11 May

Basics of Biotechnology

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Content

• Introduction
• Basics understanding of Genetic Material
• Process of manufacture of Protien
• Two core techniques that enabled birth of modern biotechnology are
• Tools of Recombinant DNA Technology
• CRISPR-CAS09
• CRISPER Technology inserted genes that allow immune cells to attack cancer cells (Nov 2022: Source – The Hindu)
• How Gene Therapy Using CRISPR can cure Cancer (Dec 2022: Source the Hindu)
• Somatic Cell Nuclear Transfer

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1) INTRODUCTION

• Definitions
  • Biotechnology is the use of biological processes, organisms, or systems to manufacture products intended to improve the quality of human life.
    • E.g., Curd, Alcohol, GM crops, test-tube baby, developing a DNA vaccine or correcting a defective gene, are all part of Biotechnology.
  • Depending on the tools and applications, it often overlaps with the (related) fields of bioengineering, biomedical engineering, bio manufacturing, molecular engineering etc.
• Two Sections of Biotechnology: The entire field of Biotechnology can be divided into two sections
  • Classical/traditional/Old Biotechnology
    • E.g.
      • Curd being prepared with the help of microbes
      • Brewing alcohol
      • Cheese, bread and vinegar
      • Penicillin
    • In all the above product only natural capabilities of the microorganisms and cells were exploited.
  • Modern Biotechnology
    • Modern biotechnology refers to manipulation of genome or innate capabilities of organisms for making it more desirable or to synthesis a valuable product.
    • E.g.
      • Genetic Engineering
    • Tissue/Cell Culture (it refers to growth of tissue or cells in an artificial medium separate from the organisms)

BASIS OF BIOTECHNOLOGY

• Most living organisms have DNA as genetic material, DNA (Deoxyribonucleic Acid).
  • Some viruses have RNA as genetic material (e.g. Tobacco Mosaic viruses, QB bacteriophage, etc.)
  • Now since all living organisms have DNA, it is possible to make changes, mix and match and this gives rise to possibility of the use of biotechnology.

2) BASICS UNDERSTANDING OF GENETIC MATERIAL

A) GENE

• It is basic physical and functional unit of hereditary. It contains the code for a molecule that has a function. They act as instructions to make molecules called proteins
• Genes are located on DNA. It is a short section of DNA. DNA can be cut and separated, forming a sort of ‘bar code’ that is different from one person to the next.
• In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases.
• The Human Genome Project has estimated that humans have between 20,000 and 25,000 genes.
• Gene Mapping
  • Determining the gene’s functionality and position of the gene in the chromosome is called gene mapping.

B) DNA (DEOXYRIBONUCLEIC ACID)

• DNA is the hereditary material in humans and almost all other organisms. Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA)
• DNA is long polymer of deoxyribonucleotides.  I.e. a deoxyribonucleotide is the monomer, or single unit, of DNA, or deoxyribonucleic acid.
• The length of the DNA is usually defined as number of nucleotides (or a pair of nucleotides referred to as base pairs) present in it.
• Human DNA is 3.3 X 109 base pairs.
• Structure of Polynucleotide Chain
  • A nucleotide has three components – a nitrogenous base, a pentose sugar, (deoxyribose in case of DNA), and a phosphate group.
  • There are two types of nitrogenous base.
    • Purines (Adenine and Guanine)
    • Pyrimidines (Cytosine, Uracil and Thymine)
  • Note: Thymine is only found in DNA and Uracil only in RNA
  • DNA bases pair up with each other, A with T and C with G, to form units called base pairs.
  • The bases in two strands are paired through hydrogen bond (H-bonds) forming base pairs (bp). Adenine forms two hydrogen bonds with Thymine from opposite strand and vice-versa. Similarly, Guanine is bonded with Cytosine with three H-bonds.
  • The structure of double helix is somewhat like a ladder, with the base pairs forming the ladder’s rungs and sugar and phosphate molecules forming the vertical sidepieces of the ladder.The two chains are coiled in right-handed fashion.

C) WHAT IS DNA FINGERPRINTING?

