CHROMOSOME BANDING

(http://geneticssuite.net/node/25)
Chromosomes display a banded pattern when treated with some stains. Bands are alternating light
and dark stripes that appear along the lengths of chromosomes.
ni!ue banding patterns are used to identify chromosomes and to diagnose chromosomal
aberrations" including chromosome breakage" loss" duplication" translocation or in#erted segments.
$ range of different chromosome treatments produce a range of banding patterns: %&bands" '&
bands" (&bands" C&bands" )*(&bands and +&bands.
,n recent years a number of chromosome banding techni!ues ha#e been de#eloped that employ
molecular cytogenetic techni!ues" for e-ample fluorescence in situ hybridi.ation (/,01).
Chromosome structure and bands
+he chromosomes of eukaryotes are composed of a combination of nuclear 2)$ and proteins.
2uring mitosis chromosomes replicate and condensed through coiling forming chromosomes
consisting of two chromatids 3oined at the centromere. 4ach chromatid condenses appro-imately
ten&thousand fold reaching ma-imal condensation at metaphase 5 2)$ of roughly 5 cm in length is
condensed to 5 micrometers in a metaphase chromosome. +hese condensed chromosomes are
#isible under the light microscope.
2uring the condensation process 2)$ is looped around protein comple-es called nucleosomes.
+his primary structure then undergoes more cycles of coiling producing the well&known metaphase
chromosome structure. 0ome of the looped segments of 2)$ are close together and condense more
than others" forming regions known as domains. +hese closely condensed domains tend to stain
more darkly than the areas where the loops are more loosely arranged.
,n the order 2iptera" which includes 2rosophila" sali#ary gland chromosomes undergo repeated
rounds of replication without cell di#ision forming highly replicated chromosomes 5 polytene
chromosomes. $ polytene chromosome in a 2rosophila sali#ary gland cell can contain as many as
fi#e thousand alternating dark and light bands. ,n these chromosomes the dark bands correspond to
highly condensed domains and the lighter bands to less condensed 2)$. ,t is in the less condensed
areas where acti#e genes can be identified. $ gene becomes acti#e by unra#elling to permit
transcription into messenger ()$. +hese unra#elled regions are obser#ed as 6puffs7 under the
microscope. +he gene becomes inacti#e by resol#ing the 6puff7 through condensation.
%&Banding
%uinacrine mustard" an alkylating agent" was the first chemical to band chromosomes #iewed under
a fluorescence microscope. %uinacrine dihydrochloride has subse!uently been substituted by
!uinacrine mustard. +he alternating bands of bright and dull fluorescence are called % bands. +he
bright bands are primary composed of 2)$ rich in adenine and thymine" while the dull bands are
rich in guanine and cytosine.
% bands are especially useful for distinguishing the human 8 chromosome and #arious chromosome
polymorphisms in#ol#ing satellites and centromeres of specific chromosomes.
'&banding
'iemsa has become the most commonly used stain in human cytogenetic analysis. nlike %&
banding" '&banding usually re!uires pre&treating chromosomes with either salt or a proteolytic
(protein&digesting) en.yme. 9hen chromosomes are pre&treated with the proteolytic en.yme trypsin
the process is called '+' banding. 'iemsa stains preferentially regions rich in adenine and
thymine. +herefore" ' bands correspond closely to % bands.
0tandard ' band staining techni!ues allow between :;; and <;; bands to be seen on metaphase
chromosomes. 9ith high resolution '&banding techni!ues" as many as two thousand different bands
ha#e been catalogued on the twenty&four human chromosomes.
(&banding
(e#erse banding ((&banding) in#ol#es the incubation of slides containing metaphase chromosomes
in hot phosphate buffer and stained with 'iemsa. +he banding pattern that results is essentially the
re#erse of ' bands. ( bands are 'C&rich. +he $+&rich regions are selecti#ely denatured by heat
lea#ing the 'C&rich regions intact. /luorochromes that are 'C specific also produce a re#erse
chromosome banding pattern. (&banding is helpful for analy.ing the structure of chromosome ends"
since these areas usually stain light with '&banding.
C&Banding
C&banding stains areas of heterochromatin" which is tightly packed and repetiti#e 2)$. C&banding
is specifically useful in humans to stain the centromeric chromosome regions and other regions
containing constituti#e heterochromatin & secondary constrictions of human chromosomes =" >" =<"
and the distal segment of the 8 chromosome long arm.
)*(&banding
)*(&banding in#ol#es sil#er staining (sil#er nitrate solution) of the ?nucleolar organi.ing region?"
which contains r()$ genes.
+&Banding
+&banding in#ol#es the staining of telomeric regions of chromosomes using either 'iemsa or
acridine orange after controlled thermal denaturation. + bands apparently represent a subset of the (
bands because they are smaller that the corresponding ( bands and are more strictly telomeric.
2$@,/2istamycin $ 0taining
2$@,/distamycin $ fluorescent staining techni!ue is a method for labelling a specific subset of C
bands. 2$@,/2istamycin $ staining is useful in identifying peri&centromeric breakpoints in
chromosomal rearrangements and in identifying chromosomes that are too small for standard
banding techni!ues.