• Chromosomes are structures found in the nucleus of cells
•Chromosomes carry all of our genes, and therefore all of
our genetic information
•Humans have 46 chromosomes, or 23 pairs, to carry our
approximately 25,000 genes
•The first 22 pairs are called autosomes
•The 23rd pair are the sex chromosomes or gonosomes
this pair will either be XX or XY
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
During mitosis, the 23 pairs of human
chromosomes begin condensing during
prophase, and reach their most condensed
state during metaphase
A karyotype analysis usually involves
blocking cells in mitosis and staining the
condensed chromosomes with Giemsa dye.
 The dye stains regions of chromosomes that are
rich in the base pairs Adenine (A) and Thymine (T)
producing a dark band
A karyotype is a pattern or picture of chromosomes. The
chromosomes are paired and arranged according to size
 Each chromosome is paired with its homologous
chromosome

 its exact match in size and structure, though the homologous
chromosomes may carry different alleles of the same gene

Then the chromosome pairs are labeled
 The autosomes are numbered 1 through 22 according to size
 The sex chromosomes are a different story
▪ The X and X or the X and Y are paired, then placed at the end, even
though they are not necessarily the smallest chromosomes
▪ These chromosomes do not receive a number - just XX or XY
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A karyotype is an actual photograph of the
chromosomes from one cell.
Karyotypes are usually done using blood
cells, fetal skin cells (from amniotic fluid or
the placenta) and occasionally bone marrow
cells
It takes at least one week to complete a
karyotype
1. Sample Collection
2. Separating cells
3. Growing Cells
4. Synchronizing Cells
5. Releasing Chromosomes from their cells
6. Staining Chromosomes
7. Analysis
a. Counting & Sorting of chromosomes
b. Looking at Chromosomes Structure

In newborns, a blood sample which contains
red bloods cells, white blood cells, serum and
other fluids is collected.

A karyotype will be done on the white blood
cells which are actively dividing (a state
known as mitosis).
 During pregnancy, the sample can either be
amniotic fluid collected during an amniocentesis
▪ The amniotic fluid contains fetal skin cells which are
used to generate a karyotype
 or a piece of the placenta collected during a
chorionic villi sampling test (CVS).
 The important thing is to choose a cell type that is
actively dividing, some cells, like nerve cells,
divide infrequently or never!

In order to analyze chromosomes, the sample
must contain cells that are actively dividing (or in
mitosis).
 In blood, the white blood cells are actively dividing
cells.
 Sometimes adult cells are treated with chemicals such
as phytohemaglutenin (PHA) to induce mitotis
 Most fetal cells are actively dividing.

Once the sample reaches the cytogenetics lab,
the non-divided cells are separated from the
dividing cells using special chemicals.
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In order to have enough cells to analyze, the
dividing cells are grown in special media or a
cell culture.
This media contains chemicals and hormones
that enable the cells to divide and multiply.
This process of “culturing” the cells can take 3
to 4 days for blood cells, and up to a week for
fetal cells


The best mitotic phase for chromosome
analysis is prometaphase or metaphase
In order to get all the cells to this specific
stage of cell division, the cells are treated
with a chemical which stops cell division at
the point where the chromosomes are the
most compact.
 cholicine
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
In order to see these compact chromosomes
under a microscope, the chromosomes have to
be out of the white blood cells.
This is done by treating the white blood cells
with a hypotonic solution that causes
 Chromosomes to spread apart for easier viewing
 And causes cells to burst

This is done while the cells are on a microscopic
slide. The leftover debris from the white blood
cells is washed away, and the chromosomes are
fixed to the slide.


Chromosomes are naturally colorless.
In order to be able to tell one chromosome from
another, a special dye called Giemsa dye is
applied to the chromosomes on the slide.
 Giemsa dye stains regions of chromosomes that are
rich in the bases adenine (A) and thymine (T).

When stained, the chromosomes look like
strings with light and dark bands.
 Each chromosome has a specific pattern of light and
dark bands which enables cytogeneticist to tell one
chromosome from another.


1st use a protease to digest any protins
Giemsa reagent consists of a mixture od dyes
including
 Aminophenothiazine dyes(basic)
 Eosin (acidic)

Produces light & dark bands
 Patterns of banding change with the stage of mitosis
 There may be as many as 1,000 bands in early
prophase, and as few as 300 on a given chromosome
in metaphase
LIGHT BANDS
DARK BANDSS
GC rich
Most genes are in light
bands
 Light bands represent
relatively open (uncoiled)
regions of DNA making
transcription easier


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
AT rich
Highly coiled regions of
DNA
 Contains few genes
 Most genes in these
regions are tissue specific,
and so not transcribed in all
cells


Once chromosomes are stained, the slide is
put under the microscope and the analysis of
the chromosomes begins.
A picture is taken of the chromosomes and at
the end of the analysis
 This is known as a “metaphase spread”
 Take a picture of the spread using a special
microscope with attached camera

When we draw chromosomes, we typically
draw them in the shape of the letter X
 What does this represent (ie, label the important
parts of the X structure)

Why, in the above metaphase spread are the
chromosomes NOT in the X shape, but rather
linear?

