RNA splicing

RNA splicing is a crucial process in molecular biology that occurs during the expression of genes in eukaryotic organisms. It is the removal of introns (non-coding regions) and the joining of exons (coding regions) in the pre-mRNA (pre-messenger RNA) molecule to produce mature mRNA (messenger RNA).

Here's how RNA splicing works:

1. Transcription: The process begins with the transcription of a gene's DNA sequence into pre-mRNA by an enzyme called RNA polymerase. During transcription, both exons and introns are transcribed.

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3. Intron Removal: In the pre-mRNA molecule, introns are interspersed between exons. The introns do not code for proteins, so they need to be removed to form a functional mRNA. Small nuclear ribonucleoproteins (snRNPs) and other protein complexes recognize specific sequences at the boundaries of introns called splice sites.

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5. Spliceosome Formation: The splice sites are recognized by the spliceosome, a large molecular complex composed of snRNPs and additional proteins. The spliceosome assembles at the intron-exon junctions.

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7. Splicing: The spliceosome catalyzes the splicing reactions. It cuts the pre-mRNA at the 5' end of the intron, creating a branched structure called a lariat, with the intron forming a loop. Then, the spliceosome cuts the pre-mRNA at the 3' end of the intron, releasing the intron as a lariat structure.

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9. Exon Joining: The exons that are now free from the intron are ligated (joined) together, resulting in a continuous mature mRNA sequence that contains only exons.

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11. Mature mRNA: The mature mRNA molecule contains the genetic information that will be used during translation (protein synthesis) in the cytoplasm. It exits the nucleus and moves to the ribosomes, where it serves as a template for the assembly of amino acids into a specific protein.

RNA splicing allows for the generation of multiple protein isoforms from a single gene, a process known as alternative splicing. This alternative splicing greatly increases the diversity of proteins that can be produced from the limited number of genes in the genome, playing a significant role in the complexity and functionality of eukaryotic organisms.

Notes on DNA fingerprinting,

DNA fingerprinting, also known as DNA profiling or genetic fingerprinting, is a powerful forensic technique used to identify and compare individuals based on their unique genetic makeup. It relies on the analysis of specific regions of an individual's DNA that are highly variable between individuals, known as Short Tandem Repeats (STRs) or Variable Number Tandem Repeats (VNTRs). These regions consist of repeated sequences of DNA bases, and the number of repeats can vary significantly between individuals.

Principles of DNA Fingerprinting:

The key principles behind DNA fingerprinting are as follows:

1. 1.Sample Collection: The process begins with the collection of biological samples, such as blood, saliva, hair follicles, or semen, from the crime scene, suspects, and victims.

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3. 2.DNA Extraction: The DNA is extracted from the collected samples using various methods to obtain a pure DNA sample.

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5. 3.Polymerase Chain Reaction (PCR): PCR is employed to amplify the specific STR regions of the DNA. Primers targeting the flanking regions of the repeat sequences are used in PCR.

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7. 4.Fragment Analysis: The amplified DNA fragments are separated and visualized using a technique like gel electrophoresis or capillary electrophoresis. These methods separate the DNA fragments based on their size, and the resulting patterns are unique to each individual.

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9. 5.DNA Profiling: The banding patterns obtained from the fragment analysis are called DNA profiles or DNA fingerprints. These profiles are unique to each individual (except for identical twins) and can be used for identification and comparison purposes.

DNA Fingerprinting Process Diagram:

Applications of DNA Fingerprinting:

1. 1.Forensic Investigations: DNA fingerprinting is extensively used in criminal investigations to match suspects with evidence collected from crime scenes, establishing links between perpetrators and victims.

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3. 2.Paternity and Maternity Testing: It can determine biological parentage in legal and personal cases, such as custody disputes or immigration cases.

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5. 3.Missing Persons and Disaster Victim Identification: DNA fingerprinting is crucial in identifying missing persons or victims in mass disasters when traditional identification methods are not feasible.

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7. 4.Historical and Anthropological Studies: DNA fingerprinting can reveal ancestral relationships, migrations, and population genetics.

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9. 5.Wildlife Conservation: It aids in studying animal populations, tracking illegal trade, and identifying individuals involved in poaching.

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11. 6.Medical Diagnostics: DNA fingerprinting can be used to diagnose genetic disorders and identify genetic predispositions for certain diseases.

Advantages of DNA Fingerprinting:

1. Highly Discriminatory: DNA fingerprinting provides a high degree of discrimination, making it highly reliable for individual identification.

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3. Accuracy: The chances of two unrelated individuals having the same DNA fingerprint are incredibly low, reducing the risk of false identifications.

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5. Rapid Analysis: With modern PCR and electrophoresis techniques, DNA fingerprinting results can be obtained relatively quickly.

