Biotech Beginnings: From Infancy to Impact

 CRISPR

CRISPR – clustered regularly interspaced short palindromic repeats – were first discovered in the sequences of DNA from Escherichia coli bacteria and described in 1987 by Ishino et al. [1] from Osaka University (Japan) who accidentally cloned an unusual series of repeated sequences interspersed with spacer sequences while analyzing a gene responsible for the conversion of alkaline phosphatase .However, the function of these sequences remained unclear.

The first experimental information about the mechanism of action of the CRISPR system was obtained in 2007 in the studies of two French food scientists, Rodolphe Barrangou and Philippe Horvath, who worked with yoghurt cultures of bacteria Streptococcus thermophilus for the Danish company Danisco.

Marraffini and Sontheimer published was the first one to describe the presence of genes associated with CRISPR repeats (named by the authors cas1-4, CRISPR-associated genes). These genes were found in close proximity to the CRISPR loci of various prokaryotes, and two of them contained motifs characteristic of helicase and nuclease, which supported the authors’ hypothesis about the non-random association of the cas genes with the CRISPR locus, and their involvement in DNA metabolism.

CRISPR-associated genes). These genes were found in close proximity to the CRISPR loci of various prokaryotes, and two of them contained motifs characteristic of helicase and nuclease, which supported the authors’ hypothesis about the non-random association of the cas genes with the CRISPR locus, and their involvement in DNA metabolism.

Emmanuelle Charpentier is the co-inventor of CRISPR. She was involved in the biochemical characterization of guide RNA and Cas9 enzymemediated DNA cleavage. She unexpectedly discovered what would form the basis of the technology within the immune system of Streptococcus pyogenes, one of the bacteria that cause the most harm to human, when she noticed that a molecule in its immune system was capable of disarming viruses by slicing up their DNA

                          

Applications:

Biomedical applications- generation of disease models and antibody production
Agriculture applications- genome engineering and development of resistant breeds/strains
Transcriptional regulation- repression, activation, and epigenetic modifications
Therapeutic application- gene therapy, antiviral defense, and drug discovery
Genome wide screening- loss of function screens, gain of function screens and knock out libraries.
Genome editing – deletion, insertion and translocation.

Flow Cytometry

Flow cytometry, developed in 1968 by Wolfgang Göhde, is a technology for rapid, multi-parametric analysis of single cells in solution. It uses lasers to produce scattered and fluorescent light signals, which are detected and analyzed by computers.

Key Components:

- Lasers: Light sources for signal generation.

- Detectors: Photodiodes or photomultiplier tubes.

- Data Analysis: Electronic signals converted to .fcs data files for analysis.

Fluorescent Reagents:

- Conjugated antibodies

- DNA binding dyes

- Viability dyes

- Ion indicator dyes

- Fluorescent expression proteins

Applications:

1. Immunophenotyping: Identifies and quantifies cell populations in blood, bone marrow, or lymph by labeling proteins with fluorescent tags. Useful for diagnosing hematological malignancies like lymphomas and leukemia.

2. Cell Sorting: Specialized flow cytometers isolate specific cells by identifying and separating them into collection tubes using fluidics and electronics.

3. Cell Cycle Analysis: Measures replication states and cell aneuploidy using fluorescent dyes to analyze the cell cycle phases.

4. Apoptosis: Differentiates between necrosis and apoptosis by analyzing morphological, biochemical, and molecular changes in cells undergoing programmed cell death.

5. Cell Proliferation Assays: Measures cellular metabolic activity and proliferation by tracking the reduction of fluorescence in labeled cells as they divide.

6. Intracellular Calcium Flux: Monitors calcium ion influx in cells to study signal transduction pathways and cellular responses to stimuli.

Flow cytometry is a vital tool in research and clinical diagnostics, providing detailed insights into cell populations and their functions.

DISCOVERY OF DNA

In 1869, a Swiss physician named Friedrich Miescher made a groundbreaking discovery while working in Tübingen. He isolated a unique substance from white blood cells (leucocytes) that he called "nuclein." This mysterious substance, now known as deoxyribonucleic acid or DNA, would later become recognized as the blueprint of life, forever changing our understanding of biology and genetics.

Miescher's groundbreaking discovery wasn't met with immediate fanfare. His meticulous nature, while ensuring the accuracy of his work, slowed its dissemination. Additionally, convincing the scientific community of the importance of this novel "nuclein" proved difficult, delaying recognition of his findings for many years.

Friedrich Miescher's journey to discovering DNA began with the ambitious goal of isolating cells from lymph nodes. However, encountering technical difficulties, he switched his focus to white blood cells, or leucocytes. It was while working with these cells that Miescher observed a previously unknown substance precipitating from the solution, a substance that would later become known as the foundation of life itself: DNA.

In the 1800s, a curious scientist named Miescher wanted to peek inside a cell's main control center, the nucleus. He started by trying to grab cells from lymph nodes, but things weren't working out. So, he switched to white blood cells. While experimenting, Miescher spotted a strange stuff clumping up in his solution. This gooey mystery, later known as DNA, would become the key to life itself!

