[DNA FINGERPRINTING]
[SIDDHAARTH K]
[XII-A]
[DNA FINGERPRINTING]
DNA Fingerprinting: A Basic Overview
DNA fingerprinting is a technique used to identify individuals based on their unique DNA
profiles. It's a powerful tool with applications in various fields, including:
* Forensic science: Identifying suspects in crimes
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* Paternity testing: Determining biological relationships
* Immigration: Verifying family ties
* Wildlife conservation: Studying genetic diversity and tracking illegal wildlife trade
How does DNA fingerprinting work?
* Sample Collection: A sample of DNA is obtained from a biological source, such as blood,
saliva, or hair.
* DNA Extraction: The DNA is extracted from the sample cells.
* Restriction Enzyme Digestion: Enzymes called restriction enzymes cut the DNA into
smaller fragments based on specific sequences.
* Gel Electrophoresis: The fragments are separated by size using a process called gel
electrophoresis.
* Visualization: The DNA fragments are visualized, often using a technique called
Southern blotting, to create a unique pattern.
Unique DNA Patterns
Each individual has a unique DNA profile due to variations in the number of repetitive DNA
sequences called VNTRs (Variable Number Tandem Repeats). These variations are
inherited from parents and are used to distinguish one person from another.
[OVERVIEW]
Key Concepts:
• 1. DNA Structure:
• DNA (deoxyribonucleic acid) is composed of two strands forming a double helix, made
up of nucleotides. Each nucleotide consists of a sugar, a phosphate group, and a
nitrogenous base (adenine, thymine, cytosine, or guanine)
2. Unique Genetic Markers:
• The human genome contains regions of repetitive sequences called Short Tandem
Repeats (STRs) or Variable Number Tandem Repeats (VNTRs). These regions vary
significantly among individuals, making them useful for identification.
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• 3. Procedure:
• Sample Collection: DNA can be extracted from blood, saliva, hair, or other biological
samples.
• DNA Extraction: The DNA is isolated from the cells.
• PCR Amplification: Polymerase Chain Reaction (PCR) is used to amplify specific regions
of DNA.
• Gel Electrophoresis: The amplified DNA is separated by size using gel electrophoresis,
allowing visualization of the unique DNA bands.
• Analysis: The resulting DNA patterns (bands) are compared against known samples for
identification.
• 4. Applications:
• Forensics: Identifying suspects in criminal cases or exonerating the innocent.
• Paternity Testing: Establishing biological relationships.
• Genetic Research: Studying genetic diversity and inheritance patterns.
• 5. Ethical Considerations:
• The use of DNA fingerprinting raises ethical issues related to privacy, consent, and
potential misuse of genetic information.
• 6. Limitations:
• While highly accurate, DNA fingerprinting is not infallible. Contamination of samples or
improper handling can lead to errors.
• Conclusion:
• DNA fingerprinting is a powerful tool in modern science, providing a reliable method
for individual identification and offering insights into genetic relationships. It combines
principles of molecular biology, genetics, and forensic science.]
[WHAT IS DNA FINGERPRINTING?]
[“DNA fingerprinting is a technique used to identify individuals by analyzing the
unique patterns in their DNA. These patterns, found in specific regions of the
genome, vary from person to person, making DNA fingerprinting useful for
personal identification, forensic investigations, and determining biological
relationships.”]
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[STEPS INVOLVED]
[The steps involved in DNA fingerprinting are:
1. Sample Collection: Biological samples (e.g., blood, saliva, hair, or skin) are
collected to obtain DNA.
Sample Collection for DNA Fingerprinting
Types of Samples:
Blood: Commonly used; drawn from a vein.
Saliva: Collected using a cheek swab.
Hair: Root with follicle provides DNA.
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Skin Cells: Shed cells can be collected.
Semen: Used in forensic case
Other Tissues: Nails, teeth, or bones.
Collection Methods:
1. Buccal Swab: Non-invasive cheek swab for cells.
2. Blood Sample: Drawn from a vein or finger prick.
3. Hair Sampling: Collect hair with the root.
4. Forensic Collection: Swabs or tweezers for evidence at a crime scene.
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Considerations:
• Ensure sterility to avoid contamination.
• Label samples accurately.
