Tools in Biochemistry | Medical Science Optional Notes for UPSC PDF Download

Tools in Biochemistry-Repeats

1. PCR

• Provide a concise overview of the fundamental procedure and clinical applications of polymerase chain reaction (2001).
• Explain the principles of 'Polymerase Chain Reaction (PCR)' technology and outline the sequential steps involved, while also delving into its clinical applications (2017).
• Explore the role of PCR in the diagnosis and management of Mycobacterium tuberculosis (2018).
• Define real-time polymerase chain reaction, highlight its distinctions from regular PCR, and elucidate how this technique aids in examining single nucleotide polymorphism (2009).

2. RFLP

• Explain the basic principle underlying the RFLP technique and outline its clinical applications (2016).

3. RIA

• In 1997, Rosalyn Yalow received the Nobel Prize in medicine for inventing a groundbreaking technique in endocrinology. Describe this technique and briefly explain its principle (2009).
• Elaborate on the principle of Radio Immunoassay (RIA) and discuss the main advantages and disadvantages of this technique.

Restriction fragment length polymorphism (RFLP) is a method developed in 1984 by English scientist Alec Jeffreys during his research on hereditary diseases.

RFLP

DNA markers play a crucial role in the genetic mapping of genomes. Four types of DNA sequences can serve as markers. An individual's genome contains numerous Variable Number Tandem Repeats (VNTRs) and Restriction Fragment Length Polymorphisms (RFLPs), each unique to the individual. The arrangement of VNTRs and RFLPs forms the foundation for DNA fingerprinting (DNA profiling). In forensic DNA analysis, the initial techniques reliant on RFLPs and VNTRs have largely given way to microsatellites (Short Tandem Repeats or STRs).

Principle

Restriction Fragment Length Polymorphism (RFLP) refers to a variation in homologous DNA sequences identifiable by the presence of fragments with different lengths following the digestion of DNA samples using specific restriction endonucleases.

The fundamental technique for detecting RFLPs involves breaking down a DNA sample with a restriction enzyme, which cuts DNA wherever a particular short sequence is present, a process termed a restriction digest. Subsequently, the resulting DNA fragments are separated by length using agarose gel electrophoresis and transferred to a membrane through the Southern blot procedure.

The membrane is then hybridized with a labeled DNA probe to determine the length of fragments that complement the probe. An RFLP is observed when the length of a detected fragment varies among individuals. Each fragment length is considered an allele and can be utilized in genetic analysis.

Clinical Applications

• In paternity or criminal cases, RFLPs are employed to ascertain the origin of a DNA sample, commonly referred to as DNA fingerprinting.
• RFLPs are utilized to determine an individual's disease status, particularly in detecting known mutations associated with:
  a. Sickle cell anemia (Chromosome 11)
  b. Cystic fibrosis (Chromosome 7)
  c. Huntington's disease (Chromosome 4)
  d. Retinoblastoma (Chromosome 13)
  e. Alzheimer's disease (Chromosome 21)
• In human population genetics, RFLPs play a role in studying geographical isolates and comparing the genetic makeup of related species.

PCR

Polymerase chain reaction (PCR) is a widely employed laboratory method designed to replicate numerous copies (in the millions or billions) of a specific segment of DNA.

The primary objective of PCR is typically to generate a sufficient quantity of the target DNA region for subsequent analysis or utilization.

For example, DNA amplified through PCR may undergo sequencing, be visualized through gel electrophoresis, or be cloned into a plasmid for additional experiments.

Steps of PCR

The essential components of a PCR reaction include Taq polymerase, primers, template DNA, and nucleotides (the building blocks of DNA).
These components are combined in a tube, along with the enzyme's necessary cofactors, and undergo repeated cycles of heating and cooling, facilitating the synthesis of DNA.

The basic steps are:

• Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands.
  This provides single-stranded template for the next step.
• Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on t he single-stranded template DNA 
• Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.
• This cycle repeats 25 - 35 times in a typical PCR reaction, which generally takes 2 - 4 hours, depending on the length of the DNA region being copied.