The first step of the analysis is counting the
chromosomes.
 Most humans have 46 chromosomes.
 People with Down syndrome have 47 chromosomes.
 It is also possible for people to have missing
chromosomes or more than one extra chromosome.

By looking at just the number of chromosomes,
it is possible to diagnose different conditions
including Down syndrome, Turners, Kleinfelters

Sort the chromosomes by
 comparing chromosome length,
 the placement of centromeres (the areas where the
two chromatids are joined),
 the location and sizes of G-bands.

The chromosomes pairs are numbered from
largest (number 1) to smallest (number 22).
 There are 22 pairs of chromosomes, called
autosomes, which match up exactly.
 2 sex chromosomes
▪ two X‘s is a female
▪ X and a Y is a male.

In addition to looking at the total number of
chromosomes and the sex chromosomes, the
cytogeneticist will also look at the structure
of the specific chromosomes to make sure
that there is no missing or additional
material, no structural abnormalities like
 Translocations
 Deletions
 Duplications

Unfortunately in high school we aren’t
allowed to stick needles in our students
and/or work with human bodily fluids
 Possible spread of infectious diseas
 Parents complain

We will practice karyotyping from the
analysis standpoint

The analysis involves comparing
chromosomes for their
 Length
 placement of centromeres (areas where the two
chromatids are joined)
 location and sizes of G-bands.
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Each chromosome has 2 arms separated by a
constriction point called the centromere
Metacentric: term used to describe
chromosomes with centromere at or near the
center of the chromosome
Submetacentric: term used to describe
chromosomes with centromere slightly
displaced from the center
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
Very submetacentric: term used to describe
chromosomes with centromere halfway
between the middle and the tip of the
chromosome
Acrocentric: term used to describe
chromosomes with centromere very near one
end

Telocentric: term used to describe
chromosomes with centromere at the very
tip of one end
 These are absent in human chromosomes

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Notice, the centromere divides the
chromosome into 2 separate regions
In general
 The shorter chromosome arm is designated p for
petite
 The longer arm is designated the q arm, because
the letter q comes after the letter p in the
alphabet
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Open your worksheet titled Key to Human
Chromosomes
It is a dichotomous key
Start by cutting chromosomes out of your
metaphase spread
Divide all chromosomes into one of the 2 class
sizes
 Class 1 = 1st 12 chromosome pairs (24), and X
chromosomes
▪ So class 1 will contain 25 chromosomes for a boy and 26
chromosomes for a girl
 Class 2 = the rest of the smaller chromosomes
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Normal female  46, XX
Normal Male  46, XY
Telomeres, centromeres, and a number of
prominent bands areused as landmarks on a
chromosome

A section of a chromosome between 2
landmarks is called a region
 Regions are numbered 1,2,3 in both directions
starting with the centromere
 Bands within a region are numbered according to
the same rule

Example: the 1st band in the 2nd region of the
p arm of chromosome #1 is indicated as
follows
 1p21
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14 refers to chromosome # 14
q refers to the q arm (the longer arm) of
chromosome 14
3 refers to region 3 of the q arm of
chromosome 14
And 2 refers to the 2nd band in the 3rd region
of the q arm in chromosome 14
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There may also be sub-bands
Decimal points are used to indicate what subband
Example: 14q32.3
 Refers to the 3rd (last) sub-band within the 2nd
band of region 3 in the q arm of chromosome 14

45, X  45 chromosomes, 1 X chromosome

47, XXY  45 Chromosomes, two X
chromsomes one Y chromosome

Use a + or – sign
 before the symbol to indicate additional or
missing whole chromosomes
 After a symbol to indicate additional or missing
segments of a chromosome


47, XY, +21  an additional 21st chromosome
46,XX, 1q+  female karyotype with 46
chromosomes showing an increase in the
length of the long arm of chromosome 1

Question marks are used to indicate
uncertainty
 45, XY, -?8
▪ 45 chromosomes
▪ XY is male
▪ Missing a chromosome, probably #8

There is special nomenclature for inversions,
deletions, translocations, insertions, etc.; but
its is beyond the scope of this class

C-Banding: stains constitutive
heterochromatin
▪ Regions of chromosomes that are highly compacted
containing highly repetitive DNA
 Results from selective removal of the
chromosomes except from regions of the C-Bands
 Used for determining presence or absence of a
centromere, as well as for studying
abnormalities of the centromeric
region
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Silver Staining for NOR
Ribosomal DNA, which codes for ribosomal
RNA, occurs in multiple copies on the short arms
of all acrocentric chromosomes
Silver stain results in the deposition of silver
grains on the active nucleolar organizer regions
(NORs)
 In other words, NORs which were actively transcribing
their rRNA’s in the preceding interphase, contain the
proteins to which thre silver grains bind
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Fluorescence In Situ Hybridization
Involves precise annealing of ssDNA probes
to complementary target sequences
Can be visualized with a fluorescent
microscope by using a probe directly labeled
with a fluorophore
Can be used to find
 If a specific gene is present (and or duplicated)
 And if it is in the correct region of the correct
chromosome
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Whole Chromosome Painting
Same idea as FISH, but you use a mixture of
many fluorophore labeled probes
 Must also use unlabeled blocking DNA to supress
highly repetitive regions common to many
different chromosomes

