IN SITU HYBRIDIZATION BANDING

1. In Situ Hybridization(ISH)

In situ hybridization (ISH) is used to map and order genes and other DNA and RNA sequences to their
location on chromosomes and within nuclei. In situ hybridization (ISH) is a means of identifying where
mRNAs are present in fixed tissue samples; as IHC identified proteins in fixed tissue, ISH identifies mRNA.

The technique is based on the principle that double-stranded DNA denatures on heating to single-
stranded DNA. ISH is performed by designing an antisense probe to your mRNA target, allowing your
probe and mRNA to bind, and visualizing where your probe is in the tissue slice. In situ is Latin for “in
location,” and so you could consider ISH and IHC to be in situ techniques.

On cooling, the single-stranded DNA reanneals with its complementary sequence into double-stranded
DNA. If an appropriately labeled fragment of a DNA sequence (a DNA probe) is denatured and added to
denatured nuclei or chromosomes on a routine, air-dried interphase preparation during the process of
reannealing, some of the labeled DNA will hybridize to its complementary sequence in the chromosomal
DNA. Detection of the labeled DNA probe under the microscope will identify the site of hybridization
and thus the region of chromosomal DNA complementary to the DNA sequence in the labeled probe. If,
for example, the DNA probe represents a sequence of more than 1 kb from a cloned gene, ISH has the
capability of assigning that gene to its chromosomal location.

ISH and IHC can be run together in the same tissue sample as an elegant control to show that your ISH
probe and your IHC antibody are specific, but the two techniques have different requirements and so
this control is difficult to pull off. To get an ISH probe, you can design it much the same way as a PCR
primer, except you’re only interested in the antisense probe. For ISH, you can use cDNA probes,
oligonucleotide probes, or RNA probes. RNA probes are reputed to be the best, followed by oligos,
followed by cDNA. RNA probes are typically generated by introducing plasmids into bacterial cultures
(like Escherichia coli) and purifying the probe. They are very sensitive (in every sense of the word) and
require everything to be RNase free at every step before probe binding (after binding, the mRNA/probe
is considered by RNases to be double stranded and therefore not a target). Oligonucleotide and cDNA
probes are not as sensitive but don’t come with the “everything must be RNase free and why won’t you
cater to my every whim” mentality that RNA probes require. Oligonucleotide probes are typically
ordered from companies, unless you have a nucleic acid synthesis machine available. cDNA probes are
generated from PCR; the PCR product is followed by asymmetric PCR (differing concentrations of
forward and reverse primers) to generate a concentrated amount of probe.

Types of ISH Probe Labeling

Once you have your probes, you must decide how to acquire a signal. Radioactive probes are the most
sensitive, but nonradioactive methods have been making great strides in recent years. For radioactive
probes, you have a couple of choices: 35S, 125I, 32P, and some others.
 The iodine and phosphorus isotopes are “hotter” and have higher reactivity and shorter half-lives
than the sulfur; sulfur seems to be the best to use unless you have a low abundance target, in which
case the room is divided—some prefer to use hotter probes, others contend that sulfur is really just the
best overall. Nonradioactive probes are currently split between biotinylated probes, digoxigenin (DIG)
probes, and tyramide probes. Biotinylated probes are amplified using an ABC kit or streptavidin,
similarly to IHC, though you can also use an anti-biotin antibody and a secondary antibody to amplify the
signal. Tyramide probes are amplified using, unsurprisingly, a tyramide amplification kit. DIG probes are
amplified using anti-DIG antibodies and other antibodies, if necessary. The draw of DIG probes is that
DIG itself is found in plants, and so you should have zero background in your animal tissue from the anti-
DIG antibodies. Biotin and tyramide are pursued because, while not as sensitive as radiation, you can
still get quite a lot of amplification out of those probes. RNA probes are more sensitive than
oligonucleotide probes, which are more sensitive than cDNA probes. Additionally, he claims that 35S is
more sensitive or equal to 33P, which is more sensitive than 32P, which is more sensitive than biotin and
DIG. He also says that frozen tissue works better than paraffinized tissue. Your mileage may vary, but
this is a good start.