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7. Admissible in Court: DNA fingerprinting is widely accepted as evidence in legal proceedings due to its accuracy and reliability.

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9. Non-invasive: In many cases, DNA can be extracted from non-invasive samples like saliva, reducing discomfort during sample collection.

Despite its advantages, DNA fingerprinting also raises ethical and privacy concerns regarding the storage and use of genetic information. Striking a balance between the benefits of forensic science and individual rights remains a significant challenge in the application of this powerful technology.

notes on ELISA what is ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used laboratory technique used to detect and quantify specific proteins, peptides, antibodies, hormones, and other molecules in biological samples. It plays a crucial role in medical diagnostics, research, and various applications in life sciences. Here, I'll provide a detailed overview of the ELISA test, its principles, types, applications, and advantages.

1. 1.Principle of ELISA:

2. ELISA is based on the principle of antigen-antibody interaction, which is specific and highly sensitive. The assay involves immobilizing the antigen of interest onto a solid surface (such as a microplate well). When a sample containing the target molecule (e.g., antibody or antigen) is added, it binds to the immobilized counterpart forming an antigen-antibody complex. A series of washing steps removes unbound molecules, and a secondary enzyme-labeled antibody (which recognizes the other part of the antigen-antibody complex) is added. The enzyme-linked secondary antibody allows the detection of the bound antigen-antibody complex using a chromogenic substrate. The signal generated is directly proportional to the amount of target molecule present in the sample.

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4. 2.Types of ELISA:

5. a. Direct ELISA: In this type, the antigen is immobilized on the surface, and the enzyme-labeled primary antibody directly binds to it. This is the simplest ELISA format but is less commonly used due to the limited sensitivity of primary antibodies labeled with enzymes.

6. b. Indirect ELISA: Here, the antigen is immobilized, and an unlabeled primary antibody is used to bind the target. Then, a secondary enzyme-labeled antibody, specific to the primary antibody, is added to amplify the signal. Indirect ELISA is more sensitive than direct ELISA.

7. c. Sandwich ELISA: Sandwich ELISA is highly sensitive and widely used for detecting antigens. The antigen is captured between two specific antibodies: one immobilized on the surface and the other enzyme-labeled. This dual binding increases specificity and sensitivity.

8. d. Competitive ELISA: In competitive ELISA, the sample antigen and a labeled antigen compete for binding to a limited amount of specific antibodies. The more antigen in the sample, the less labeled antigen binds, leading to a lower signal.

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10. 3.ELISA Procedure:

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    • Coating: The antigen is immobilized onto a microplate well surface.
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    • Blocking: Non-specific binding sites on the plate are blocked to reduce background noise.
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    • Sample and Controls: The test samples, along with positive and negative controls, are added to the wells.
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    • Incubation: The plate is incubated to allow the antigen-antibody reaction to occur.
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    • Washing: Unbound molecules are removed by washing the plate.
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    • Secondary Antibody: The enzyme-labeled secondary antibody is added to the wells.
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    • Substrate: A chromogenic substrate is added that reacts with the enzyme, producing a measurable signal (usually color change).
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    • Reading: The absorbance or fluorescence of each well is measured, and the results are analyzed.
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11. 4.Applications of ELISA:

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    • Medical Diagnostics: ELISA is used for diagnosing various infectious diseases, such as HIV, hepatitis, and COVID-19, by detecting specific antibodies or antigens in patient samples.
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    • Autoimmune Diseases: It helps detect autoantibodies present in conditions like rheumatoid arthritis, systemic lupus erythematosus, etc.
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    • Hormone Assays: ELISA is employed in measuring hormone levels (e.g., insulin, cortisol, etc.) in endocrinology and fertility testing.
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    • Allergy Testing: It can identify allergen-specific IgE antibodies responsible for allergic reactions.
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    • Cancer Biomarker Detection: ELISA can detect tumor markers to aid in cancer diagnosis and monitoring.
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    • Drug Detection: It is used in toxicology to measure drug levels or detect drug abuse.
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    • Environmental Monitoring: ELISA is applied in detecting contaminants and pollutants in food, water, and soil samples.

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13. 5.Advantages of ELISA:

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    • High Sensitivity: ELISA can detect low concentrations of antigens or antibodies, making it highly sensitive.
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    • Specificity: The antigen-antibody interaction is highly specific, reducing the chances of false positives or negatives.
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    • Quantification: ELISA can quantify the amount of the target molecule present in the sample.
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    • High Throughput: ELISA can be easily adapted to analyze numerous samples simultaneously, making it suitable for large-scale studies.
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    • Versatility: ELISA can be used to detect a wide range of molecules, from small peptides to large proteins.

In summary, the ELISA test is a versatile and powerful technique used in various fields of research, clinical diagnostics, and industry. Its ability to detect and quantify specific molecules with high sensitivity and specificity has made it an indispensable tool in the scientific community.