Unit-IV Biochemical Techniqugvgvenjs.pdf

vgchb

1. BBG 4
th
Semester – 2023 Batch
Subject: Biochemical Techniques
Subject Code: B23BC0401
UNIT-4
Molecular Techniques
Dr. Vijayakumar Govindaraj
School of Allied Health Sciences

2. Syllabus -UNIT IV
Molecular Techniques: Primer design, Polymerase chain reaction, RFLP, RAPD.
Cloning, Cre/lox genetic recombination, Hydrolysis probe-based qPCR, RNA
preparation and cDNA synthesis, SYBR based RTPCR, Protein Sequencing DNA
Finger printing, Cell culture ,hybridoma technology, Microarray and its
applications.

3. HISTORY
•1983 : Kary Mullis, a scientist working for the Cetus Corporation came up with
the idea for the polymerase chain reaction.
•1985 : The polymerase chain reaction was introduced to the scientific
community.
•1987 : Was awarded with noble prize for this discovery.
Polymerase Chain Reaction (PCR)

4. Principle of PCR
•By applying heat double stranded DNA is made to single
stranded.
•Two oligonucleotide strands or primers are used that are
complementary to 3’ end of each strand of DNA.
•The primer attach to 3’ end of each strand of DNA.
•Taq polymerase helps to extend the DNA by incorporating
nucleotides.

5. Stages of PCR
1. Exponential Amplification: With every
cycle, amount of product is doubled. The
reaction is very sensitive.
2. Levelling of Stage : Reaction slows as DNA
polymerase loses activity.
3. Plateau: No more product accumulate due
to exhaustion of reagents and enzymes.

6. PCR Technique
•PCR targets and amplifies
a specific region of a DNA
strand.
•It is an in-vitro technique to
generate large quantities
of a specified DNA (only
small quantity of DNA is
used).
STEPS IN PCR
DENATURATION ANNEALING EXTENSION

7. PCR Requirements
•Template DNA : A segment of DNA to
be amplified.
•Extracted from the sample.
• Reaction buffer (Tris-HCl, ammonium
ions, KCl), magnesium ions, bovine
serum albumin).
•This buffer provides the ionic strength and
buffering capacity needed during the reaction.
•Monovalant and Divalant cations :
Magnesium Chloride, Potassium.
•Works as co-factor for enzyme

8. •Primers : small pieces of artificially made DNA strands.
•Complimentry to 3’ end of target DNA.
•20-30 nucleotides.
•Two primers :
a)A forward primer
b)A reverse primer
•DNA polymerase : It combines at the end of the primer and then sequentially adds
new nucleotides to the DNA strand at 3′ end complementary to the target DNA
•Taq Polymerase- has the unique characteristic to work efficiently in higher
temperature.
•extracted from the bacteria Thermus aquaticus.
•Deoxynucleotide Triphosphates (dNTPs): dATP, dCTP, dGTP, and dTTP.
•raw material or the basic building blocks of the new DNA strands.
•PCR Machine : A thermal cycler

9. Steps in PCR
•Denaturaturation:
•At 94-98° C , the double-stranded DNA
melts and opens into two pieces of
single-stranded DNA.
•1 to 2 minutes are given in this
process.

10. ANNEALING :
•At medium temperatures,
around45-60°C, the primers
pair up (anneal) with the single-
stranded "template“.
•On the small length of double-
stranded DNA (the joined primer
and template), the polymerase
attaches and starts copying the
template.

11. Extension:
•At72°C (161.6 F), the polymerase
works best.
•The complementary nucleotide are
attached from 3’ end to 5’ end of DNA.
•Exponential increase in no. of genes in
each cycle.
•At least 30 cycles of all three steps is
done in each PCR.

12. Analysis of PCR Product
1.Agarose gel electrophoresis: Tells:
•Any band present in agarose gel electrophoresis .
•Any other band of different size.
•Is there a smear pattern
•Single sharp band of expected size is present or not.
2. Cloning of Product : Done when gene is present in
very tiny amount.
3. Sequencing of Product : By automated sequencer
machine to analyse sequence of DNA formed as PCR
product.

