz e a ll o f th e h u m a n c h r o m o s o m e s a t o n e tim e b y " p a in tin g " e a c h p a ir o f c h r o m o s o m e s in a d iffe r e n t flu o r e s c e n t c o lo r . in te r p h a s e m e ta p h a s e One color per chromosome w w w .g e n o m e .g o v /1 0 0 0 0 2 0 8 http://www.bio.unc.edu/courses/2003spring/ biol050 h/biolog2.jpg M e ta p h a s e v s in te rp h a s e M ito tic m e ta p h a s e is o n ly ~ 2 % o f th e c e lls ! Must culture and trap by cholchicine http://www2.m rc-lmb. cam.ac.u k/personal/sl/Html/Graphics/CellCycle.gif One probe gives a pair of dots/bands BAC probe w w w .c a n in e -g e n e -p r o je c t.d e / F is h .h tm l 1 Where on the chromosome are you? BAC probe on stretched metaphase Detecting aberrations in metaphase ncgr.ac.cn/ chinese/news7.htm What things cause aberrations? E.g. Hexavalent chromium! Hexavalent chromium! C h ro m a te tre a te d lu n g c e lls • W h e n d o u b le s tr a n d b r e a k s fo r m , H is to n e g a m m a -H 2 A X (H 2 A X ) r a p id ly a c c u m u la te s o n th e m a n d la b e ls th e b r e a k fo r r e p a ir . • A n H 2 A X -s p e c ific flu o r e s c e n t a n tib o d y , a p p e a r s a s a g lo w in g g r e e n d o t. • E a c h d o t r e p r e s e n ts a d o u b le s tr a n d b r e a k in th e D N A u n tre a te d lu n g c e lls F ig u re 6 B . A s p e c tra l k a ry o ty p e o f th e m e ta p h a s e in fig u re 6 A . T h is k a ry o ty p e s h o w s a tra n s lo c a tio n b e tw e e n c h ro m o s o m e s 2 a n d 5 . http://www.u sm. m aine. e du/toxicology/research/chromium. php Hexavalent chromium! Gel electrophoresis http://www.u sm. m aine. e du/toxicology/research/chromium. php R ita lin C h ro m a te tre a te d lu n g c e lls • C o m e t in d ic a te s tr a ilin g b ro k e n D N A fra g m e n ts Electrophoretic migration u n tre a te d lu n g c e lls http://www.u sm. m aine. e du/toxicology/research/chromium. php 2 Nucleoplamic bridges br A tr a n s lo c a tio n b r e a k (t4 :? ) M a rk e rs o f c h ro m o s o m e re a rra n g e m e n ts (dicentrics, rings etc) 1 1 2 2 t4 t4 4 Chromosome painting, or fluorescence in situ hybridization (FISH) Figure. An example of FISH-treated metaphase chromosomes Here, chromosomes 1, 2, and 4 were labeled yellow with FISH and the other chromosomes were stained red. Translocations between yellow and red chromosomes are detected. The left picture represents a normal cell (the numbers in the figure indicate chromosome numbers) and the right picture is an example of reciprocal translocation with two bi-color chromosomes (indicated by two arrows). w ww.rerf . or.j p/ Gene/eng/fig/fishf.htm A n e w tr a n s lo c a tio n th a t r e a r r a n g e s th e A M L 1 g e n e in a p a tie n t w ith T -c e ll a c u te ly m p h o b la s tic l e u k e m ia . M ik h a il F M , S e r r y K A , H a te m N , M o u r a d Z I, F a r a w e la H M , E l K a ffa s h D M , C o ig n e t L , N u c ifo r a G . C a n c e r G e n e t C y to g e n e t 2 0 0 2 ; 1 3 5 (1 ): 9 6 -1 0 0 A n e w tr a n s lo c a tio n th a t r e a r r a n g e s th e A M L 1 g e n e in a p a tie n t w ith T -c e ll a c u te l y m p h o b la s tic l e u k e m ia . M ik h a il F M , S e r r y K A , H a te m N , M o u r a d Z I, F a r a w e la H M , E l K a ffa s h D M , C o ig n e t L , N u c ifo r a G . C a n c e r G e n e t C y to g e n e t 2 0 0 2 ; 1 3 5 (1 ): 9 6 -1 0 0 Metaphase FISH