• DNA fingerprinting, also called DNA typing, DNA profiling, genetic fingerprinting, genotyping, or identity testing is a method of isolating and identifying variable elements in the base pair sequence of DNA.
• This technique was developed in 1984 by British geneticist Alec Jeffreys, after he noticed that certain sequences of highly variable DNA (known as minisatellites), which don’t contribute to the function of genes, are repeated within genes.
• It was also noticed that each individual has a unique pattern of minisatellites (the only exceptions being multiple individuals form a single zygote, such as identical twins).
• DNA fingerprinting is a technique that simultaneously detects lots of mini satellites in the genome to produce a pattern unique to an individual. This is a DNA Fingerprint.
• How is DNA fingerprint created?
  • Obtaining a sample of cells: such as skin, hair, or blood cells which contain DNA.
    • Extract and purify DNA from these cells.
    • PCR is used to amplify the desired fragments of DNA many times over creating thousands of copies of the fragments.
    • Once an adequate amount of DNA has been produced using PCR, the exact sequence of nucleotide pairs in a segment of DNA can be determined by using one of several biomolecular sequencing methods.
• Application of DNA Fingerprinting:
• Identification: It is a forensic technique used to identify individuals/ dead bodies by characteristics of their DNA.
  • Solving legal disputes:
    • Physically connect a piece of evidence to a person or rule out someone as a suspect.
    • To determine paternity and other relationships
  • Medical applications:
    • Match tissue of organ donors with those of people who need transplant ▪ Identify diseases that are passed down through your family
    • Help find cure for those diseases, called hereditary diseases.
• Problems:
  • Sources of errors: Sample contamination, faulty preparation procedures, and mistakes in interpretation of results are major sources of error.

D) DNA BARCODING

• DNA Barcoding is a tool for rapid species identification based on DNA sequence. It uses as short section of DNA from a specific gene or genes.
  • The way barcodes on a product, uniquely identifies a commercial product, in the same way, short gene segments – known as DNA barcodes – are unique for each species.
  • DNA barcoding has emerged as a global standard for fast and reliable genetic species identification of animals, plants and fungi.
• Different gene regions are used to identify the different organismal groups using barcoding:
  • For e.g., for animals (birds, butterflies, fish) and some protists – a short DNA sequence of COI gene found in mitochondrial DNA is used.
  • Similarly, Species identification of land plants is enabled by the combination of two different chloroplast gene regions – matK and rbcL.
  • Fungi species can be determined by the ITS region.
• The ultimate goal of DNA barcoding is to build a publicly accessible reference database with species-specific DNA barcode sequences.
• Various methods of DNA Barcoding: Barcoding can be done from tissue from a target specimen, from a mixture of organisms (bulk samples), or DNA present in environmental samples (e.g. water or soil). The methods barcoding will differ in each of these cases:
  • Tissue Samples
  • Bulk Samples: This sample contains several organisms from the taxonomic group under study.
    • E.g. – Aquatic macroinvertebrate samples collected by kick-net, or insect samples collected with a Malaise trap.
  • eDNA samples: The environmental DNA (eDNA) method is a non-invasive approach to detect and identify species from cellular debris or extracellular DNA present in environmental samples (e.g., water or soil).
    • The main difference between bulk samples and environmental samples is that the bulk sample usually provides a large quantity of good-quality DNA.
  • Applications of DNA Barcoding:
    • Identifying plant leaves (even when flowers and fruits are not available)
    • Identifying pollen collected on the bodies of pollinating animals
    • Identifying insect larvae which may have fewer diagnostic characteristics than adults
    • Investigating the diet of an animal based on its stomach content

E) CHROMOSOMES

• In the nucleus of each cell, the DNA molecule is packaged into thread-like structure called chromosomes.
• Each chromosome is made up of DNA tightly coiled many times around protein called histones that support the structure.
• The adjacent figure shows the relation between chromosome and DNA molecule