13. An oligo primer is a synthetic strand of nucleotides (usually 18–24 bases long) that binds to a specific
complementary sequence on a DNA template to allow DNA polymerase to start replication.
Oligo Primer
Types of Primers:
1.Forward Primer: Binds to the start of the sense strand.
2.Reverse Primer: Binds to the start of the antisense strand.
(Both are used in PCR to amplify a specific DNA region)
Features of a Good Primer:
1.Length: 18– 25 nucleotides.
2.Melting Temperature (Tm): 50–65°C.
3.GC Content: 40–60% (more GC = higher Tm).
4.No Secondary Structures: Avoid hairpins, dimers.
5.Specificity: Matches only the target sequence.
Applications:
•PCR (Polymerase Chain Reaction): For amplifying DNA.
•RT-PCR: Converts RNA into cDNA before amplification.
•DNA Sequencing: Initiates sequencing reaction.
•Gene Cloning: Helps to insert DNA fragments into
vectors.
•Mutation Detection: Detects single nucleotide changes.

14. Primer Designing using Primer3 Tool

15. 15
Primer Designing using Primer3 Tool

16. 16
Primer Designing using Primer3 Tool

17. 17

18. 18
Primer Designing using Primer3 Tool

19. RFLP (Restriction Fragment Length Polymorphism)
What is RFLP?
RFLP is a technique that exploits variations in homologous DNA sequences. These variations can lead to
the presence or absence of restriction enzyme recognition sites, thereby producing DNA fragments of
different lengths when digested with restriction enzymes.
Principle of RFLP:
1.DNA Sequence Variations: Differences in DNA sequences among individuals (like insertions, deletions,
or point mutations) may alter restriction enzyme sites.
2.Restriction Enzyme Digestion: Restriction enzymes cut DNA at specific recognition sequences.
3.Fragment Separation: The resulting DNA fragments are separated by gel electrophoresis.
4.Southern Blotting: The DNA fragments are transferred onto a membrane and hybridized with a labeled
DNA probe that binds to a specific sequence.
5.Detection: The pattern of bands (fragment lengths) varies depending on the polymorphisms and can be
visualized using X-ray film or other detection systems.

20. Steps Involved in RFLP:
1.DNA Isolation: Extract genomic DNA from cells (blood,
tissue, etc.).
2.Digestion with Restriction Enzymes: Incubate DNA with
specific restriction enzymes.
3.Gel Electrophoresis: Separate DNA fragments by size on
an agarose gel.
4.Southern Blotting: Transfer DNA fragments to a nylon or
nitrocellulose membrane.
5.Probe Hybridization: Add a labeled DNA probe that binds
to complementary sequences.
6.Visualization: Use autoradiography or
chemiluminescence to detect hybridized fragments.
Applications of RFLP:
•Genetic Mapping and Gene Localization
•Detection of Mutations and Genetic Disorders
•DNA Fingerprinting in forensic science
•Paternity Testing
•Plant and Animal Breeding (genetic diversity studies)
•Pathogen Identification

21. RAPD (Random Amplified Polymorphic DNA)
What is RAPD?
RAPD (Random Amplified Polymorphic DNA) is a PCR-based technique used to detect genetic
polymorphisms. RAPD is a method that amplifies random segments of genomic DNA using short, single
primers of arbitrary nucleotide sequence (usually 10 bases long) under low-stringency PCR conditions. It
reveals polymorphisms (differences in DNA sequences) based on the presence or absence of amplified
products.
Principle of RFLP:
1.Uses short, random primers that bind to multiple sites on the genome.
2.If two primer binding sites are close and in opposite orientations, PCR will amplify the region between
them.
3.Variation in DNA sequences among individuals may result in gain or loss of primer binding sites, causing
presence or absence of amplification.
4.These differences are visualized as distinct banding patterns on an agarose gel.

22. Steps Involved in RAPD:
1.DNA Isolation: Extract genomic DNA from the sample.
2.PCR Amplification:
1.Use a single short (10-mer) primer.
2.Set low annealing temperatures to allow non-specific
primer binding.
3.Gel Electrophoresis:
1.Separate the amplified DNA fragments on agarose gel.
2.Stain the gel (e.g., with ethidium bromide or GelRed) .
4.Visualization: Observe the banding pattern under UV light.
Applications of RAPD:
•Genetic diversity analysis in populations, plants, animals, and
microbes.
•Species identification and phylogenetic studies.
•Genotyping and fingerprinting of cultivars or strains.
•Linkage mapping in breeding programs.
•Monitoring genetic stability in tissue culture.