You can order probes with these signal-generating molecules attached, or you can attach them yourself
using different kits. Either way is costly. Be aware that some kits and some companies will attach
molecules via a dideoxynucleotide base; this means you can attach only one molecule, and your
signal/noise better be awesome for you to get anything for your time. It is better to have a regular
nucleotide, so that you can add many signal molecules to your probe. If you use multiple types of signal
molecule, you can perform multiplex ISH, using a combination of different probes (biotin, DIG,
fluorescein) and antibodies (anti-biotin, anti-DIG, anti-fluorescein) to generate signals (or different
fluorescent molecules on the probes, but this doesn’t allow as much amplification or long probe
storage). As with fluorescent immunoblot or IHC, make sure that your fluorophores emit nonoverlapping
wavelengths of light.

Visualizing Your ISH

The process of visualizing your data depends on what kind of probe you use. For radioactive probes,
you can submit your tissue to liquid emulsion followed by photography, or autoradiography film. Use of
the film has the same issues here that it does for immunoblotting; you need a minimum amount of
signal to get the silver grains to react, and there is a point where the film can’t detect any further
changes.

For nonradioactive probes, you have many of the same options you have for IHC: fluorescence and
colorimetric staining. Colorimetric stains require that you include an enzyme at some point in your
experiment, whether conjugated to streptavidin or a secondary antibody (I’ve not heard of someone
attaching HRP or AP directly to their probe, and you won’t get much amplification that way). For
fluorescence, you can attach a fluorophore to your probe,streptavidin, or secondary antibody.
Afterward, you can visualize your colorimetric or fluorescent ISH reactions using the same methods of
microscopy that you used for IHC.
2.Fluorescence In Situ Hybridization (FISH)

FISH (fluorescence in situ hybridization) technique FISH

deploys DNA probes specific for each chromosome (or
subchromosomal region or single locus). These probes are fragments of DNA or RNA, usually 100–1,000
bases long, used to detect the presence of nucleotide sequences that are complementary to the
sequence in the probe. The probes are labeled with modified nucleotides that fluoresce under particular
conditions. By using different fluorochromes, a karyotype can be “painted” as desired. A 24 different
probes and fluorochromes produce a visually striking karyotype that has powerful uses in cytogenetics.
It allows, for instance, direct karyotyping of a metaphase spread without the laborious cutting and
arranging process usually employed.

Principles of fluorescence in situ hybridization (FISH).

(a) The basic elements of FISH are a DNA probe and a target sequence. (b) Before hybridization, the DNA
probe is labeled by various means, such as nick translation, random primed labeling, and PCR. Two
labeling strategies are commonly used: indirect labeling and direct labeling .For indirect labeling, probes
are labeled with modified nucleotides that contain a hapten, whereas direct labeling uses nucleotides
that have been directly modified to contain a fluorophore. (c) The labeled probe and the target DNA are
denatured. (d) Combining the denatured probe and target allows the annealing of complementary DNA
sequences. (e) If the probe has been labeled indirectly, an extra step is required for visualization of the
nonfluorescent hapten that uses an enzymatic or immunological detection system. Whereas FISH is
faster with directly labeled probes, indirect labeling offers the advantage of signal amplification by using
several layers of antibodies, and it might therefore produce a signal that is brighter compared with
background levels.

Soon after Gall and Pardue's work, fluorescent labels quickly replaced radioactive labels in hybridization
probes because of their greater safety, stability, and ease of detection (Rudkin & Stollar, 1977). In fact,
most current in situ hybridization is done using FISH procedures (Trask, 2002; Speicher & Carter, 2005).

Detecting a DNA sequence can be compared to looking for a needle in a haystack, with the needle being
the DNA sequence of interest and the haystack being a set of chromosomes. This search is made much
easier if the investigator has a powerful "magnet"—in this case, a fluorescent copy of the DNA sequence
of interest. Hybridization occurs when the "magnet" meets the "needle"; this requires both a probe and
a target, as shown in Figure 1. In the figure, the probe sequence, often a piece of cloned DNA, is shown
in red. The target DNA—chromosomes on a glass slide—is shown in blue (in the right column). Hydrogen
bonds that join the two strands of the DNA helix are represented by black lines.

