Tools and Techniques: Southern Blotting, DNA fingerprinting, Polymerase Chain Reaction & Fluorescenc | Botany Optional for UPSC PDF Download

Southern Blotting 

Principle

• Southern blotting is a technique used to identify specific DNA fragments from a mixture of similar molecules. 
• It involves separating DNA fragments based on their size and charge using gel electrophoresis. The separated fragments are then transferred onto a nylon membrane, where a specific DNA probe is used to detect the desired DNA. 
• The probe is a short, single-stranded DNA that is labeled with a marker for easy detection after hybridization. 
• The technique is named after its developer, Edward M. Southern, and is a type of RFLP (restriction fragment length polymorphism). 

Procedure/Steps

1. Restriction Digest: The DNA is cut into fragments using a specific restriction enzyme, and the number of fragments is amplified by PCR.
2. Gel Electrophoresis: The DNA fragments are separated based on size using gel electrophoresis.
3. Denaturation: The gel is treated with alkali or acid to denature the double-stranded DNA fragments, separating the DNA strands.
4. Blotting: The separated DNA strands are transferred onto a positively charged nylon membrane through a process called blotting.
5. Baking and Blocking: The membrane is baked to fix the DNA, and then treated with casein or BSA to block non-specific binding sites.
6. Hybridization with Labeled Probes: The DNA on the membrane is treated with a labeled probe that contains complementary sequences to the gene of interest. The probe binds to the complementary DNA on the membrane.
7. Visualization by Autoradiogram: The membrane-bound DNA labeled with the probe is visualized using an autoradiogram, which provides a pattern of bands.

Application of Southern Blotting

• Detection of DNA in a given sample.
• DNA fingerprinting.
• Paternity testing, criminal identification, and victim identification.
• Isolation and identification of desired genes.
• Restriction fragment length polymorphism analysis.
• Identification of mutations or gene rearrangements in DNA sequences.
• Diagnosis of genetic diseases.
• Identification of infectious agents.

DNA fingerprint

The human body is a remarkable biological entity composed of trillions of cells, each harboring a genetic blueprint known as DNA (deoxyribonucleic acid). Remarkably, nearly 99.9 percent of the DNA across different individuals is identical, highlighting the remarkable genetic similarity shared among human beings. However, it is the remaining 0.1 percent of DNA that sets each person apart, barring the exception of identical twins. This seemingly small fraction equates to approximately three million base pairs, where variations in genetic sequences occur. These distinctions are the building blocks of our uniqueness and can be harnessed to differentiate one individual from another.

The Role of Minisatellites

Within the diverse landscape of human DNA, minisatellites play a pivotal role in establishing individual identity. Minisatellites are relatively short sequences, typically consisting of 10 to 60 base pairs, and they exhibit a higher degree of variation from one person to the next compared to other parts of the genome. This variability manifests as differences in the number of repeated units, often referred to as 'stutters,' within the minisatellite sequence. The discovery of minisatellites in 1980 marked a significant breakthrough in genetics and paved the way for the development of DNA fingerprinting.

The Birth of DNA Fingerprinting

The concept of DNA fingerprinting was conceived and introduced by Professor Sir Alec Jeffreys in 1984. He recognized that variations in human DNA, particularly within minisatellites, could serve as unique identifiers for individuals. DNA fingerprinting is a sophisticated technique that simultaneously detects numerous minisatellites within the genome, culminating in the creation of a distinct genetic pattern, aptly named a DNA fingerprint. The likelihood of two individuals sharing the same DNA fingerprint, without being identical twins, is exceedingly low. Similar to one's physical fingerprint, a DNA fingerprint is an inherent trait, unique from birth.

The Genesis of the First DNA Fingerprint

• The journey to producing the first DNA fingerprint began with the extraction of DNA from a human sample, typically sourced from blood. Molecular 'scissors,' known as restriction enzymes, were employed to cleave the DNA, generating a multitude of DNA fragments with varying lengths. These fragments were subsequently sorted by size through a technique called gel electrophoresis.
• Gel electrophoresis involved loading the DNA fragments into wells at one end of a porous gel, which acted as a molecular sieve. The application of an electric current propelled the negatively-charged DNA through the gel, with shorter fragments migrating more swiftly than longer ones. By the time the current ceased, the DNA fragments had sorted themselves by size, with the smallest molecules farthest from the original sample location.
• The next step involved transferring these DNA fragments from the delicate gel onto a sturdy nylon membrane and then 'unzipping' them to yield single DNA strands. Subsequently, the nylon membrane was exposed to radioactive probes—small fragments of minisatellite DNA tagged with radioactive phosphorus. These probes selectively adhered to complementary minisatellite sequences within the genome. Upon exposure to radioactivity, more than 30 dark bands materialized on X-ray film, forming the distinctive DNA fingerprint.