Visualization of PCR results

• Typically, the outcomes of a PCR reaction are visualized through gel electrophoresis.
• Gel electrophoresis is a method in which DNA fragments are propelled through a gel matrix via an electric current, segregating the DNA fragments based on their sizes.
• Typically, a standard, known as a DNA ladder, is incorporated to enable the determination of the size of fragments in the PCR sample.
• DNA fragments of identical length create a "band" on the gel, visible to the naked eye when the gel is stained with a DNA-binding dye.

Applications of PCR

Variations of PCR

As a versatile technique, PCR is adapted to suit specific requirements in various situations.

• Nested PCR
• Inverse PCR
• Anchored PCR
• RT-PCR (Reverse transcription PCR)
• Asymmetric PCR
• Real-time quantitative PCR
• Random amplified polymorphic DNA (RAPD)
• Amplified fragment length polymorphism (AFLP)
• Rapid amplification of cDNA ends (RACE)

Real Time PCR

Real-time polymerase chain reaction (Real-Time PCR) is a molecular biology laboratory technique built upon the principles of polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule in real-time, during the PCR process, as opposed to the endpoint measurement in conventional PCR. Real-time PCR is conducted in a thermal cycler equipped to illuminate each sample with a specified wavelength of light and detect the emitted fluorescence from the excited fluorophore.

Two common methods for detecting PCR products in real-time PCR are:

• Non-specific fluorescent dyes that intercalate with any double-stranded DNA.
• Sequence-specific DNA probes, consisting of oligonucleotides labeled with a fluorescent reporter, allowing detection only after hybridization of the probe with its complementary sequence.

Nested vs Inverse PCR

CBNAAT/Gene expert/RIF

Nucleic Acid Amplification Tests (NAATs) stand as the preferred initial diagnostic approach, ensuring swift confirmation across all types of tuberculosis (TB).

(a) The Xpert MTB/RIF assay, a fully automated, real-time nucleic acid amplification technology operating on the GeneXpert® diagnostic platform, holds distinct advantages. Capable of concurrently detecting TB and rifampin resistance in under 2 hours, it demands minimal biosafety measures and training. The World Health Organization (WHO) recommends its global utilization as the primary diagnostic test for both adults and children exhibiting signs or symptoms of active TB. Additionally, it is suggested as the initial diagnostic test for individuals living with HIV where TB is suspected and as the first test for suspected TB meningitis cases. The sensitivity of this test is approximately 98% in AFB-positive cases and around 70% in AFB-negative specimens.

(b) The innovative Xpert® MTB/RIF Ultra assay (Ultra) exhibits an overall sensitivity 5% higher than its predecessor, particularly benefiting smear-negative, culture-positive cases (increased by 17%) and HIV-infected individuals (increased by 12%). Notably, "trace calls" in this new assay, indicating the detection of nonviable bacilli or bacilli fragments, require evaluation based on risk/benefit considerations.

(c) The TB-LAMP assay, founded on loop-mediated isothermal amplification (LAMP) technology for temperature-independent DNA amplification, is straightforward to use and interpreted through a visual display. Requiring minimal laboratory infrastructure and possessing limited biosafety prerequisites, it presents an accessible option.

Radioimmunoassay (RIA)

Principle of Radioimmunoassay: 

The method integrates three fundamental principles.

• An immune reaction, specifically the binding of antigen and antibody.
• A competitive binding or competitive displacement reaction, ensuring specificity.
• Measurement of radio emission, contributing to sensitivity.

The primary advantage of RIA lies in its remarkably high sensitivity. This test can accurately determine minute quantities, such as nanograms, of antigens and antibodies in the serum. It is capable of analyzing concentrations as low as nanomolar and picomolar levels of hormones in biological fluids.

Applications of RIA include testing various biologic fluids for substances like ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins, and glucocorticoids. Additionally, it is employed in therapeutic drug monitoring for substances such as barbiturates, morphine, and digoxin. In diagnostic procedures, RIA is used for detecting infections, including HIV, Hepatitis A, B, etc.

However, the limitations of RIA encompass the cost associated with equipment and reagents, the short shelf-life of radiolabeled compounds, and challenges related to the disposal of radioactive waste.