This document provides an overview of FISH (fluorescence in situ hybridization), SNPs (single nucleotide polymorphisms), and ESTs (expressed sequence tags). FISH allows visualization of specific DNA sequences on chromosomes. SNPs are variations in a single DNA nucleotide that can provide information about disease risk. ESTs are short DNA sequences that can be used to identify genes being expressed in a cell. All three are important tools in molecular biology and genetics research.

1. FISH, SNP, and
EST
Presented by:
Md Tausif Alam
B.Sc(Hons.) BT/MB – V Semester

2. Table of contents
ESTs are short segments of cDNA
(complementary DNA) representing
portions of expressed genes.
FISH is a powerful molecular biology
technique used to visualize and map
the location of specific DNA
sequences on chromosomes.
SNP is a type of genetic variation that
occurs when a single nucleotide (A, T, C, or
G) at a specific position in the genome
differs among individuals.
FISH, SNP, and EST are indispensable
tools in molecular biology and genetics
research.
01
03
02
04
FISH SNP
EST CONCLUSION

3. FISH
01
FISH- Fluorescence In Situ Hybridization allows the direct
study of a sequence located on the chromosome. It has been
used for insertion and selection studies. Let’s see what FISH
is and how it works.

4. • Fluorescence in situ hybridization (FISH) is a molecular cytogenic techniques that uses
fluorescent probes that bind to only those part of the chromosome with a high degree of
sequence complementarity.
• It was developed by biomedical researchers in the early 1980s.
• It is a technique used to detect the presence or absence and location of specific gene
sequences.
• FISH is a process which vividly paints chromosome or portions of chromosome with
fluorescent molecules.
• It identifies chromosomal abnormalities & aid in gene mapping, toxicological studies, and
analysis of chromosome structural aberrations.

5. PRINCIPLE OF FISH
● The basic element of FISH are a DNA probe and a target sequence.
● Before hybridisation the DNA probe is labelled indirectly with a
hapten or directly labelled via incorporation of a fluorophore.
● The labelled probe and the direct DNA are denatured.
● Combination of denatured probe and target allows the annealing of
complementary DNA sequence.
● If the probe has been labelled indirectly an extra step is require for
visualization of the non-fluorescent hapten that uses an enzymatic
or immunological detection system. Finally, the signals are
evaluated by fluorescence microscopy.

7. PROBES
• Probe is a nucleic acid that can be labelled with a marker which
allows identification and quantitation.
• It will hybridize to another nucleic acid on the basis of base
complementarity.
• These probes can be as small as 20-40 base pair or be up to 1000 bp.
• A part of DNA (or RNA) that is complementary to certain sequence
on target DNA (i.e. DNA of the patient)
• Plasmid, phage DNA, cosmid
• PCR-product (amplification of certain segment of chromosomal
DNA)

8. PROBES TYPES
• There are three main types of probes used in fluorescence in situ
hybridization (FISH):
1. Locus-specific probes: These probes are designed to bind to a specific
region of a chromosome. They are often used to identify genes or to
detect chromosomal abnormalities.
2. Repetitive sequence probes: These probes are designed to bind to
repetitive sequences found in the middle of each chromosome. They are
often used to determine whether an individual has the correct number
of chromosomes.
3. Whole chromosome probes: These probes are collections of small
probes labelled with different fluorescent dyes, each of which binds to a
different sequence along a given chromosome. They are often used to
map the whole chromosome or to study the expression of genes in
specific chromosomes.

9. FISH PROCEDURE
• Fixation: The cells or tissues are fixed on a slide to prevent them from
moving or degrading. This is usually done by treating the cells with a
solution of formaldehyde or methanol.
• Denaturation: The DNA is denatured by heating it to a high temperature.
This separates the DNA into two single strands.
• Hybridization: The probes are hybridized to the denatured DNA. This is
done by incubating the slides in a solution that contains the probes. The
probes will bind to the DNA sequences that they are designed to bind to.
• Washing: The slides are washed to remove the probes that have not
hybridized to the DNA.
• Detection: The slides are examined under a fluorescent microscope. The
probes will fluoresce in different colors, depending on the label that they
are attached to.

12. APPLICATION OF FISH
• Cytogenetics: FISH can be used to study the structure and organization of
chromosomes. This can be used to identify chromosomal abnormalities, such as
deletions, duplications, and translocations.
• Gene mapping: FISH can be used to map the location of genes on chromosomes.
This can be used to identify the genes that are involved in diseases or to study the
evolution of genes.
• Prenatal diagnosis: FISH can be used to diagnose chromosomal abnormalities in
fetuses. This can be done by analysing cells from the amniotic fluid or from
chorionic villus samples.
• Cancer research: FISH can be used to detect cancer cells. This can be done by
analysing cells from a biopsy or from a blood sample.
• Developmental biology: FISH can be used to study the expression of genes during
development. This can be used to understand how genes control the development
of different tissues and organs.

