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A. Chromosome Banding.
Chromosome banding techniques are based on denaturation and/or
enzymatic digestion of DNA, followed by incorporation of a DNA-binding dye. This results in
chromosomes staining as a series of dark and light bands.
1. G-Banding.
G-banding uses trypsin denaturation before staining with the Giemsa dye and
is now the standard analytical method in cytogenetics.
a.
Giemsa staining produces a unique pattern of
dark bands (Giemsa positive; G bands)
which consist of heterochromatin, replicate in the late S phase, are rich in A-T bases,
and contain few genes.
b.
Giemsa staining also produces a unique pattern of
light bands (Giemsa negative; R
bands)
which consist of euchromatin, replicate in the early S phase, are rich in G-C
bases, and contain many genes.
2. R-Banding.
R-banding uses the Giemsa dye (as above) to visualize
light bands (Giemsa
negative; R bands)
which are essentially the
r
everse of the G-banding pattern. R-banding
can also be visualized by G-C specific dyes (e.g., chromomycin A
3
, oligomycin, or
mithramycin).
3. Q-Banding.
Q-banding uses the fluorochrome
q
uinacrine (binds preferentially to A-T
bases) to visualize
Q bands
which are essentially the same as G bands.
4. T-Banding.
T-banding uses severe heat denaturation prior to Giemsa staining or a combi-
nation of dyes and fluorochromes to visualize
T bands
,
which are a subset of R bands,
located at the
t
elomeres.
5. C-Banding.
C-banding uses barium hydroxide denaturation prior to Giemsa staining to
visualize
C bands
,
which are constitutive heterochromatin, located mainly at the cen-
tromere.
B. Fluorescence in situ Hybridization (FISH).
■
The FISH technique is based on the ability of single stranded DNA (i.e., a DNA probe) to
hybridize (bind or anneal) to its complementary target sequence on a unique DNA
sequence that one is interested in localizing on the chromosome.
■
Once this unique DNA sequence is known, a fluorescent DNA probe can be constructed.
■
The fluorescent DNA probe is allowed to hybridize with chromosomes prepared for
karyotype analysis and thereby visualize the unique DNA sequence on specific chromo-
somes.
C. Chromosome Painting.
■
The chromosome painting technique is based on the construction of fluorescent DNA
probes to a wide variety of different DNA fragments from a single chromosome.
■
The fluorescent DNA probes are allowed to hybridize with chromosomes prepared for
karyotype analysis and thereby visualize many different loci spanning one whole chro-
mosome (i.e., a chromosome paint). Essentially, one whole particular chromosome will
fluoresce.
D. Spectral Karyotyping or 24 Color Chromosome Painting.
■
The spectral karyotyping technique is based on chromosome painting whereby DNA
probes for all 24 chromosomes are labeled with five different fluorochromes so that each
of the 24 chromosomes will have a different ratio of fluorochromes.
■
The different fluorochrome ratios cannot be detected by the naked eye but computer
software can analyze the different ratios and assign a pseudocolor for each ratio.
■
This allows all 24 chromosomes to be painted with a different color. Essentially, all 24
chromosomes will be painted a different color.
■
The homologs of each chromosome will be painted the same color, but the X and Y chro-
mosomes will be different colors, so 24 different colors are required.
E. Comparative Genome Hybridization (CGH).
■
The CGH technique is based on the competitive hybridization of two fluorescent DNA
probes; one DNA probe from a normal cell labeled with a red fluorochrome and the
other DNA probe from a tumor cell labeled with a green fluorochrome.
Chapter 10
Chromosomal Morphology Methods
95
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■
The fluorescent DNA probes are mixed together and allowed to hybridize with chromo-
somes prepared for karyotype analysis.
■
The ratio of red to green signal is plotted along the length of each chromosome as a dis-
tribution line.
■
The red/green ratio should be 1:1 unless the tumor DNA is missing some of the chromo-
somal regions present in normal DNA (more red fluorochrome and the distribution line
shifts to the left) or the tumor DNA has more of some chromosomal regions than present
in normal DNA (more green fluorochrome and the distribution line shifts to the right).
A.
The appearance of chromosomal DNA can vary considerably in a normal resting cell (e.g.,
degree of packaging, euchromatin, heterochromatin) and a dividing cell (e.g., mitosis and
meiosis). It is important to note that the pictures of chromosomes seen in karyotype analy-
sis are chromosomal DNA at a particular point in time i.e., arrested at metaphase (or
prometaphase) of mitosis.
B.
Early metaphase karyograms showed chromosomes as
X
-shaped because the chromosomes
were at a point in mitosis when the protein
cohesin
no longer bound the sister chromatids
together but the centromeres had not yet separated.
C.
Modern metaphase karyograms show chromosomes as
I
–shaped because the chromosomes
are at a point in mitosis when the protein cohesion still binds the sister chromatids together
and the centromeres are not separated. In addition, many modern karyograms are
prometaphase karyograms where the chromosomes are
I
-shaped.
A.
A chromosome consists of two characteristic parts called
arms
.
The short arm is called the
p
(petit) arm
and the long arm is called the
q (queue) arm.
B.
The arms of G-banded and R-banded chromosomes can be subdivided into
regions
(count-
ing outwards from the centromere),
subregions (bands), sub-bands
(noted by the addition of a
decimal point), and
sub-sub bands
.
C.
For example, 6p21.34 is read as: the short arm of chromosome 6, region 2, subregion (band)
1, sub-band 3, and sub-sub band 4. This is not read as: the short arm of chromosome 6,
twenty-one point thirty-four.