F) RNA

• RNA stands for ribonucleic acid. It is a molecule with long chain of nucleotides. A nucleotide contains a nitrogenous base, a ribose sugar, and a phosphate.
• Like DNA, RNA is also vital for living cells.
• Shape and structure
  • It comes in a variety of different shapes.
  • Unlike double-stranded DNA, RNA is a single-stranded molecule in many of its biological roles and has a much shorter chain of nucleotides.
  • However, RNA can, by complementary base pairing, form intra-strand (i.e., single-strand) double helixes, as in TRNA
• Functions of RNA
  • Carrying genetic material in some viruses
  • The main job of RNA is to transfer the genetic code needed for the creation of proteins from the nucleus to the ribosome. The process prevents DNA from having to leave the nucleus. This keeps the DNA and genetic code protected from damage. Without RNA, proteins could never be made.
  • Some RNAs act as enzymes. Such RNA enzymes are called ribozymes and they exhibit many of the features of a classical enzyme.
• MRNA, RNA, and TRNA
  • RNA is central to protein synthesis.
    • First a type of RNA called messenger RNA (mRNA) carries information from DNA to structure called ribosomes.
    • These ribosomes are made from proteins and ribosomal RNA (rRNAs).
    • These all come together and form a complex that can read messenger RNAs and translate the information they carry into proteins. This requires the help of transfer RNA or TRNA.
  • RNA is formed from DNA by a process called transcription. This uses enzymes like RNA polymerase.
  • Transcriptome is the set of all messenger RNA molecules in one cell or a population of cells.
    • Because transcriptome includes all mRNA transcripts in the cell, the transcriptome reflects the genes that are being actively expressed at any given time.

Biotechnology makes it possible to move gene which is responsible for some particular feature from one organism to another.

3) PROCESS OF MANUFACTURE OF PROTIEN

• Protein synthesis starts when mRNA moves from nucleus to a ribosome on the surface of RER.
• The two sub-units of ribosomes come together and combine with MRNA. They lock onto the MRNA and start the protein synthesis.
• Ribosome builds the amino acid chain. The process is simple. First, you need an amino acid. Another nucleic acid that lives in the cell is transfer RNA. It is bonded to amino acids floating around the cell. With mRNA offering instructions, the ribosome connects to a tRNA and pulls of one amino acid. The tRNA is then released back into the cell and attached to another amino acid.
• When the protein is complete RER pinches off a vesicle. That vesicle, a small membrane bubble, can move to the cell membrane or the Golgi apparatus. Some of the protein will be used in the cell and some will be sent out into intercellular-space.

1) RNA INTERFERENCE TECHNOLOGY

• RNA Interference Technology (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules.
• It is also known as co-suppression, post-transcriptional gene silencing (PTGS), and quelling.
• Here mechanisms are developed to degrade mRNA molecules. This decreases their activity by preventing translation, via gene silencing.
• Functions/Applications
  • RNA interference is a vital part of the immune response to viruses and other foreign genetic material, especially in plants where it may prevent the self-propagation of transposons.
  • RNA interference has an important role in defending cells against parasitic nucleotide sequences – virus etc.
  • It can be useful to study the function of a gene in experimental biology in cell culture.

4) TWO CORE TECHNIQUES THAT ENABLED BIRTH OF MODERN BIOTECHNOLOGY ARE:

1) GENETIC ENGINEERING

• Technique to alter the chemistry of genetic material (DNA and RNA), to bring about desired modifications into host organisms and thus change the phenotype of the host organisms.
  • Phenotype: The set of observable characteristics of an individual resulting from the interaction of its genotype with the environment.
  • Genotype: the genetic constitution of an individual organism.
  • Jelly fish glow at night. If we want other living organism to glow at night, we can extract the gene which is responsible for this glow and put it in the new host organism.
• Advantage of genetic engineering over traditional hybridization process
  • Traditional hybridization processes used in plant and animal breeding, very often lead to inclusion and multiplication of undesirable genes along with desired genes.
  • The techniques of genetic engineering which include creation of recombinant DNA, use of Gene Cloning, and gene transfer, overcomes the above limitation and allows us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organisms.