The first step in the process is to make either a fluorescent copy of the probe sequence or a modified
copy of the probe sequence that can be rendered fluorescent later in the procedure . Next, before any
hybridization can occur, both the target and the probe sequences must be denatured with heat or
chemicals. This denaturation step is necessary in order for new hydrogen bonds to form between the
target and the probe during the subsequent hybridization step. The probe and target sequences are
then mixed together, and the probe specifically hybridizes to its complementary sequence on the
chromosome. If the probe is already fluorescent, it will be possible to detect the site of hybridization
directly. In other cases, an additional step may be needed to visualize the hybridized probe. Hybrids
formed between the probes and their chromosomal targets can be detected using a fluorescent
microscope.

Using FISH to Identify the Positions of Genes

Two micrographs show FISH

experimental results. Panel A shows red
bands on two chromosomes in a spread
of metaphase chromosomes, which appear
as white and grey banded structures on a
black background. In panel B, a FISH probe is used to map a breakpoint of a translocation. The
chromosomes appear as blue and gray banded structures on a black background, and three
chromosomes are marked with a red signal.

Cytogenetic analyses of sequence-integrated clones.

(a) Using FISH, fluorescent signals are observed at cytogenetic bands (grey) where fragments of a
sequence-tagged bacterial artificial chromosome hybridize (red). (b) A clone selected on the basis of
band location is used in FISH analysis to map the breakpoint of a translocation involving chromosomes
11 and 19 in a patient with multiple congenital malformations and mental retardation. The clone spans
the breakpoint on chromosome 19; thus, the red signal is split between the derivative 11 and derivative
19 chromosomes and is present on the normal chromosome 19.

FISH provides a powerful tool for identifying the location of a cloned DNA sequence on metaphase
chromosomes. Figure 2a shows the results of a typical FISH experiment, in which a cloned DNA
sequence was hybridized to normal metaphase chromosomes. Red bands are detected at hybridization
sites on two homologous chromosomes, which can be identified by their characteristic banding patterns.
Closer examination shows that each red band actually consists of two spots, corresponding to the two
sister chromatids in a mitotic chromosome. A skilled cytogeneticist would be able to use these
hybridization data together with the banding pattern to place the probe sequence within a few
megabases of other known genes on the chromosome.

Historically, FISH and other in situ hybridization results played a primary role in mapping genes on
human chromosomes. Results from these experiments were collected and compiled in databases, and
this information proved useful during the annotation phase of the Human Genome Project (HGP). Now
that the HGP is complete, investigators rarely use in situ hybridization simply to identify the
chromosomal location of a human gene. (In species for which the genome has not been sequenced,
however, FISH and related in situ hybridization methods continue to provide important data for
mapping the positions of genes on chromosomes.) Currently, human FISH applications are principally
directed toward clinical diagnoses.

Diagnosing Chromosomal Abnormalities Using Karyotypes and FISH

FISH and other in situ hybridization procedures are important in the clinical diagnosis of various
chromosomal abnormalities, including deletions, duplications, and translocations. Figure 2b shows one
example in which investigators used FISH together with standard karyotyping to analyze a patient
translocation. The hybridization probe corresponded to a segment of chromosome 19 that was
suspected to include the translocation breakpoint. Three areas of hybridization are apparent in the
fluorescent image. One spot corresponds to the patient's normal copy of chromosome 19 (nl19), and the
other two spots correspond to the altered, or derived (der), versions of chromosomes 11 and 19 that
were produced during the translocation. Thus, investigators were able to use the data both to narrow
down the breakpoint region on chromosome 19 and to identify the second chromosome involved in the
translocation.

3. Chromosomal In Situ Hybridization(CISS)

DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes
in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization.
The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both
the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the
specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12)
remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase
nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown
origin (47,XY; +r).
 The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its
autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small
translocations and fragments in human lymphocyte metaphase spreads after irradiation with 60Co-
gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-
library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the
short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in
interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case
with a known balanced translocation t(7;13) in the father. The same translocation was observed in
amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations
will be particularly important, in the future, in cases that cannot be solved reliably by conventional
chromosome banding alone.