• Store samples properly to maintain DNA integrity.
2. DNA Extraction: The DNA is isolated from the collected cells.
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DNA Extraction Process
1. Cell Lysis:
Use a lysis buffer (containing detergents and salts) to break open the cell membranes,
releasing the cellular contents, including DNA.
2. Protein Removal:
Add a protease enzyme or a mixture of phenol and chloroform to digest proteins and
separate them from the DNA, ensuring a cleaner sample.
3. Precipitation:
Add cold alcohol (ethanol or isopropanol) to the mixture, which causes the DNA to
precipitate out of the solution due to its insolubility in alcohol.
4. Centrifugation:
Spin the mixture in a centrifuge to separate the DNA, which forms a pellet at the bottom
of the tube, from the liquid (supernatant) containing impurities.
5. Washing:
Wash the DNA pellet with cold alcohol to remove residual salts and impurities, improving
the purity of the DNA.
6. Dissolving:
Resuspend the purified DNA pellet in a buffer (like TE buffer) or sterile water, making it
ready for storage or further analysis, such as PCR or gel electrophoresis.
This process yields high-quality DNA suitable for various applications, including DNA
fingerprinting.
3. PCR Amplification: Specific regions of the DNA, especially Short Tandem Repeats
(STRs), are copied multiple times using Polymerase Chain Reaction (PCR).
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WHAT IS PCR AMPLIFICATION?
PCR Amplification
What: Polymerase Chain Reaction (PCR) is a technique used to amplify specific segments
of DNA, creating millions of copies from a small initial sample.
PCR Amplification: How (Detailed)
1. Denaturation:
The reaction mixture, containing the DNA sample, primers, nucleotides, and a buffer
solution, is heated to 94-98°C for 20-30 seconds. This high temperature disrupts the
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hydrogen bonds between complementary base pairs, causing the DNA strands to separate
into two single strands.
2. Annealing:
After denaturation, the temperature is lowered to about 50-65°C for 20-40 seconds. This
temperature allows the primers, which are designed to match specific sequences at the
start and end of the target region, to bind (anneal) to their complementary sites on the
single-stranded DNA. The specific choice of primers is crucial for the accuracy of the
amplification.
3. Extension:
The temperature is then raised to around 72°C for 30-60 seconds. This is the optimal
temperature for the heat-stable DNA polymerase, typically Taq polymerase, to function.
The enzyme begins adding nucleotides complementary to the template strand, starting
from the primer and extending the new DNA strand. This step synthesizes new DNA,
effectively doubling the amount of target DNA in the sample.
4. Repeat:
The three steps—denaturation, annealing, and extension—are repeated for 25-35 cycles in a
thermal cycler. Each cycle results in the exponential amplification of the target DNA
sequence. For example, after 30 cycles, a single copy of DNA can yield over a billion
copies, making PCR an incredibly powerful method for DNA amplification.
5. Final Extension (optional):
After the cycles, a final extension step at 72°C for 5-10 minutes can be added to ensure
that any remaining single-stranded DNA is fully extended and to complete the synthesis of
all DNA strands.
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This detailed process of PCR allows for the precise and rapid amplification of specific DNA
sequences, making it essential in research, diagnostics, forensics, and genetic studies.
4. Digestion with Restriction Enzymes (if applicable): In some methods, DNA is cut into
fragments using enzymes.
WHAT IS RESTRICTION ENZYMES ?
A restriction enzyme is a protein that cuts DNA at specific sequences, known as
recognition sites, typically 4-8 base pairs long. These enzymes are used in molecular
biology for DNA manipulation, such as cutting and cloning DNA segments.
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Mechanism of Restriction Enzymes in DNA
1. Recognition:
Restriction enzymes scan the DNA and bind to a specific short sequence of nucleotides,
called a recognition site. These sites are usually palindromic, meaning the sequence reads
the same forward and backward.
2. DNA Cleavage:
Once the enzyme binds to its recognition site, it cuts the DNA at or near this sequence.
The enzyme breaks the phosphodiester bonds between nucleotides in the DNA backbone,
leading to either a blunt end (cut straight across both strands) or sticky ends (staggered
cuts, leaving overhangs).