13. LIMITATION OF FISH
Here are some specific examples of the limitations of FISH:
• Low sensitivity: FISH may not be able to detect small chromosomal abnormalities,
such as deletions or duplications. This is because the probes used in FISH are
typically designed to bind to large DNA sequences.
• False positives: In some cases, FISH can produce false positive results. This can
happen if the probes hybridize to non-target DNA sequences or if the hybridization
conditions are not optimized.
• Technical difficulty: FISH can be a technically challenging technique. It requires
careful preparation of the cells or tissues, as well as accurate hybridization and
washing steps.
• Cost: FISH can be a relatively expensive technique. This is because it requires
specialized equipment and expertise.

14. SNPs
02
A single nucleotide polymorphism is a
genomic variant at a single base position
in the DNA. Scientists study if and how
SNPs in a genome influence health,
disease, drug response and other traits.

15. • Single nucleotide polymorphism or SNP are most common type of genetic variation
among peoples.
• Each SNP represents a difference in single DNA building block, called a nucleotide.
• It is a DNA sequence variation occurring when a single nucleotide A, T, C, and G in the
genome differs between member of a species.
• For example, two sequenced DNA fragments from different individuals, AAGCCTA to
AAGCTTA, contain a difference in single nucleotide

16. SOME FACTS
• In human being 99.9% bases are same, remaining 0.1% makes a
person unique (different attributes/ characteristics/ traits, how
person looks, disease he or she develops).
• The variation can be :
i. Harmless (change in phenotype)
ii. Harmful (diabetes, cancer, heart disease, Huntington’s disease,
and haemophilia)
iii. Latent (variation found in coding and regulatory regions, are not
harmful on their own, and the change in each gene only becomes
apparent under certain condition e.g. susceptibility to lung
cancer)

17. SNPs FACTS
• SNPs are found in coding and (mostly) non-coding regions.
• Occurs with a very high frequency about 1 in 1000 bases to 1 in 100
to 300 bases.
• The abundance of SNPs and the ease with which they can be
measured make these genetic variations significant.
• SNPs in coding region may alter the protein structure made by
that coding region.
• Occurs once in every 300 nucleotides on average roughly there are
10 million SNPs in human genome.

18. SNPs TYPES
• Synonymous SNPs: do not change the amino acid sequence of the protein
that is produced. This is because multiple codons (sets of three nucleotides)
can code for the same amino acid.
• Nonsynonymous SNPs: change the amino acid sequence of the protein
that is produced. This may or may not result in a change in the protein’s
function.
• Silent SNPs: These SNPs do not change the amino acid sequence of the
protein that is produced, but they may affect the splicing of the RNA
transcript.
• Missense SNPs: These SNPs change the amino acid sequence of the protein
that is produced, but they may not affect the protein's function.
• Nonsense SNPs: These SNPs change the amino acid sequence of the
protein that is produced, and they result in a premature stop codon. This
prevents the protein from being produced.

20. • SNPs (Single Nucleotide Polymorphisms) play a crucial role in the development and
susceptibility to various diseases. These genetic variations can impact an individual's risk of
developing certain diseases, their response to medications, and their overall health. Here are
some key roles of SNPs in disease:
1. Disease Susceptibility and Risk Assessment: Certain SNPs have been associated with an
increased or decreased risk of developing specific diseases. For example, particular SNPs may be
linked to an increased risk of cardiovascular diseases, diabetes, cancer, autoimmune disorders,
and neurological conditions.
2. Cancer Susceptibility: Many cancer-related SNPs have been identified, some of which increase
the likelihood of developing specific types of cancer. These SNPs may affect genes involved in
cell growth, DNA repair, and apoptosis.
3. Inheritance Patterns: SNPs contribute to the inheritance of disease risk from parents to
offspring. Inherited SNPs can confer genetic predisposition to certain diseases and be passed on
through generations.
ROLE OF SNPs IN DISEASE