4.Whole Chromosome Painting Probes (WCP)

Chromosome painting refers to the hybridization of fluorescently labeled, chromosome-specific,

composite probe pools to cytological preparations. Chromosome painting allows highly sensitive and
specific visualization of individual chromosomes in metaphase or interphase cells and the identification
of both numerical and structural chromosomal aberrations in human pathology. In addition to human
chromosome-specific probe pools. ASI offers a comprehensive range of whole chromosome painting
probes for human, mouse and rat chromosomes. These probes are designed for use in fluorescence in
situ hybridization analysis. Whole Chromosome Paint (WCP) probes are labeled with FITC, Rhodamine
and Aqua. A combination of two or three colors can be visualized simultaneously. They are ready to use
in hybridization solution and supplied in an economical 10 test format, or as a complete paintbox set.
The painting probes are visible using single band pass filters or a standard triple DAPI/FITC/Texas Red
filter. Due to their excellent stability, they are shipped at room temperature and should be stored at
-20℃ upon arrival. All ASI probes are manufactured in compliance with ISO 9000:2003 under rigorous
quality control and strict GMP standards.

5.Direct Visual Hybridization (DIRVISH)

The direct visual hybridization (DIRVISH) protocol is to stretched DNA fibers, it is possible to label the
intragenic and intergenicsequences with different fluorescent dyes and to count the total number of
visual pigment genes using direct microscopic inspection.

The procedure referred to as direct visual hybridization (DIRVISH) DNA mapping involves the
simultaneous hybridization of multiple probes and the fluorescent colors, red, green and blue to
produce images that convey high‐resolution mapping information. The images appear as long strings of
fluorescent signals positioned as they are in the genome. A visual multi‐color map is generated within 2
days. Cosmid probes span a distance of 10 μm or more and have been observed to contain patterns
within the strings of signals. We have developed computer imaging programs to scan through the strings
of signals and plot the intensities. Scans through multiple signal strings for one cosmid probe revealed
consistent patterns. We have interpreted the patterns as the result of suppression of repetitive DNA
sequence hybridization. These patterns may prove useful as fingerprints for regions of DNA.
6.Primed In Situ Labelling (PRINS)

Primed in situ labeling (PRINS) is a fast and sensitive technique for sequence specific in situ detection
of DNA. An unlabeled oligonucleotide probe is hybridized and used as primer for chain elongation in situ
catalyzed by a DNA polymerase. Thus, the oligonucleotide primer binds by sequence-specific base
pairing to its target sequence, which is subsequently labeled when labeled nucleotides are incorporated
by the DNA polymerase, using the oligonucleotide as primer and the cellular DNA as template. Using
unlabeled probes (primers) in the PRINS reaction means that high concentrations can be used, because
probe bound to cell-structures cannot function as a primer and therefore does not give rise to
background signals—only probe hybridized correctly to DNA can function as primer for chain elongation.
Thus, using high probe concentrations the hybridization is very fast. A PRINS reaction normally runs for
only 5–30 min. Owing to the speed of the reaction the morphology of chromosomes is very well
preserved, which makes it possible to obtain chromosome banding of good quality after a PRINS
reaction.

7. Fluorescence Immunophenotyping and Interphase Cytogenetics (FICTION)

FICTION technique can be applied on interphase cells, which is of great advantage especially for
tumor cytogenetics. In general, conventional unstained peripheral blood or bone marrow smears,
imprints and cytospin slides from fresh tissue and cryostat sections are applicable for FICTION. In order
to preserve cellular antigens, preparing high quality slides is one of the critical steps for successfully
performing FICTION. Freshly prepared slides should be air dried overnight at room temperature.
Thereafter, slides can be processed immediately or stored airtight at -80°C for years.