analysis using the BAC probe RP11-104M2 hybridized to the patient's metaphase shows one normal green signal on the intact chromosome 4 (dashed arrow) and two smaller green signals on d er(21) (arrowhead) and on der( 4) (arrow) as a result of the t(4;21)(q28;q22) Oncogenesis T he predicted AML1-FGA7 chimeric proteins contain a limited number of amino acid residues fused to AML1 in a situation similar to that reported for AML1-EAP fusion that is a product of t(3;21). It is possible that the expression of a constitutively shortened AML1 could compete with full-length AML1 and act as a dominant negative inhibitor of the promoters that the core binding factor (CBF) activates. Metaphase FISH analysis using the BAC probe RP11-104M2 labeled with FITC (green) hybridized to a normal metaphase cell confirms the chromosomal localization of the probe (gene) to 4q28. a tla s g e n e tic s o n c o lo g y. o r g / G e n e s /F G A 7 ID 5 2 5 .h tm l C h a n g e s in c h ro m o s o m e # tris o m y 8 http://atlasgeneticsoncology . org/Anomalies/A nomliste. html Interphase vs metaphase DOTS vs BANDS 3 sep probes www.c hrombios . com/Karyotyping/Mouse_ES1.html IN T E R P H A S E IS C H E A P E R A N D E A S IE R vs c olchicine-arrested metaphases 3 Interphase vs metaphase 2 probes on same chromosome A b b o t t la b s t ( 1 1 ; 1 8 ) ( q 2 1 ; q 2 1 ) D u a l C o lo r , D u a l F u s io n T r a n s lo c a t io n P r o b e These probes are for detecting Chromosome rearrangements involving MALT1 (Mucosa associated lymphoid tissue lymphoma translocation gene 1) on chromosome 18q21. In t(11;18)(q21;q21), a translocation involving the MALT1 gene, a gene fusion is produced on the der(11) chromosome between a 5' portion of the API2 (11q21) gene and a 3' portion of the MALT1 gene (18q21). The resulting API2/MALT1 fusion gene encodes an abnormal, but functional chimeric protein. The green probe spans the API2 gene while the red probe spans the MALT1 gene. The pattern seen in a translocation heterozygote is shown here. http://international.abbottmolecular.com/LSIAPI2MALT1t(11;18)(q21;q21)DualColorDualFusionTranslocationProbe_6739.aspx Small probes can overlap at this resolution (e.g. a cosmid). INVERSION W illia m s - B e u r e n S y n d r o m e ( W B S ) ( O MIM #194050) = very interesting. Inversions, deletions, Lenhoff, contiguous gene, autism etc. 3 probes on same chromosome NO PROBE SPANS A BREAK!! R B W W B R B W R R W B tel cen FISH showing t(15;17). FISH study of this translocation using a cosmid p robe in which the green and red signals represent the sequences in normal chromosomes 15 (green) and 17 (red); the fusion product is shown as a mixture of signals (yellow).[4] www.meds.com/leukemia/guide/guide_chapter4.html cen Inversion Normal Fig 1: Inversions of chromosome 7 in Williams-Beuren syndrome. Bottom three dots show normal order: green, yellow, pink. Top three dots show WB inversion: yellow, pink, green. h ttp ://w w w .g e n o m e n e w s n e tw o r k .o r g /a r tic le s /0 4 _ 0 3 /c h r o m 7 . s h tm l h ttp ://w w w .u to ro n to .c a /o s b o rn e /re s e a rc h .h tm l A n e u p lo id y s c r e e n in g Detecting changes in chromosome # Using probes for chromosomes 13, 18, 21, X and Y, to rapidly screen samples of amniotic fluid for the common trisomies. Results usually available in 48 hours. P A T IE N T NORM AL CONTROL www.humangenetics. org.u k/ molcyto.htm 