23. Cloning by PCR
Cloning by PCR is a method used to amplify and insert a specific DNA fragment (gene of
interest) into a vector (plasmid) for further use in experiments such as protein expression,
sequencing, or genetic studies.
PCR cloning involves using the Polymerase Chain Reaction (PCR) to generate a specific DNA
fragment and then ligating it into a cloning vector.
Steps in PCR Cloning
1.Design Primers
Create primers specific to the gene of interest, often with added restriction sites.
2.PCR Amplification
Amplify the target DNA using PCR.
3.Purify PCR Product
Clean the amplified DNA to remove unwanted components.
4.Prepare Vector
Linearize the plasmid using restriction enzymes or use a ready-to-clone vector.
5.Ligation
Insert the PCR product into the vector using DNA ligase or TA/TOPO cloning method.
6.Transformation
Introduce the ligation mix into competent E. coli cells.
7.Selection and Screening
Grow colonies on antibiotic plates and confirm insert using colony PCR or digestion.

24. Cre/lox Genetic Recombination System
The Cre/lox system is a powerful genetic tool used to control gene expression, delete DNA sequences, or insert specific
genes in a targeted, tissue-specific, or time-specific manner in cells and organisms. It originated from bacteriophage P1
(a virus that infects bacteria).
Key Components:
1. Cre Recombinase
•Cre (Causes REcombination) is a 38 kDa enzyme.
•It is a site-specific recombinase that recognizes loxP sites
and catalyzes recombination between them.
•Derived from bacteriophage P1, it doesn’t need any
cofactors or specific sequences outside the loxP site.
2. loxP Site
•A loxP site is a 34-base pair DNA sequence made of:
•Two 13 bp palindromic sequences (inverted
repeats),
•Flanking an 8 bp asymmetric core spacer region
(which gives directionality).
How Cre/lox Recombination Works
The effect of recombination depends on the orientation
and position of the loxP sites:

25. 1.Design Primers:
One set flanks the loxP sites.
One set within the floxed region (to detect presence/absence).
One set specific to the Cre gene (to confirm Cre presence).
2.Extract genomic DNA from tissue or cells.
3.Run PCR using designed primers.
4.Analyze PCR products by agarose gel electrophoresis:
Size shift in the band indicates successful recombination.
No product or shorter band suggests deletion between loxP sites.
PCR-Based Workflow to Confirm Cre/lox Recombination
Example:
Before recombination: PCR product = 1.5 kb (with floxed exon)
After recombination: PCR product = 0.4 kb (exon deleted)
Applications of Cre/lox + PCR
Tissue-specific gene knockouts (using tissue-specific Cre promoters)
Time-controlled gene expression (using inducible Cre, e.g., Cre-ERT2)
Lineage tracing in developmental biology
Functional gene studies in vivo

26. RNA extraction is a fundamental laboratory technique used to isolate ribonucleic acid (RNA) from biological samples such as
tissues, cells, or blood. The goal is to obtain pure, intact RNA free from contaminants like proteins, DNA, and enzymes that
could degrade RNA (e.g., RNases).
RNA Extraction
Major Steps involved in RNA Extraction
1.Cell Lysis
Break open cells using a lysis buffer with chaotropic agents (e.g.,
guanidinium) to release RNA and inactivate RNases.
2.Homogenization
Physically disrupt the sample (e.g., grinding or using a homogenizer)
to reduce viscosity and shear DNA.
3.Phase Separation
Add chloroform and centrifuge to separate layers:
1.Top aqueous phase: RNA
2.Middle: DNA
3.Bottom: Proteins
4.RNA Precipitation
Add isopropanol or ethanol to the aqueous phase to precipitate RNA,
then centrifuge to collect the RNA pellet.
5.Washing
Wash the RNA pellet with 70–75% ethanol to remove impurities.
6.Resuspension
Dissolve the clean RNA pellet in RNase- free water or buffer for
storage or use.
Quality Control of Extracted RNA
Quantification:
•Spectrophotometry (e.g., NanoDrop):
•A260/A280 ratio should be ~2.0 for pure RNA.
•A260/A230 ratio indicates salt and organic contamination (ideal ~2.0–2.2).
Integrity:
•Gel electrophoresis – shows 28S and 18S rRNA bands in eukaryotic RNA.
•Bioanalyzer or TapeStation – gives RIN (RNA Integrity Number).