For the FICTION procedure, slides are fixed in acetone and then immunophenotyped by a direct or
indirect fluorescence detection system. Immunophenotyping is followed by a fixation step to preserve
the fluorescent immunostaining during the harsh hybridization procedure. After fixation, standard direct
or indirect fluorescence in situ hybridization (FISH) is performed using appropriately modified DNA
probes. Using appropriate filter sets, the immunophenotype and the hybridization Signals can be
evaluated simultaneously under a fluorescence microscope

CHROMOSOME IDENTIFICATION
1.G Banding

G-banding is the most commonly performed banding method and produces a recognizable pattern of
alternating dark and clear (light) bands, which distinguishes each chromosome pair.

G-banding allows each chromosome to be identified by its characteristic banding pattern. The banding
pattern can distinguish chromosomal abnormalities or structural rearrangements, such as
translocations, deletions, insertions, and inversions. G-banding has been divided into regions, bands,
and subbands.

In general, the bands have a lower G-C content than the interbands, where genes tend to be located.
Harsher treatment of chromosome (87°C for 10 min) before Giemsa staining can produce a pattern
called R banding, which is opposite to the G-banding pattern. The R banding can stain the euchromatin
region.

The region flanking the centromere often displays a considerable amount of heterochromatin. When the
entire chromosome is condensed, the centromeric heterochromosome is not visible in a mitotic
chromosome. The centromere, however, can be seen by a staining that generates C-bands . The C
banding or centromere banding results from the alkali treatment of chromosome. Centromere staining
is absent in G-band patterns. C bands are associated with heterochromatin along the chromosomes and
around the centromeres

Figure explanation:C-banding in
CHO and human chromosomes. Left:
C-banded metaphase of CHO9 cell line.
This cell line contains eight normal and
twelve rearranged autosomes with
only one X chromosome. Giemsa-
stained C- band regions are visualized in
yellow (reflected light microscopy). Right: C-banded karyotype of a human peripheral lymphocyte
metaphase showing centromeric, pericentric (Chromosomes 1, 9, and 16), and distal Yq heterochromatic
blocks.

2. C-banding stain

A selective chromosome banding stain used in human cytogenetics, employing Giemsa stain after most
of the DNA is denatured or extracted by treatment with alkali, acid, salt, or heat; only heterochromatic
regions close to the centromeres and rich in satellite DNA stain, except for the Y chromosome its long
arm usually stains throughout.
C-banding is a technique of chromosomal staining in which chromosomes are exposed to alkaline and
then acid conditions, in order to reveal bands of constitutive HETEROCHROMATIN that are identified
with Giemsa stain.

C-banding, reveals the AT-rich centromere, which consists of constitutive heterochromatin. This
technique involves acid treatment, hot saline incubation, and alkali treatment of the chromosomes.
These treatments depurinate the DNA and break the DNA backbone, which then cause the extraction of
the DNA from certain regions of the chromosomes. C-bands are produced due to this differential
extraction of the DNA. It was observed that the DNA in the C-bands is more resistant to extraction than
the DNA in the other regions of the chromosomes. This is due to the stronger interaction of the proteins,
which protects the DNA from extraction, with the DNA in the C-bands than in the other regions of the
chromosome

3. Flourescent Banding

Fluorochromes are organic molecules that are capable of undergoing fluorescence. These molecules
contain large conjugated systems such as aromatic or heterocyclic groups and are characterized by rigid
and planar structures.

There are several parameters that are important in describing the fluorescence of a fluorochrome.
These include the excitation and emission wavelengths, and quantum efficiency or yield. The excitation
wavelength shows how much energy is required to excite the fluorochrome while the emission
wavelength shows the energy of the photon emitted by the fluorochrome. The emission wavelength is
usually longer than the excitation wavelength. Meanwhile, the quantum efficiency or yield, Φ, shows the
probability of the excited fluorochrome to undergo fluorescence.