Evolution to DNA Profiling

• Modern DNA profiling, also known as STR (Short Tandem Repeat) analysis, has supplanted traditional DNA fingerprinting. Unlike minisatellites, STRs are shorter sequences, typically comprising two to five base pairs. They are repeated throughout the human genome, such as the sequence 'TATATATATATA.' This advanced technique has revolutionized the field of genetics by enabling the analysis of minute DNA quantities obtained from various biological samples, including blood, saliva, and hair.
• In contrast to the original method, DNA profiling dispenses with restriction enzymes and employs the polymerase chain reaction (PCR) to amplify specific STR sequences. PCR, an automated process, generates multiple copies of a designated DNA segment, even from partially degraded samples. In STR analysis, fluorescently-labeled primers attach to both ends of the target STR sequence, facilitating their identification after PCR. Electrophoresis is then utilized to segregate the fragments based on size, with a laser illuminating the fluorescently-tagged STRs and producing a profile of colored peaks, each corresponding to a specific STR sequence.

The Power of DNA Profiles in Forensics

• DNA profiles have emerged as invaluable tools in forensic science due to their ability to identify individuals with minute traces of biological material left at crime scenes. In the United Kingdom, a complete DNA profile comprises 11 STR sequences, along with a sex determiner. This profile helps confirm whether the DNA belongs to a man or a woman. Furthermore, new profiles now include an additional five STR sequences, promoting cross-border consistency within Europe.
• In the United States, the Federal Bureau of Investigation (FBI) recommends testing 13 STR sequences, with many states expanding their testing to enhance inter-state investigations. A match between a crime scene profile and an individual's profile signifies a potential suspect, while matches between different crime scene profiles can unveil repeat offenders. DNA evidence, when combined with other legal evidence, strengthens criminal prosecutions.

Storage of DNA Profiles

The United Kingdom pioneered the establishment of a national DNA database in 1995, housing DNA profiles primarily linked to serious crimes. The Protection of Freedom Act 2013 mandated the removal of 1,766,000 DNA profiles obtained from innocent adults and children. Today, many countries maintain national DNA databases, enabling the efficient storage and retrieval of genetic information for investigative purposes.

DNA Profiling and Kinship

As humans inherit half of their DNA from each parent, DNA profiling plays a pivotal role in confirming familial relationships. It is frequently utilized to establish or disprove biological parentage and has aided in the identification of crime victims and reuniting separated families.

Conclusion

DNA fingerprinting, the brainchild of Professor Sir Alec Jeffreys, has evolved into the cutting-edge science of DNA profiling. This remarkable technique enables the identification of individuals with unprecedented accuracy, making it an indispensable tool in forensic investigations, kinship determination, and countless other applications in the realm of genetics. DNA profiles are not merely scientific marvels but powerful instruments that safeguard justice, promote family bonds, and unveil the unique genetic code that defines each of us.

Polymerase chain reaction (PCR)

Polymerase Chain Reaction (PCR) has been a game-changer in molecular biology ever since Kary Mullis introduced it in 1983. This in vitro enzymatic process has the remarkable ability to amplify specific segments of DNA or RNA from diverse living organisms. It is a versatile and cost-effective technique with applications spanning clinical medicine to forensic science. 

The PCR Principle

At its core, PCR aims to amplify particular segments of DNA. It does so by replicating DNA in vitro through a series of temperature-dependent steps.
These steps are as follows:

• Denaturation: This initial step involves heating the DNA to a high temperature (typically 92°C-96°C) for approximately 2 minutes. During this phase, the double-stranded DNA separates into single-stranded DNA.
• Annealing: After denaturation, the temperature is lowered to a range of 45°C-55°C, allowing primers to anneal or bind to each of the single DNA strands.
• Extension: The temperature is then raised to around 72°C, enabling DNA polymerase to add complementary dNTPs to the 3’ end of the primer, synthesizing a new DNA strand.

These three steps are reiterated 20-30 times using an automated thermocycler. This repetitive process results in an exponential accumulation of specific DNA fragments, with the number of DNA strands doubling in each cycle. The formula used to estimate this amplification is Amplification = 2ⁿ, where n represents the number of PCR cycles.
This rapid and precise amplification of DNA is a significant advantage of PCR. It allows the amplification of DNA sequences from any source, hundreds of millions of times, within a matter of hours. This is in stark contrast to traditional recombinant DNA technology, which is considerably more time-consuming.

Procedure or Steps of PCR

PCR consists of three fundamental steps, as previously mentioned. These steps are essential for the successful amplification of DNA. However, their efficiency and specificity can be influenced by various factors, including primer design, template DNA amount, and the choice of DNA polymerase.

PCR consists of three basic steps:

• Denaturation
  • Two strand of DNA separates (melt down) to form single stranded DNA
  • This step is generaly carried out at 92C-96C for 2 minutes.
• Annealing
  • Annealing of primer to each strand is carried out at 45C-55C
• Extension
  • DNA polymerase adds dNTPs complementary to templates strands at 3’end of primer.
  • It is carried out at temperature of 72C.