The first dna isolation protocol (by Friedrich Miescher)

1.   1.       Miescher ensured the source material (pus on surgical bandages) was fresh and uncontaminated, discarding anything decomposed (Miescher, 1871d).

2.       Samples meeting the requirements were used to isolate leucocytes.

3.       Miescher separated leucocytes from bandaging material and serum (Miescher, 1869a, Miescher, 1871d).

4.       Washing pus with NaCl or alkaline solutions caused a “slimy swelling” of cells, hindering further processing (His, 1897b).

5.       Miescher successfully isolated distinct leucocytes using a dilute solution of sodium sulfate (one part cold saturated Glauber's salt (Na2SO4·10 H2O) solution and nine parts water).

6.       Leucocytes were filtered out through a sheet to remove cotton fibers.

7.       Washing solution was allowed to stand for 1–2 hours to let cells sediment; cells were inspected microscopically to ensure no damage.

8.       Miescher separated nuclei from cytoplasm, a novel procedure at the time.

9.       Cells were rinsed 6–10 times with diluted (1:1000) hydrochloric acid over several weeks at wintry temperatures to remove most protoplasm.

10.   Residue included isolated nuclei and nuclei with little cytoplasm; nuclei were confirmed by the inability to be stained yellow by iodine solutions.

11.   Nuclei were shaken with a water and ether mixture to dissolve lipids; clean nuclei remained in the water phase, while nuclei with cytoplasm collected at the water/ether interface.

12.   Nuclei were filtered and examined microscopically, resulting in completely pure nuclei.

13.   Isolated nuclei were extracted with alkaline solutions; highly diluted (1:100,000) sodium carbonate caused significant swelling and translucency.

14.   Miescher isolated a yellow solution of a substance from these nuclei.

15.   Adding acetic or hydrochloric acid in excess precipitated an insoluble flocculent (DNA), which could be dissolved again with alkaline solutions.

16.   This protocol allowed Miescher to isolate nuclein in appreciable purity and quantities but not enough for subsequent analyses.

17.   Miescher improved the protocol to purify sufficient amounts of nuclein for his experiments on its elementary composition.

Miescher developed new protocols to isolate the nuclei from cells, used hydrochloric acid and ether to extract pure nuclei, and identified a yellow substance precipitate (DNA). His first protocol did not yield enough material for further analysis, leading him to develop a second protocol utilizing pepsin to isolate DNA from proteins.

APPLICATIONS:
Oncology Marker Testing

Genetic Disorder Screening

Infectious Disease Screening

Forensic testing (crime scene investigation)

ELISA

Enzyme immunoassays (EIAs) use enzymes to detect and quantify immunologic reactions. ELISA, a common heterogeneous EIA in clinical analyses, involves attaching an antigen or antibody to a solid phase to separate bound and free reactants.

Typical ELISA Steps:

1. Add sample containing antigen (Ag) to a solid-phase antibody (Ab).

2. Bind, wash, and add enzyme-labeled antibody to form a "sandwich complex" (solid-phase Ab-Ag-Ab enzyme).

3. Wash away unbound antibody and add enzyme substrate.

4. Measure the product, proportional to antigen quantity.

Alternatively, specific antibodies can be quantified by binding the antigen to the solid phase and using an enzyme-labeled antibody specific to the analyte antibody. ELISA assays can detect antibodies to viruses and autoantigens, useful for screening and point-of-care testing.

Development:

ELISA was developed by Engvall and Perlman, and Van Weemen and Schuurs as a modification of radioimmunoassay (RIA). Initially used to measure IgG in rabbit serum and human chorionic gonadotropin in urine, it has become a routine research and diagnostic method. The first ELISA used chromogenic reporters for observable color changes, with advancements leading to fluorogenic, quantitative PCR, and electrochemiluminescent reporters.

Four Major Types of ELISA:

Direct ELISA:

- Antigen-coated plate; screening antibody.

- Steps: Coat antigens, block unbound sites, add enzyme-conjugated primary detection antibody, add substrate.

- Advantage: Eliminates secondary antibody cross-reactivity, rapid.

- Disadvantage: Low sensitivity, high cost.

Indirect ELISA:

- Antigen-coated plate; screening antigen/antibody.

- Steps: Similar to direct ELISA but uses two antibodies (primary detection and secondary enzyme-linked).

- Advantage: Higher sensitivity, less expensive, more flexible.

- Disadvantage: Risk of cross-reactivity.

Sandwich ELISA:

- Antibody-coated plate; screening antigen.

- Steps: Coat capture antibody, block, add antigen, add primary detection antibody, add secondary enzyme-conjugated antibody, add substrate.

- Advantage: Highest sensitivity.

- Disadvantage: Time-consuming, expensive, requires matched pair antibodies.

Competitive ELISA:

- Tests for the presence of a specific antibody in serum.

- Steps: Combine enzyme-conjugated antibody and test serum antibody, add to antigen-coated wells, add substrate.

- Advantage: Less sample purification, measures a wide range of antigens, useful for small antigens, low variability.

- Disadvantage: Low specificity, not suitable for dilute samples.

Applications:

- Detect and measure antibodies in blood.

- Estimate levels of tumor markers and hormones.