D.
In addition, locations on an arm can be referred to in anatomical terms:
proximal
is closer to
the centromere and
distal
is farther from the centromere.
E.
The chromosome banding patterns of human G-banded chromosomes have been standard-
ized and are represented diagrammatically in an idiogram.
F.
A
metacentric chromosome
refers to a chromosome where the centromere is close to the mid-
point, thereby dividing the chromosome into roughly equal length arms.
G.
A
submetacentric chromosome
refers to a chromosome where the centromere is far away from
the midpoint so that a p arm and q arm can be distinguished.
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Chapter 10
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2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
Y
A
B
C
D
E
FIGURE 10-1. Karyotypes and chromosomal morphology. (A)
G-banding of metaphase chromosomes with only minimal
separation of the sister chromatids are shown arranged in a karyotype. Chromosomes 1 through 3 consist of the largest
metacentric chromosomes. Chromosomes 4 and 5 are slightly smaller and submetacentric. Chromosomes 6 through12 are
arranged in order of decreasing size with the centromere moving from a metacentric position to a submetacentric posi-
tion. Chromosomes 13 through 15 are medium sized and acrocentric. Chromosomes 16 through18 are smaller and meta-
centric. Chromosomes 19 and 20 are even smaller and metacentric. Chromosomes 21 and 22 are the smallest chromo-
somes and acrocentric. The X chromosome is similar to chromosomes 6 through 12. The Y chromosome is similar to
chromosomes 21 and 22. (B) Karyotype of Down syndrome. G-banding of metaphase chromosomes with only minimal sep-
aration of the sister chromatids are shown arranged in a karyotype. Note the three chromosomes 21 (circle). (C) FISH for
Down syndrome.
FISH using a probe for chromosome 21 (red dots) shows that each cell contains three red dots indicat-
ing trisomy 21. The green dots represent a control probe for chromosome 13. (D) FISH for sex determination. FISH using a
probes for the X chromosome (green) and the Y chromosome (red) shows that a cell that contain one green dot and one
red dot indicating the male sex. The two blue areas represent a control probe for chromosome 18. (E) Chromosome paint-
ing.
Chromosome painting using paints for chromosome 4 (green) and chromosome 14 (red) shows a chromosomal
rearrangement between chromosomes 4 and 14 (chromosome with green and red staining; arrow). (continued)
H.
A
telocentric chromosome
refers to a chromosome where the centromere is at the very end of
the chromosome so that only the q arm is described.
I.
An
acrocentric chromosome
refers to a chromosome where the centromere is near the end of
the chromosome, so that the p arm is very short (just discernible).
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98
BRS Genetics
F
G
H
1
2
3
1
11
4
12
FIGURE 10-1.
(continued) (F) Spectral karyotyping of a chronic myelogenous leukemia cell line demonstrating a complex
karyotype with several structural and numerical chromosome aberrations. (F1)
A metaphase cell showing the G-banding
pattern. (F2) The same metaphase cell as in F1 showing the spectral display pattern. (F3) The same metaphase cell as in
F1 and F2 arranged as a karyotype and stained with the spectral karyotyping colors. Arrows indicate structural chromo-
some aberrations involving two or more different chromosomes. (G) Spectral karyotyping. Spectral karyotyping using
paints for chromosome 1 (yellow) and chromosome 11 (blue) shows a balanced reciprocal translocation between chro-
mosomes 1 and 11, t(1q11p). A balance translocation means that there is no loss of any chromosomal segment during the
translocation. This forms two derivative chromosomes each containing a segment of the other chromosome from the
reciprocal exchange. (H) Spectral karyotyping. Spectral karyotyping using paints for chromosome 4 (blue) and chromo-
some 12 (red) shows an unbalanced reciprocal translocation between chromosomes 4 and 12, t(4q12q). An unbalanced
translocation means that there is loss of a chromosomal segment during the translocation. In this case, the chromosomal
segment 12 is lost.
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99
1.
Which one of the following is a suitable
specimen for cytogenetic analysis?
(A)
placenta in formalin
(B)
frozen (not cryopreserved) blood plasma
(C)
frozen (not cryopreserved) amniotic fluid
(D)
peripheral blood
2.
Which one of the following is the appro-
priate specimen for cytogenetic analysis
where the patient is a child with dysmorphic
features and unexplained mental retarda-
tion?
(A)
peripheral blood
(B)
skin
(C)
bone marrow
(D)
cheek cells
3.
A cytogenetics laboratory report states
that a patient has a deletion of a chromo-
some distal to 5p15.31. Which of the follow-
ing best describes what this means?
(A)
There is a deletion of a portion of the
long arm of chromosome 5 with the
breakpoint at band p15.31.
(B)
There is a deletion of a portion of the
short arm of chromosome 5 with the
breakpoint at band p15.31.
(C)
There is a deletion of a portion of the
long arm of chromosome 15 at band
5p31.
(D)
There is a deletion of a portion of the
short arm of chromosome 15 at band
5p31.
4.
Which one of the following is often the
preferred stage for more detailed cytogenetic
analysis?
(A)
meiotic prometaphase
(B)
meiotic metaphase
(C)
mitotic prometaphase
(D)
mitotic metaphase
5.
In a balanced reciprocal translocation in
which two chromosomes exchange pieces, a
breakpoint in which one of the following
would be most likely to cause gene disrup-
tion and thus an abnormal phenotype?
(A)
Giemsa negative G-band
(B)
Giemsa positive G-band
(C)
Giemsa negative R-band
(D)
C-band
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