21. • SNPs are also being used in cancer diagnosis. For example, SNPs can be used to:
1. Identify people who are at risk for developing cancer: SNPs that are linked to cancer can be
used to identify people who are at an increased risk of developing cancer. This information can
be used to help people make informed decisions about their health, such as whether to get
screened for cancer or to make lifestyle changes to reduce their risk.
2. Track the inheritance of cancer: SNPs can be used to track the inheritance of cancer within a
family. This information can be used to help people make informed decisions about their health,
such as whether to get genetic testing or to talk to their doctor about their family history of
cancer.
3. Diagnose cancer: SNPs can be used to diagnose cancer in people who are already showing
symptoms. This is done by looking for SNPs that are associated with specific types of cancer.
• E.g. The Breast Cancer Association Consortium (BCAC) is a large research project that is
studying the role of SNPs in breast cancer. The BCAC has identified over 1,000 SNPs that are
associated with breast cancer. These SNPs can be used to identify women who are at an
increased risk of developing breast cancer.
SNPs AND CANCER

22. METHODS OF IDENTIFICATION OF SNPs
 Sanger Sequencing
• Traditional Sanger sequencing is the gold standard for SNP identification.
• Involves DNA amplification and chain termination sequencing using dye-labeled nucleotides.
• SNPs are identified by comparing the sequence of the target DNA with a reference sequence.
 PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism)
• SNPs are detected by PCR amplification of the target region, followed by digestion with specific restriction
enzymes that recognize SNP sites.
• The presence or absence of restriction sites indicates the SNP genotype.
 Next-Generation Sequencing (NGS)
• NGS platforms can detect SNPs in a high-throughput manner.
• SNPs are identified by aligning sequence reads to a reference genome and identifying nucleotide variations
at specific positions.
 Mass spectrometry
• Mass spectrometry is a method that is used to identify molecules by their mass. This method can be used
to identify SNPs by looking for differences in the mass of the DNA molecules at specific positions in the DNA
sequence.

23. • Personalized medicine: SNPs are being used to identify people who are at risk for developing
certain diseases, such as cancer, heart disease, and diabetes. This information can be used to
personalize medicine and develop treatments that are tailored to the individual's genetic
makeup. For example, a person who has a SNP that is associated with an increased risk of cancer
may be advised to get screened for cancer more often than someone who does not have the SNP.
• Drug discovery: SNPs are being used to identify genes that are involved in drug response. This
information can be used to develop new drugs that are more effective and have fewer side
effects. For example, a drug that is effective in people who do not have a certain SNP may not be
effective in people who do have the SNP.
• Genetics research: SNPs are being used to study the genetic basis of diseases and to understand
how genes interact with each other. This information can be used to develop new treatments and
prevention strategies for diseases. For example, researchers are studying how SNPs that are
associated with cancer interact with each other to increase the risk of cancer.
USE AND IMPORTANCE OF SNPs

24. ESTs
03
ESTs are short (200–500 nucleotides)
DNA sequences that can be used to
identify a gene that is being expressed
in a cell at a particular time.

25. • ESTs are short (200-500 nucleotides) DNA sequences that are derived from messenger RNA (mRNA).
• ESTs are created by reverse transcription, which is the process of using RNA as a template to
synthesize DNA.
• ESTs are typically generated from cDNA libraries, which are collections of DNA clones that are
complementary to mRNA.
• ESTs can be used to identify genes that are being expressed in a particular cell or tissue.
• ESTs can also be used to assemble full-length cDNA sequences and to map genes to their
chromosomal locations.
• ESTs have been used to identify genes in a wide variety of organisms, including humans, animals,
plants, and bacteria.
• ESTs have also been used to study gene expression patterns in different tissues and under different
conditions.

26. Here are some of the limitations of ESTs:
• ESTs are often incomplete, as they may only represent a portion of the coding sequence of a gene.
• ESTs can be difficult to assemble into full-length cDNA sequences, as they may be from different
transcripts of the same gene.
• ESTs can be misleading, as they may represent pseudogenes, which are genes that are no longer
functional.

27. CONCLUSION
• FISH (Fluorescence in situ hybridization) is a technique that uses fluorescent probes to
identify specific DNA sequences on chromosomes. FISH can be used to map genes to
chromosomes, to identify chromosomal abnormalities, and to study gene expression patterns.
• SNPs (single nucleotide polymorphisms) are DNA sequence variations that occur when a
single nucleotide (A, T, C, or G) is different between two individuals. SNPs can be used as
genetic markers to track the inheritance of genes, to identify populations of individuals with
similar genetic profiles, and to study the effects of genes on disease.
• ESTs (expressed sequence tags) are short DNA sequences that are derived from messenger
RNA (mRNA). ESTs can be used to identify genes that are being expressed in a particular cell or
tissue, to assemble full-length cDNA sequences, and to map genes to their chromosomal
locations.

28. THANK
YOU!