3. Fragment Formation:
After cleavage, the DNA is broken into smaller fragments. If the enzyme produces sticky
ends, these can later pair with complementary sequences, facilitating the joining of DNA
fragments from different sources.
4. Function in Nature:
In bacteria, restriction enzymes serve as a defense mechanism against invading viruses
(bacteriophages) by cutting up the foreign DNA, preventing the virus from replicating.
This precise cutting ability makes restriction enzymes essential tools in genetic
engineering and molecular biology....
Types of Restriction Enzymes
1. Type I:
#Cuts DNA at random sites far from the recognition sequence.
#Requires ATP and other cofactors.
2. Type II:
#Cuts DNA at specific sites within or near the recognition sequence.
#No ATP required; most widely used in genetic research (e.g., EcoRI).
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3. Type III:
#Cuts DNA a short distance from the recognition site.
#Requires ATP but functions less precisely than Type II.
4. Type IV:
#Recognizes and cuts modified DNA (e.g., methylated).
#Used in bacterial defense against modified viruses.
Type II is the most important for precise DNA manipulation in molecular biology.
5. Gel Electrophoresis: The amplified or digested DNA is placed in a gel and separated by
size when an electric current is applied. This results in a pattern of DNA bands.
Gel electrophoresis is a technique used to separate DNA, RNA, or proteins based on
their size and charge by applying an electric field to a gel matrix. The molecules move
through the gel at different rates, allowing for visualization and analysis.
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#Gel Electrophoresis Process
1. Prepare the Gel:
Agarose or polyacrylamide gel is made and placed in a gel tray with wells for loading
samples.
2. Load DNA Samples:
DNA samples, mixed with a loading dye, are placed into the wells of the gel.
3. Apply Electric Current:
The gel is submerged in a buffer solution, and an electric current is applied. DNA, being
negatively charged, moves towards the positive electrode.
4. Separation of DNA Fragments:
Smaller DNA fragments move faster and farther through the gel, while larger fragments
move more slowly.
5. Staining the Gel:
A DNA-binding dye (like ethidium bromide) is used to stain the gel, allowing the separated
DNA fragments to be visualized under UV light.
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6. Analyze Results:
The DNA fragments appear as bands, and their sizes are determined by comparing them to
a DNA ladder (size standard).
6. Visualization: The DNA bands are stained or labeled to make them visible, typically
under UV light.
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7. Comparison and Analysis: The pattern of DNA bands is compared to reference samples
to identify or match individuals.
After gel electrophoresis in DNA fingerprinting, the next processes typically involve:
1. Visualization:
The gel is examined under UV light to view the stained DNA bands.
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2. Documentation:
Images of the gel are captured for analysis and record-keeping.
3. Analysis:
The band patterns are compared to determine similarities or differences between
samples. This can involve measuring the band sizes or using software for more precise
comparisons.
4. Interpretation:
Based on the banding patterns, conclusions are drawn regarding identity, relationships, or
forensic evidence (e.g., matching a suspect's DNA to a crime scene sample).
5. Reporting:
A formal report is generated summarizing the findings, which may be used in legal or
scientific contexts.
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These steps help establish unique DNA profiles for comparison in applications like
forensics, paternity testing, and genetic research.
Uses of DNA Fingerprinting
1. Forensic Science:
Identifying suspects or victims in criminal cases by matching DNA from crime scenes with
potential offenders.
2. Paternity Testing:
Establishing biological relationships by comparing the DNA of a child with that of a mother
and potential father.
3. Ancestry and Genealogy:
Tracing lineage and ancestry through comparisons of genetic markers among individuals.
4. Biodiversity Studies:
Assessing genetic diversity and population structure in wildlife conservation and ecological
studies.
5. Medical Diagnostics:
Identifying genetic disorders or predispositions to diseases through analysis of specific DNA
markers.
6. Agricultural Biotechnology:
Developing genetically modified organisms (GMOs) by tracking the inheritance of specific
traits.
7. Identification of Remains:
Assisting in the identification of human remains in disaster scenarios or historical contexts
through DNA analysis.
8. Immigration Cases:
Confirming family relationships for immigration purposes.
These applications highlight the versatility of DNA fingerprinting in various fields,
including law, medicine, and research. These steps allow scientists to create a unique DNA
profile for each person.
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