In chromosome staining, fluorochromes are used when the chromosomes are to be studied with a
fluorescent microscope. They are also capable of producing bands in the chromosomes. Although
fluorochromes are less stable than visible light dyes such as giemsa, as discussed earlier, they offer
several advantages in banding. Giemsa requires the use of trypsin, which removes proteins from the
chromosomes, in order for banding to occur. On the other hand, fluorochromes do not require this
pretreatment process. Hence, the extraction of proteins from the chromosomes is avoided. This is
particularly important in studying the chromosome structure. Furthermore, fluorochromes allow
chromosomes to be simultaneously banded and hybridized in situ with probes [8, 20]. Modern
cytogenetic techniques that makes use of fluorescence, such as comparative genomic in situ
hybridization (CGH), multicolor fluorescence in situ hybridization (M-FISH), and spectral karyotyping
(SKY), require coordinated chromosomal banding analysis in order to be completely useful.

4. R-Banding

R-banding reveals the GC-rich euchromatin and produces positive bands that correspond to the
negative G-bands. Banding is produced by incubating the chromosomes in an ionic solution at a high
temperature (~ 87degrees Celsius) followed by staining with giemsa. The incubation process causes the
denaturation of the AT regions of the chromosomes because of the low melting point of these regions (~
65 degrees Celsius) as compared to that of the GC regions (~ 105 degrees Celsius)

A simplified R banding technic is described which provides excellent delineation of major regions, easy
identification of all chromosomes, and an accurate comparison of homologue lengths. The technic is
simple, requiring only an initial incubation in buffer at 85 degrees C followed by acridine orange staining.
The best presentation of the R banded chromosomes was obtained by printing in black and white from
color transparency film. Variations in the length of the short arms of the acrocentric chromosomes are
clearly and consistently defined. Satellites are not demarcated and appear as part of the short arm.
Consistent banding was obtained, and the technic is suitable for use in routine clinical cytogenetic work.

5.CpG Island

CpG islands are defined as stretches of DNA 500–1500 bp long with a CG: GC ratio of more than 0.6,
and they are normally found at promoters and contain the 5′ end of the transcript (reviewed in Cross
and Bird, 1995).

CpG islands are found in certain regulatory regions of the genome, including the promoter regions of
many housekeeping genes such as the phosphoglycerate kinase gene. Since DNA methylation is involved
in the repression of gene expression, it is usually not seen in association with housekeeping genes,
which are expressed in all tissues. In tissue-specific genes, CpG islands are much less common, probably
because these genes are frequently methylated, resulting in potential loss of CpG dinucleotides due to
mutation. There are about 30000 CpG islands in the human genome. The fact that many are associated
with genes suggests that CpG islands might be useful in locating genes within DNA sequences.

6.BrdU (Bromodeoxyuridine / 5-bromo-2'-deoxyuridine)

It is an analog of the nucleoside thymidine used in the BrdU assay to identify proliferating cells.

BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells
within a living animal. During the BrdU assay, BrdU is incorporated into replicating DNA and can be
detected using anti-BrdU antibodies.

After BrdU labeling, an additional DNA hydrolysis step (sometimes referred to as a DNA denaturing step)
may be required after fixation and permeabilization to allow the anti-BrdU antibody access to the BrdU
within the DNA.

There are several methods for labeling cells in vivo with BrdU. Two commonly used methods are
intraperitoneal injection and oral administration.

Intraperitoneal injection

 Dilute BrdU in PBS to make a sterile solution of 10 mg/mL.

  For mice, as a general rule, inject the BrdU solution to a concentration of 100 mg/kg.

After treatment with BrdU, the animals can be sacrificed according to your lab's approved procedures.

 Fix and process tissue according to standard immunohistochemistry (IHC) protocols. However,
before continuing with immunostaining, refer to the DNA hydrolysis step below.

Note: BrdU incorporation into rapidly dividing tissues, such as the small intestine, will be detectable as
soon as 30 minutes post-injection. However, for most tissue, you may need to wait up to 24 hours. The
exact treatment time and dosage will need to be optimized for your tissue of interest.

Oral administration

Oral administration of BrdU is a non-invasive procedure and therefore useful for extended BrdU
administration, although it may introduce variability into experiments due to lack of control over an
animal’s water consumption.

 Dilute BrdU to 0.8 mg/mL in drinking water. Prepare this fresh and change daily.
 After treatment with BrdU, the animals can then be sacrificed according to standard protocols.
 Fix and process tissue according to standard IHC protocols. However, before immunostaining
refer to the DNA hydrolysis step below.

