Comparative chromosome painting in fish using human sex ...
Comparative chromosome painting in fish using human sex ...
Comparative chromosome painting in fish using human sex ...
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Punetnhnlw~bhnsdmodsltishfmh wator and agsm dehydrated m wnea oi<br />
study slnw dplold chrmnolame numbar ethanol .a menhond abwe<br />
s lesser and <strong>sex</strong> chrommomes have not<br />
been as far reparced(Mannaand Rmd,<br />
H9b*tk"L<br />
1973. S h a and Amd. 19811 Sa<br />
In abtu hybnduatton af <strong>fish</strong> and huehrommwes<br />
~dentfied on the basia oi man <strong>chromosome</strong> wps oerformed mul.<br />
comparative FISH, can be utllucd far<br />
asangeeUs from mmahlre or adult fieh<br />
and aho for sennp of sprm IPhilLpe,<br />
2W11<br />
tail conr!etlng of 3.6-pl hybridization<br />
Matariplrr nnd methods<br />
buffer. 0.6~1 each ofX and Y praba md<br />
0.6~1 tnpla &Ued water waa a~~hed<br />
Chromo~omrpnpamtbnr<br />
mumlip. mdaeded mth rubkcamat<br />
Danaturationoiprobe md duamoms<br />
Wms performed mmulrameaualy on a hot<br />
plate at 74'C far 5 muur. Slides were m.<br />
abated wnnight <strong>in</strong> mom chamber at<br />
37'C. The mbkr eement and mver ahp<br />
were removed end the slldsa were<br />
waahed by mnrbahngm aolutian of0.35<br />
Chrsmaaome spreads m Channa<br />
pumtolw (n=lOI were pnplvedimm hdnay<br />
tissue, w<strong>in</strong>g eolctunnshypotomc.<br />
methanol - acebe aad - mr drpg tseh.<br />
mque Metaphaaeehmm-rs fmm the<br />
normal <strong>human</strong> mda were obta~ned by<br />
ualnsstandard lymphwe ruifure techniques<br />
and chrme~lmerl~desasre pre<br />
pared by above-mentioned technique<br />
The chmmosome slides to be ued form<br />
arb hybndisahoo wen -red at -2O'C<br />
Whole chmmoaome pslnt IWCP) probes<br />
for <strong>human</strong> Xand Y chmmaaame, dmctly<br />
labelled with two duTamnt fluomphara.<br />
ware prauredfmmVym. Frantm, for the<br />
pre~snt study<br />
The ahdea were thawed lust befm<br />
use and treated wth 70% acsbe acid for<br />
2 m<strong>in</strong>utes, followed by dehydration <strong>in</strong><br />
senna of 70, 90 and 100% ethanol for 3<br />
mui, each, at m m temprat-<br />
h'P40 lauppl~ad by Vpls lac. France) at<br />
74 * 1'C far 3 mutea fallowed by w-<br />
tahng m aoluuon dNP (0.1%) at rmm<br />
tWFemtm for 23 m um. Slidevert<br />
than dehydrated m seriw of ethanol at<br />
roam temperature. Dried slidea were<br />
ata<strong>in</strong>ed and mounted with l0pl DAPI-11<br />
with antifade followed by wal<strong>in</strong>g with<br />
tnmaparentrub~~ m d the*<br />
ofmversltp.<br />
Detection and oirualization of<br />
h9ridpmbe~.<br />
The slidea wore obaerved under<br />
PnIrrcrtmmL.<br />
The dehydrated audned ~Ldem we18<br />
<strong>in</strong>cubated In 1.25% ao&e ppm soluboo<br />
Nikon fluomseant rmmgmp w<strong>in</strong>g ormge'pn<br />
ator Tbe FISH imase~wrn<br />
captd by wled CCD camera and mnvertcd<br />
O computer lmw by CyLmaion<br />
far 20 rmnuree at 37'C followed by wash. Unu 4.1 aabm (Apphed un-) for<br />
<strong>in</strong>s m howh hate buffered &s IPBSi dvna '<br />
and ddob~c-dided water ~hdca am<br />
mcubated for lomutea at 4'C m M y RB.ult4 r<strong>in</strong>d<br />
prepared precooled solutlon of Coutenr~mmgbyDAP1f~dLt.ted<br />
pardomaldehyde 10.87%) prepared m ident~ficat~on of chremosomea of C.<br />
PBS Slzdes were then removed and pvnetotva and all the apeimeu wan<br />
warhed with PBS foUowed by distilled found to possess diploid <strong>chromosome</strong>
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