4 H o w m a n y w a y s c o u ld t h is b e in t e r p r e t e d ? Trisomy 18 T r is o m y, t r a n s lo c a t io n , in v e r s io n , P o s s ib le d u p lic a tio n N e e d to k n o w c e n tr o m e r e # ; o r d o o th e r k a r y o ty p in g http://www.b iomedcentral.com/content/figures/1471-2407-6-249-2.jpg CENTROMERE PROBES CENTROMERE T r is o m y 8 ( + 8 ) . F IS H . C e n tro m e re p ro b e fo r c h ro m o s o m e 8 (g re e n ). T h re e c e lls e a c h w ith tr is o m y 8 ( th r e e g r e e n s ig n a ls each) ( i n te rp h a s e p r e p a r a tio n ) U s e 2 p ro b e s in d iffe r e n t p a rts o f c h r 2 1 to d is tin g u is h tr is o m y f r o m a b e r r a tio n Chromosome painting lights up the extra chromosome 21 in the cells of people with Down syndrome (pink and light blue dots). http://www.a ccessmedicine. com/search/s earchAMResultImg. aspx? r ootterm= t risomy+ x+syndrome&r ootID= 42751&s earchType= 1 http://www.sciencemuseum.org.uk/exhibitions/genes/156.asp A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation Nature Genetics 24, 167 - 170 (2000) How do you know when you have the DNA that contains the gene of interest? A b b e r a t io n s a n d F IS H & /o r S o u t h e r n b lo t s . Figure 1 . Genomic region of the X chromosome containing th e X ;2 tra n s lo c a tio n b re a k p o in t a n d T M 4 S F 2 (X p 1 1 .4 te tra s p a n in ) . 107D22 a , P h y s ic a l m a p o f th e X p 1 1 .4 lo c u s a n d g e n o m ic s tr u c tu r e o f T M 4 S F 2 . T h e p o s itio n o f th e b r e a k p o in t is in d ic a te d b y a v e r tic a l d o tte d lin e . T h e e ig h t e x o n s a r e in d ic a te d ( fille d b o x e s ) a n d th e ir m a p p in g s h o w e d th a t T M 4 S F 2 s p a n s a t le a s t 1 3 0 k b . S T S s a n d E S T s a r e in d ic a te d ( to p ) a n d th e o r d e r o f E S T s t h a t m a p in th e o v e r la p p in g r e g io n b e tw e e n Y A C c lo n e s 7 7 0 D 1 2 a n d 9 5 7 F 5 w a s d e fin e d h e r e th r o u g h th e ir p o s itio n o n th e B A C c o n tig . E S T A A 9 1 1 3 2 4 9 w a s r e p o r te d in th e E S T d a ta b a s e , b u t its p o s itio n in th is r e g io n w a s d e d u c e d th r o u g h s e q u e n c in g o f th e e n d s o f B A C c lo n e 1 1 0 L 0 3 . A ll B A C c lo n e s w e r e fr o m th e C E P H B 7 5 1 B A C lib r a r y . b, Metaphase spread from the patient with the X;2 translocation showing FISH signals obtained with BAC clone 107D22 (red) and with a c hromosome-X−s p ecific p robe (green). C h r o m o s o m e s X , d e r X a n d d e r 2 w ith h y b r id iz in g s ig n a ls a r e in d ic a te d . N o te th e h y b r id iz a tio n s ig n a ls o n b o th d e r X a n d d e r 2 c h r o m o s o m e s , in d ic a tin g th a t th is B A C c lo n e s p a n s th e b r e a k p o in t X cen 2 X cen 5 Summary • P r o b e s c a n b e u s e d t o id e n tify c h a n g e s in g e n e o r c h ro m o s o m e n u m b e r, o r abberations. • T h e le v e l o f r e s o lu tio n = th e s iz e o f th e p ro b e . • S o u th e r n b lo ts i n te r r o g a te v e r y d e fin e d d o m a in s • F IS H in te r r o g a te s la r g e r d o m a in s 6 ...
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This note was uploaded on 12/13/2011 for the course BIOSCI 137 taught by Professor Staff during the Fall '11 term at UC Irvine.

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