27. cDNA Synthesis
What is cDNA Synthesis?
cDNA (complementary DNA) is DNA synthesized from an RNA template using the enzyme reverse transcriptase. It
is typically used to analyze gene expression (via RT-PCR/qPCR) or to clone eukaryotic genes in prokaryotic systems.
Why is cDNA Synthesis Important?
RNA is unstable and cannot be amplified by DNA polymerases.
cDNA is a stable DNA copy of RNA (mostly mRNA).
It allows us to study gene expression, perform RT-PCR, or create expression libraries.
Steps in cDNA Synthesis
1.Isolate RNA
Extract high- quality, DNase-treated RNA from cells or tissues.
2.Add Primers
Use oligo(dT) (for mRNA), random hexamers, or gene-specific primers to bind RNA.
3.Denature RNA
Heat the RNA-primer mix to ~65°C to remove secondary structures, then cool for primer binding.
4.Reverse Transcription
Add: Reverse transcriptase enzyme, dNTPs, RT buffer, RNase inhibitor. Incubate at 37 –55°C for
30–60 min to synthesize cDNA.
5.Terminate Reaction: Heat to 70°C for 10– 15 min to inactivate the enzyme.
6.Store or Use cDNA: Use the cDNA immediately or store at –20°C for downstream applications (e.g., qPCR,
cloning).

28. Definition: Quantitative PCR (qPCR), also known as real-time PCR, is a
molecular biology technique used to amplify and quantify DNA or RNA
sequences in real time. qPCR is a modification of conventional PCR that
allows detection and quantification of DNA (or cDNA from RNA) as it is
being amplified, cycle by cycle, using fluorescent dyes or probes.
Key Principles
1.Amplification: Like traditional PCR, qPCR amplifies DNA using
cycles of denaturation, annealing, and extension.
2.Fluorescence Detection: A fluorescent signal increases in direct
proportion to the amount of PCR product formed.
3.Real-Time Monitoring: Fluorescence is measured at each cycle,
providing quantitative data on the target nucleic acid.
Steps in qPCR Workflow
1.Sample Preparation:
Extract DNA or RNA from the sample.
For RNA, convert it to complementary DNA (cDNA) using
reverse transcriptase (this is called RT-qPCR).
2.PCR Reaction Setup:
DNA template or cDNA.
Forward and reverse primers.
DNA polymerase.
dNTPs, buffer, and fluorescent dye or probe.
3.Amplification Cycles:
Denaturation (~95°C): DNA strands separate.
Annealing (~50–65°C): Primers bind to the target sequence.
Extension (~72°C): DNA polymerase synthesizes new DNA
strands.
4.Fluorescence Monitoring:
Fluorescence increases as more DNA is amplified.
The machine records fluorescence at the end of each cycle.
5.Quantification:
The Ct (threshold cycle) value is the cycle at which
fluorescence exceeds background.
Lower Ct = more starting template.
Data can be analyzed using standard curves or relative
expression (e.g., ΔΔCt method).
qPCR

29. Fluorescence Dye based RT-PCRFluorescence Probe based RT-PCR
Major Types of qPCR
Applications of qPCR
•Gene expression analysis
•Detection of pathogens (e.g., SARS- CoV-2)
•Genotyping and mutation analysis
•Quantification of DNA/RNA
•miRNA and lncRNA studies
•Cancer biomarker validation

30. Protein Sequencing Methods
Feature Classical Method (Edman Degradation) Modern Method (Mass Spectrometry- Based)
Principle
Sequential removal and identification of N-terminal
amino acids
Measures mass- to-charge ratio (m/z) of peptide
fragments
Main Technique Used Edman degradation Mass spectrometry (e.g., MALDI-TOF, ESI-MS/MS)
Sample Type Requires pure peptide or small protein Can work with complex protein mixtures
Sequence Information Direct sequence of amino acids from N-terminus
Sequence inferred from mass differences of
fragments
Length Limitations Efficient for peptides up to 30–50 amino acids No strict limit; can handle large proteins
Speed Slow and labor-intensive Rapid and high- throughput
Post-Translational
Modifications
Difficult to detect
Can identify modifications (e.g., phosphorylation,
glycosylation)
Automation Limited automation Highly automated
Accuracy High for short peptides
High with proper fragmentation and database
matching
Sample Quantity NeededRequires relatively large amounts of pure sampleRequires very small amounts (picomoles or less)
N-terminal Blocked ProteinsCannot be sequenced if N- terminus is blocked Still analyzable via MS-based techniques
Use in Proteomics Not widely used in proteomics Widely used in proteomics studies
Database Dependency No database needed for basic sequencing
Requires protein sequence databases for
identification