Note: for mice, 225 mg/kg per day of BrdU (calculated by measuring water consumption volumes per
animal) should achieve sufficient BrdU labeling. However, the exact dose should be optimized for
individual experimental conditions.

Examples:

DNA hydrolysis

Preparation:

 Sodium borate buffer: 3.8g sodium borate (MW=381.4) + 100 ml distilled water. Adjust pH with
NaOH.

Cells

 Incubate cells in 1–2.5 M HCL for 10 minutes to 1 hour at room temperature. The exact HCl
concentration and incubation time should be optimized for your experiment. If using a shorter
incubation time, incubating at 37oC may be more effective than room temperature.
 Optional step: remove the HCl and neutralize with 0.1 M sodium borate buffer pH 8.5 for 30
minutes at room temperature.
 Wash three times in PBS.
 Continue with immunostaining according to standard immunocytochemistry (ICC) protocols.
Tissue sections

Note: if using paraffin-embedded sections, ensure they are de-waxed before proceeding. Read our
deparaffinization protocol here.

 Incubate sections in 1–2 M HCL for 30 minutes to 1 hour. The exact HCl concentration and
incubation time should be optimized for your experiment. If using a shorter incubation time,
incubating at 37oC may be more effective than room temperature.
 Optional step: neutralize sections by incubating in 0.1 M sodium borate buffer pH 8.5 for 10
minutes at room temperature.
 Wash three times in PBS for about 5 seconds per wash.
 Continue with immunostaining according to standard IHC protocols.

Note: some researchers have reported that they don’t perform the HCl hydrolysis step and simply
perform heat-induced epitope retrieval before continuing with immunostaining.

7.and 8.B-pulse and T-pulse

In the “T pulse”procedure, BrdU is made available at the beginning of the cell cycle and then replaced
with thymidine the for last 5–6 h before the harvest. With the RBG technique (R-bands by BrdU and
Giemsa), the active or early replicating chromosome regions that inactive X chromosome, stain dark. The
“B pulse” is the opposite. Thymidine,made available at the beginning of the cell cycle, is replaced with
BrdU the last 5–6 h before harvest. Subsequent Giemsa staining will result in early-replicating
chromosome regions appearing dark because they have incorporated thymidine, while the inactive or
late-replicating chromosome regions appear pale due to theBrdU-incorporation.Banding patterns: The
equivalent of Q- and G- or R-banding patterns is achieved depending on whether a B or T pulse is used. If
a B-pulse is used, a Q or G-banding pattern is achieved and if a T-pulse is used, an R-banding pattern is
achieved. Subtle changes in pattern toward the earliest R-bands or latest G-bands can be achieved by
shortening the length of the BrdU pulse. A short T-pulse at the very end of the S-period can produce
what are referred to as T-bands (bright or dark bands at the terminal ends of some chromosome arms).
These bright bands with a T-pulse also correspond to early replicating, GC-rich regions, whereas dull
bands correspond to late-replicating AT-rich regions. The exception to this is the latereplicating X
chromosome whose bright bands do not differ in AT: GC content from the less intensely stained bands
at the same locations on the early-replicating X.

9.RE Banding

The sequence-specific DNA cleavage activity of restriction endonucleases (REases), combined with other
enzymatic activities that amplify and ligate nucleic acids, have enabled modern molecular biology. After
more than half a century of research and development, the applications of REases have evolved from
the cloning of exogenous DNA and genome mapping to more sophisticated applications, such as the
identification and mapping of epigenetic modifications and the high-throughput assembly of
combinatorial libraries. Furthermore, the discovery and engineering of nicking endonucleases (NEases)
has opened the door to techniques such as isothermal amplification of DNA among others.

Restriction enzymes are traditionally classified into four types on the basis of subunit composition,
cleavage position, sequence specificity and cofactor requirements. However, amino acid sequencing has
uncovered extraordinary variety among restriction enzymes and revealed that at the molecular level,
there are many more than four different types.