- Track disease outbreaks.

- Detect past exposures.

- Screen donated blood for viral contaminants.

- Detect drug abuse.

ISSR

Inter Simple Sequence Repeat (ISSR) is a PCR-based technique that amplifies DNA segments between two identical microsatellite regions oriented in opposite directions. It uses primers (16–25 bp) targeting multiple genomic loci. ISSR markers are stable, reproducible, and generate high polymorphism, making them valuable in genetic studies.

Advantages:

- High reproducibility and stability

- High polymorphism generation

- Cost-effective and simple

Applications:

- Fingerprinting

- Phylogenetic analysis

- Population structure analysis

- Varietal/line identification

- Genetic mapping

- Marker-assisted selection

Examples in Mulberry (Morus spp.):

- Analyzing phylogenetic relationships among varieties

- Identifying taxonomic positions

- Associating markers with leaf yield traits

·       Genetic Diversity:

- Swertia chirayita: 19 primers, 315 bands, 98.7% polymorphism

- Glycyrrhiza uralensis: 14 primers, 249 polymorphic bands (92.2%)

- Humulus lupulus: RAPD (42.3%), STS (71%), ISSR (32.6%), AFLP (57.6%)

- Psychotria ipecacuanha: 193 bands, 97.4% polymorphic

- Benincasa hispida: 5 primers, 26 markers, 11 polymorphic

- Cashew germplasm: RAPD (51/60), ISSR (58/67)

- Tribulus terrestris: AFLP (82.9%), SAMPL (94.7%), ISSR (73.6%), RAPD (59%)

- Mactra veneriformis: 20 primers, 240 loci, 97.9% polymorphic

- Rhodiola chrysanthemifolia: 13 primers, 116 fragments, 89.7% polymorphic

- Momordica charantia: RAPD (76/208), ISSR (94/125)

- Vanilla planifolia: RAPD (83.24%), ISSR (86.11%)

·       Authentication of Medicinal Plants:

- Flammulina velutipes: 8 primers, 104 bands, 81 polymorphic

- Eucalyptus spp.: Developed SSR markers

- Capsicum annum: Used ISSR-PCR and FISSR-PCR for DNA profiling

·       Identification of Plants:

- Diplodus spp. & Dentex dentex: 8 primers, 97 fragments, 97.9% polymorphic

- Radish cultivars: RAPD (85.44%), ISSR (85.2%), SRAP (85.41%)

- Tomato species: 14 primers, 9 distinguishable species

- Eggplant and Solanum species: 34 primers, 99.1% polymorphic

·       Germplasm Authentication:

- Gerbera jamesonii: 15 primers, 12 monomorphic, 3 polymorphic

- Vernicia fordii: 12 primers, 110 bands, 90 polymorphic

- Jute species: STMS, ISSR, RAPD showed high polymorphism

·       Genotyping of Plants:

- Junisperus populations: Analyzed using ITS sequences, RAPDs, ISSRs, terpenoids

- Chondrus crispus: 22 primers, 163 loci, 27.0-55.8% polymorphic

- Cucumber: Genetic linkage map with 116 SRAPs, 33 RAPDs, 11 SSRs, 9 SCARs, 3 ISSRs, 1 STS

- Coffea arabica: Nuclear genome polymorphism (4.36%), mitochondrial genome polymorphism (41%) 

PCR (Polymerase Chain Reaction)

PCR, developed by Kary Mullis in the 1980s, is a technique to amplify specific DNA sequences. It utilizes DNA polymerase's ability to synthesize a complementary DNA strand from a template strand using primers.

Steps:
1. Denaturation: At 94-98°C for 20-30 seconds, double-stranded DNA is separated into single strands.

2. Annealing: Temperature is lowered to allow primers to bind to complementary DNA sequences.

3. Extension: At 72°C, Taq polymerase synthesizes the new DNA strand by adding nucleotides in the 5’-3’ direction.

Applications:

- Diagnostics: Amplifies viral genomes to detect infections (e.g., SARS-CoV-2 via RT-PCR).

- Genetic Disorder Detection: Identifies genes associated with genetic disorders from patient or fetal samples.

- Oncogene Detection: Detects mutations associated with cancers such as leukemia and lymphoma.

- Forensics: Amplifies DNA from crime scenes for comparison with suspects.

- Molecular Biology Research: Sequencing genomes and studying gene expression alterations.

- Recombinant DNA Technology: Amplifies genes for insertion into vectors for cloning.

- Ancient DNA Analysis: Studies ancient DNA samples (e.g., mammoths, Egyptian mummies).

- Phylogenetics: Analyzes relationships from ancient DNA samples.

- Disease Monitoring: Tracks disease spread and detects new mutations.

- Prenatal Testing: Tests for genetic disorders and carrier status.

- qPCR (Real-Time PCR): Quantifies DNA or gene expression levels in real-time.

Advantages:

- High sensitivity and specificity

- Rapid results

- Simple and easy to perform

- Produces millions of copies of the target DNA for various analyses

PCR has revolutionized research, diagnostics, forensics, and genetic studies, providing a crucial tool for amplifying and analyzing DNA sequences.