Types of PCR

PCR is a versatile technique with various specialized applications. Some notable types of PCR include:

• Standard PCR: Used for general DNA amplification.
• Reverse Transcriptase PCR (RT-PCR): Specifically for RNA amplification.
• Real-Time PCR: Allows for quantification of DNA or RNA in real-time.
• Multiplex PCR: Amplifies multiple target sequences simultaneously.
• High Fidelity PCR: Generates high-fidelity DNA copies.
• Solid Phase PCR: Conducted on a solid substrate.

Applications of PCR

The applications of PCR span across several domains, including:

• Forensic Science: PCR plays a pivotal role in DNA fingerprinting, paternity testing, and criminal identification.
• Medical Diagnosis: It is used for molecular identification of microorganisms and diagnosis of genetic diseases.
• Evolutionary Biology: PCR aids in the study of evolutionary processes.
• Paleontology: It contributes to fossil studies.
• Gene Cloning and Expression: PCR is invaluable for gene manipulation.
• Gene Sequencing: It is a crucial component of sequencing efforts.
• Vaccine Production: PCR contributes to the production of vaccines through recombinant DNA technology.
• Drug Discovery: PCR supports drug development.
• Mutation Studies: It is used to study genetic mutations.
• Human Genome Project: PCR played a significant role in the Human Genome Project.

In conclusion, Polymerase Chain Reaction (PCR) is a groundbreaking molecular biology technique with a multitude of applications. Its ability to rapidly amplify DNA or RNA fragments has revolutionized various fields of science and medicine, making it an indispensable tool for researchers and diagnosticians alike. The precision, speed, and specificity of PCR continue to drive advancements in genetics and biotechnology.

Fluorescence In Situ Hybridization (FISH)

In the realm of biotechnology, one groundbreaking technique has emerged as a beacon of innovation: Fluorescence In Situ Hybridization, commonly known as FISH. This article delves into the intricacies of FISH, shedding light on its applications, techniques, and limitations.

Uncovering the Unseen: FISH in Action

FISH is a molecular biology technique employed to detect subtle chromosomal abnormalities that evade conventional microscope analysis.
It primarily serves two crucial purposes:

• Chromosome Counting and Verification: FISH is a powerful tool to ascertain the number of specific chromosomes within each cell and confirm any visible rearrangements under microscopic examination.
• Targeted Chromosome Analysis: FISH allows researchers to zoom in on a specific region of a chromosome, facilitating the identification of genetic anomalies. This precision is achieved by utilizing a specialized chemical that emits a vivid fluorescence signal upon binding to a particular chromosome region.

The Probing Process

• FISH operates on the principle of utilizing probes—short DNA strands labeled with fluorescent markers. These probes are meticulously designed to complement specific segments of a chromosome. When subjected to heat-induced denaturation, these probes hybridize with their complementary sequences in the patient's DNA. Importantly, a probe fails to hybridize if a small deletion is detected in the target region, thus serving as a diagnostic indicator. Conversely, the presence of duplications results in an increased number of probe hybridizations.

The FISH procedure entails several critical steps:

• Denaturing of the Chromosomes
• Denaturing of the Probes
• Hybridization
• Fluorescence Staining
• Examination of Slides or Storage in Darkness

Detecting Deletions and Duplications

FISH employs two types of probes, each with a distinct role in the detection process:

• Control Probes (Usually Green): These probes bind to sequences not affected by the deletion, ensuring a signal on both chromosomes.
• Deletion Probes (Typically Red): These probes target sequences within the deletion region. As deletions usually affect only one of the chromosome pair, these probes bind to the undamaged chromosome but fail to attach to the deleted one, resulting in a single signal.

Varieties of Probes

FISH harnesses three different types of probes, each serving a unique purpose:

• Locus-Specific Probes: These probes target specific chromosome regions, aiding in the identification of gene locations on particular chromosomes.
• Centromeric Repeat Probes: Alphoid or centromeric repeat probes identify the presence of the correct number of chromosomes and are used alongside locus-specific probes to detect missing genetic material from specific chromosomes.
• Whole Chromosome Probes: Comprising a collection of smaller probes, these are useful for scrutinizing chromosomal abnormalities, such as the fusion of one chromosome's segment with another.

Sample Sources for FISH

FISH analysis primarily relies on blood samples obtained from both adults and children. It also plays a crucial role in prenatal diagnostics, utilizing placental samples from chorionic villus sampling (CVS) or amniotic fluid from amniocentesis. This approach enables the detection of aneuploidy and deletions in utero.

Exploring the Boundaries: Limitations of FISH

While FISH has revolutionized genetic analysis, it does come with certain limitations. FISH probes are readily available for well-characterized duplication and deletion syndromes. Detecting duplications can be challenging due to the complexity of interpreting extra probe attachments.

FISH in Clinical Practice

The versatility of FISH extends to various realms of clinical practice:

• Pre-Implantation Diagnostics
• Prenatal Diagnostics
• Tumor Diagnostics
• Postnatal Diagnostics
• Centromere Mutations Study

In conclusion, Fluorescence In Situ Hybridization stands as a hallmark technique in the field of biotechnology, opening new vistas in genetic analysis. Its precision and versatility continue to drive innovative applications in clinical practice, making it an indispensable tool for researchers and clinicians alike.