31. What is DNA Fingerprinting?
DNA fingerprinting involves analyzing specific regions of the genome that are highly variable among individuals. These
regions are mostly non-coding sequences known as Variable Number of Tandem Repeats (VNTRs) or Short Tandem Repeats
(STRs).
DNA Fingerprinting
Steps Involved in DNA Fingerprinting:
1. Sample Collection: DNA is extracted from biological samples such as:
2. DNA Extraction: The DNA is isolated from the cells using chemical or enzymatic methods. Care is taken to prevent
contamination.
3. Amplification by PCR (Polymerase Chain Reaction): Specific STR regions of DNA are amplified using PCR. These STRs
differ in length between individuals.
4. DNA Fragment Separation: Amplified DNA fragments are separated based on size using gel electrophoresis or capillary
electrophoresis. Smaller fragments move faster and farther than larger ones.
5. Visualization: DNA bands or peaks are visualized using: Staining (e.g., ethidium bromide) for gels/ Fluorescent tags in
automated systems.
6. Analysis of STR Profiles
•The pattern of bands or peaks (representing STR alleles) is compared between individuals.
•Each individual inherits one allele from each parent, so they usually show two bands at each STR locus.

32. Applications of DNA Fingerprinting
1.Forensic Science
•Identifying suspects in criminal cases.
•Exonerating the innocent.
•Matching DNA from crime scenes.
2.Paternity and Maternity Testing
•Determining biological relationships.
3.Identification of Remains
•Disaster victim identification.
•Missing persons.
4.Wildlife and Conservation Biology
•Tracking genetic diversity.
•Identifying species and subspecies.
5.Medical Diagnostics
•Detecting genetic disorders or
predispositions.

33. Cell Culture

34. What is Cell Culture?
Cell culture is the process of growing cells outside their natural environment under controlled conditions, usually in a lab
using a sterile nutrient-rich medium. These cells may be obtained from animals, humans, plants, or microbes.
Types of Cell Culture:
1. Primary Cell Culture
•Cells are taken directly from tissues (like skin, liver, or kidney).
•They grow for a limited time (finite lifespan).
•Closely mimic the original tissue.
•Example: Fibroblasts from skin biopsy.
2. Secondary Cell Culture / Subculture
•Cells are transferred (passaged) to new vessels to continue
growth. Maintains culture longevity.
3. Cell Lines
•Derived from a single cell type and can grow indefinitely
(immortalized).
•Examples: HeLa (human cervical cancer cells); CHO (Chinese
hamster ovary cells)
4. Stem Cell Culture
•Includes pluripotent and multipotent cells used for
regeneration studies.
Basic Requirements for Cell Culture:
A. Sterile Environment
•Prevents contamination using:
•Laminar flow hoods
•Autoclaved instruments
•Sterile gloves and techniques
B. Culture Medium
•Provides nutrients, growth factors, and
hormones.
•Common media: DMEM, RPMI, MEM,
FBS (serum) as supplement.
C. Incubation Conditions
•Temperature: 37°C for mammalian cells
•pH: 7.2–7.4 (maintained with buffer
system like CO₂)
•Humidity: ~95%
•CO₂: Usually 5%

35. Steps in Cell Culture:
1.Isolation of Cells:
•From tissues via enzymatic digestion (e.g., trypsin, collagenase).
2.Seeding in Culture Vessels:
•Placed into flasks, Petri dishes, or multi-well plates.
3.Growth and Monitoring:
•Cells attach to the surface and begin dividing.
•Observed under an inverted microscope .
4.Subculturing (Passaging):
•Once confluent (~80–90% of surface covered), cells are detached (e.g., trypsinized) and transferred to new vessels.
5.Cryopreservation (Optional):
•Cells are frozen in liquid nitrogen with cryoprotectants like DMSO for long-term storage.

36. Application of Cell Culture

37. Hybridoma
Technology

38. Definition:
Hybridoma technology is a technique that involves fusing a specific antibody-
producing B-cell (from a mouse immunized with an antigen) with a myeloma (cancer)
cell to create a hybrid cell called a hybridoma. This hybrid cell has the ability to divide
indefinitely and produce a single type of antibody.
History:
Monoclonal antibody was produced by the technique
described by the scientiest George Kohler and Milstein in
1975.
They were awarded Noble Prize in 1984 for their work.