Type I Enzymes

Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut
DNA at random far from their recognition sequences. Originally thought to be rare, we now know from
the analysis of sequenced genomes that they are common. Type I enzymes are of considerable
biochemical interest, but they have little practical value since they do not produce discrete restriction
fragments or distinct gel-banding patterns.

Type II Enzymes

Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They
produce discrete restriction fragments and distinct gel banding patterns, and they are the only class
used in the laboratory for routine DNA analysis and gene cloning. Rather than forming a single family of
related proteins, Type II enzymes are a collection of unrelated proteins of many different sorts. Type II
enzymes frequently differ so completely in amino acid sequence from one another, and indeed from
every other known protein, that they exemplify the class of rapidly evolving proteins that are often
indicative of involvement in host-parasite interactions.
References:

M.A. Ferguson-Smith, in Encyclopedia of Genetics, 2001

John T. Corthell Ph.D., in Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls, 2014

https://www.sciencedirect.com/topics/neuroscience/in-situ-hybridization

© 2005 Nature Publishing Group Speicher, M. R. et al. The new cytogenetics: blurring the boundaries
with molecular biology. Nature Reviews Genetics 6, 784 (2005).

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Khrystylle Ivy Castillo

BMLS II-2

Proteinsythesis
Protein synthesis refers to the construction of proteins by the living cells. Comprising two primary parts
(transcription and translation), the process of protein synthesis involves ribonucleic acids (RNA),
deoxyribonucleic acid (DNA), enzymes, and ribosomes.

Proteins are important organic compounds present in living organisms. They are essential in almost all
cell functions. Specific proteins are involved with particular functions. Proteins are made up of long
chains of amino acids, which are either arranged in a linear pattern, or folded to form a complex
structure.

Based on the structural complexity, structure of proteins is classified into four types – primary,
secondary, tertiary, and quaternary. Also, the types of amino acids play a crucial role in determining the
expression of genes in this process.

Protein synthesis is a biological procedure performed by living cells to manufacture proteins in a step-
by-step manner. Many times, it is used to denote translation, which otherwise is a primary part in the
protein synthesis process. When studied in detail, the synthesis process is very complex. The process
itself begins with production of different amino acids, out of which some are derived from food sources

Protein synthesis comprises two major parts – transcription and translation. The process involves
ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and a set of enzymes. All types of ribonucleic acids,
namely messenger ribonucleic acid (mRNA), ribosomal ribonucleic acid (rRNA), and transfer ribonucleic
acid (tRNA) are required for protein synthesis.

Transcription

It is the first part in the process of protein synthesis. It takes place in the cell nucleus, where
deoxyribonucleic acid (DNA) is housed in the chromosomes. As we all know, DNA is a double helix
structure. From two parallel strands, one acts as a template to produce mRNA. As an initiation step of
transcription, RNA polymerase binds itself to a particular site (promoter region) in one of the DNA
strands that will act as a template.

Following its attachment to a DNA template strand, the polymerase enzyme synthesizes a mRNA
polymer under the direction of the template DNA. The mRNA strand continues to elongate until the
polymerase reaches a ‘terminator region’ in the template.

Hence, the transcription part encompasses three steps – initiation, elongation, and termination. The
newly transcribed mRNA is released by the polymerase enzyme, which then migrates to the cytoplasm
to complete the process of protein synthesis.

Translation

It is the second part in the process of synthesis of proteins. Contrary to transcription that occurs in the
nucleus, translation takes place in the cell cytoplasm. This part is initiated as soon as the transcribed
mRNA enters the cytoplasm.

The ribosomes present in the cytoplasm immediately attach to the mRNA at a specific site, called the
start codon. An amino acyl tRNA also binds at the mRNA strand. This phase is called initiation.

In this part, the ribosomes and tRNA get attached to the mRNA, which reads the coded information
present in the strand. Accordingly, protein synthesis of a specific amino acid sequence takes place.
Overall, the process of protein synthesis involves transcription of DNA to mRNA, which is then translated
into proteins. This process requires proper coordination of RNA, DNA, enzymes, and ribosomes. The
stepwise procedure of protein synthesis is also known as ‘central dogma’ in molecular biology.