Allows for
 Chromosome enumeration
 Identification of chromosomal structural
rearrangements


Particularly useful in solid tumor and linical
genetic research because it has the ability to
aid in the analysis of complex and cryptic
translocations
Also helpful in research that is evaluating the
extent of chromosomal breakage and
translocations following exposure to
mutagenic chemicals or radiation

A karyotype allows a cyto-geneticist or lab
technician to examine the chromosomes and
see if there is anything extra or missing, or if
the structure of the chromosomes is grossly
different than usual
Trisomy 21 is the presence
of 3 chromosome 21’s.
 Trisomy 21 causes the
condition commonly
known as Down syndrome
 The extra chromosome
leads to the specific
characteristics of Down
syndrome, some of which
are very familiar

 not all individuals with
Down syndrome will show
the exact same
characteristics there is a
great deal of variability

Individuals with Down syndrome have a typical facial appearance

All have some degree of mental retardation, but for most it is mild
or moderate
 Can learn to read, write, do some math, and live day-to to-day with
minimal assistance from others
 Others require a lot of attention and care

There are several possible health concerns, including
 heart problems, hearing loss, feeding concerns, and others

Individuals with Down syndrome are now living longer than in the
past – into their 50’s and 60’s

We are now discovering that those individuals who survive to this
age are at very high risk of developing Alzheimer Alzheimer’s
disease

Trisomy 13 is the presence of 3 chromosome 13’s, and causes the
condition sometimes known as Patau syndrome

Trisomy 13 is a very serious condition with only about 5% of babies
with the disorder survive past their first year
 Most pregnancies involving Trisomy 13 end in miscarriage

All individuals with Trisomy 13 have severe mental retardation

Other characteristics commonly seen in people with Trisomy 13
include





a small head
extra fingers and/or toes
and a cleft lip or cleft palate
Trouble breathing, especially in their sleep
seizures

Trisomy 18 is the presence
of 3 chromosome 18’s, &
causes the condition
sometimes known as
Edwards syndrome

Only about 10% of babies
with the disorder survive
past their first year.
 A majority of babies who
survive are female.
 Most pregnancies involving
Trisomy 18 end in
miscarriage


Children with Trisomy 18 usually have
breathing problems, difficulty eating, and
many have seizures. Some have serious heart
conditions.
All individuals with Trisomy 18 have severe
mental retardation.Most babies with Trisomy
18 are very small and have certain
recognizable facial features. They also tend to
overlap their fingers in a very distinct pattern



An individual with the genotype 47, XXY is
male 􀁺 The person has 47 chromosomes just
like someone with Trisomy 21, 13, or 18, but
does not have a typical “trisomy,” as he has
two of one chromosome and one of another
One could say, though, that such an
individual has a sex chromosome trisomy
The extra X chromosome leads to features of
the condition commonly known as Klinefelter
syndrome


Affects about 1 in 1000 males
Most males are taller than average and may have a
different distribution of body fat (e.g. more than usual
in the hips or chest)
 Also tend to have sparse facial and body hair
 Some have a degree of mental retardation, but many have
normal intelligence
 The most common feature is infertility
▪ It is estimated about 2% of infertile men have Klinefelter Syndrome

Since the characteristics of the syndrome are not
always obvious, many males with Klinefelter will
never be diagnosed
An individual with the genotype 45, X is
phenotypically female
 The person has 45 chromosomes instead of the
usual 46 Instead of a trisomy, this would be
called a monosomy X
 One copy of an X chromosome = monosomy

 Monosomy X is the only monosomyknown to be
compatible with life
 Having only one copy of an X chromosome leads to
the features of the condition known as Turner
syndrome


Affects about 1 in 5000 newborn females
Physical features
 Swelling of the hands and feet
 webbing of the neck
 broad chest

May also have features which affect their health,
including
 heart disease
 a“horseshoe-shaped”kidney
 Individuals with Turner syndrome will not typically
have mental retardation, but may have specific
learning disabilities

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

Why would the presence (or absence) of a
chromosome lead to certain characteristics in a
person?
Why wouldn’t the physical, mental, and
behavioral features be exactly the same for two
people with the same extra (or missing)
chromosome?
Why are babies with trisomy 21 more likely to
survive than babies with trisomy 13 or 18?
Why do you think monosomy X (Turner
syndrome) is the only monosomy in which the
individual survives?