39. Monoclonal antibodies vs Polyclonal antibodies
Monoclonal antibodies Polyclonal antibodies
•Monoclonal antibodies refers to a
homogenous population of antibodies
that are produced by a single clone of
plasma B cells.
•A homogeneous antibody population.
•Interact with particular epitope on the
antigen.
•Possess less cross reactivity.
•Used as therapeutic drug.
•Polyclonal antibodies refers to a
mixture of immunoglobulin molecules
that are secreted against the antigen.
•A heterogeneous antibody population.
•Interact with different epitope on the
same antigen.
•Possess comparatively high cross
reactivity.
•Used as general research application.

40. Steps in Hybridoma Technology:
1.Immunization:
•A mouse is immunized with a specific antigen to stimulate B-cell
production.
2.Isolation of Spleen Cells:
•After a few weeks, the spleen (rich in B-lymphocytes) is removed from
the mouse.
3.Fusion:
•Spleen B-cells are fused with immortal myeloma cells using a chemical
like PEG (polyethylene glycol).
4.Selection (HAT Medium):
•Fused cells are cultured in HAT (Hypoxanthine-Aminopterin-Thymidine)
medium, which only allows the growth of hybridomas—not unfused B-
cells (which die naturally) or myeloma cells (which lack the salvage
pathway enzymes).
5.Screening:
•The hybridomas are screened to identify those producing the desired
antibody.
6.Cloning:
•Positive hybridomas are cloned (usually via limiting dilution) to ensure
monoclonality.
7.Production & Purification:
•The selected hybridoma is cultured, and monoclonal antibodies are
harvested and purified.

41. 1. Diagnostic Applications
•Clinical diagnostics: Monoclonal antibodies are used in ELISA, Western blotting, immunofluorescence, and
immunohistochemistry to detect biomarkers of diseases like cancer, infections, and autoimmune disorders.
•Rapid diagnostic kits: Used in home pregnancy tests, malaria and dengue tests, COVID-19 antigen tests.
2. Therapeutic Applications
•Treatment of diseases: Therapeutic monoclonal antibodies are used to treat cancers (e.g., Rituximab for lymphoma),
autoimmune diseases (e.g., Infliximab for rheumatoid arthritis), and infections (e.g., Palivizumab for RSV).
•Targeted drug delivery: Monoclonal antibodies can deliver cytotoxic drugs specifically to tumor cells, reducing side
effects.
•Immunotherapy: Used in checkpoint inhibitors for cancer immunotherapy (e.g., anti-PD-1, anti-CTLA-4 antibodies).
3. Research Applications
•Cell and molecular biology: Used to identify and locate proteins within cells, study receptor-ligand interactions, and
trace specific cell types.
•Flow cytometry and cell sorting: Monoclonal antibodies help in tagging specific cell surface markers for cell population
analysis.
Major Applications of Hybridoma Technology

42. What is a DNA Microarray?
A DNA microarray is a solid surface (usually a glass slide or silicon chip) onto which thousands of DNA fragments
(probes) are immobilized in a precise grid. These fragments represent known genes or gene segments.
When labeled nucleic acid samples (targets) from a cell or tissue are applied to the microarray, they will hybridize (bind)
to their complementary DNA sequences. This interaction is detected using fluorescence.
DNA Microarray

43. Steps in DNA Microarray Technique
1. RNA Isolation
•Extract total RNA from the cells or tissues of interest
(e.g., healthy vs cancerous cells).
2. cDNA Synthesis and Labeling
•Reverse transcribe RNA into complementary DNA
(cDNA).
•Label the cDNA with fluorescent dyes (commonlyCy3
-greenandCy5-red).
3. Hybridization
•The labeled cDNA is applied to the microarray chip.
•Complementary sequences between the cDNA and
the probes on the chip hybridize.
4. Scanning
•The chip is scanned with a laser scanner.
•The intensity of fluorescence at each spot is
measured.
6. Data Analysis
•Fluorescent signals are analyzed to determine the
expression level of each gene.
•Ratios of Cy3/Cy5 fluorescence are compared for
differential gene expression between samples.

44. Application Purpose
Gene Expression Profiling
Comparing gene activity across different conditions (e.g.,
cancer vs normal)
Genotyping Detecting single nucleotide polymorphisms (SNPs)
Mutation Detection Identifying mutations in specific genes
Drug Response Studies Predicting how patients respond to certain drugs
Pathogen Detection Identifying microbial or viral DNA in clinical samples
Comparative Genomic Hybridization Detecting chromosomal abnormalities