2) MAINTENANCE OF STERILE (MICROBIAL CONTAMINATION-FREE) AMBIENCE IN CHEMICAL ENGINEERING PROCESS

• To enable growth of only the desired microbe / eukaryotic cell in large quantities for the manufacture of biotechnological products like antibiotics, vaccines, enzymes etc

5) TOOLS OF RECOMBINANT DNA TECHNOLOGY

Genetic engineering or recombinant DNA technology can be accomplished only if we have key tools, i.e., restriction enzymes, polymerase enzymes, ligases, vector and the host organisms.

1) RESTRICTION ENZYMES

• A restriction enzyme or restriction endonuclease is an enzyme that cuts DNA at a near specific recognition nucleotide sequence known as restriction sites.
  • To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.
• Restriction endonuclease are used in genetic engineering to form ‘recombinant’ molecule of DNA, which are composed of DNA from different sources/genomes.
• When cut by same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together (end-to-end) using DNA ligases.

2) CLONING VECTOR

• They are used to transfer the foreign DNA to host DNA.
• Vectors used at present are engineered in such a way that they help easy linking of foreign DNA.

3) DNA LIGASE

• It is a specific type of enzyme, a ligase that facilitates the joining of DNA together by catalyzing the formation of a phosphodiester bond.

4) HOST ORGANISMS

• The organism where the gene would be inserted.
• Techniques such as micro-injection are used. Here recombinant DNA is directly injected into nucleus of an animal cell.
• In other methods suitable for plants, the cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA in a method known as biolistic or gene gun.
• Another method is using ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the recombinant DNA into the host.

6) CRISPR-CAS09

• What is Gene Editing:
  • Gene editing is the process of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases, or “molecular scissors”. These nuclease or enzymes create site-specific double strand breaks (DSBs) at desired locations The induced double strand breaks are repaired through end joining or recombination, resulting in targeted mutation.
• What is (CRISPR/CAS9)?
  • CRISPR-CAS9 is a new genome editing tool, which is simpler, faster, cheaper, more versatile and more accurate than the previous techniques of editing DNA and has wide range of potential applications.
  • Background: The inspiration for CRISPR:
    • The inspiration of developing CRISPR CAS9 came from the CRISPR system used by several bacterias to fight against bacteriophages.
    • CRISPR (Clustered Regularly Interspaced Short Palindromic Sequence) are short DNA sequences found in the genome of Prokaryotic organisms such as bacteria, which are reminders of various bacteriophage (virus) attacks that the bacteria successfully defended against. Cas9 enzyme (part of the bacteria’s defence mechanism) uses these flags to precisely target and cut any foreign DNA, thus protecting the bacteria from future attacks by similar bacteriophages.
  • Emmanuelle Charpentier of France and Jennifer Doudna of the US won the Nobel Chemistry Prize in 2020 for developing CRISPR-Cas9. This was the first time a Nobel Science prize has gone to a women-only team.

NOTE: Prof. Charpentier, 51, and Prof. Doudna, 56, were just the sixth and seventh women to receive the Nobel Prize in Chemistry.

• How does CRISPR-CAS9 work? (Clustered Regularly interspaced short palindromic repeats)
  • https://www.youtube.com/watch?v=UKbrwPL3wXE&ab_channel=MayoClinic
  • The first task is to identify the particular sequence of genes that is cause of problem and thus have to be deleted.
  • Once this is done, an RNA molecule (called guideRNA) is programmed to locate this sequence of DNA stand, just like the ‘find’ or ‘search’ function of a computer.
  • After this, a special protein called Cas9 (CRISPR associated Protein 9), which is often described as ‘genetic scissors / molecular scissors’, is used to break the DNA strand at specific points so that bits of DNA can then be added or removed.
    • A DNA strand, when broken, has a natural tendency to re-attach and heal itself. But if the auto-repair mechanism is allowed to continue, the bad sequence can regrow. So, scientists intervene during the auto-repair process by supplying the correct sequence of genetic codes, which attaches to the broken DNA strand.
  • The entire process is programmable, and has remarkable efficiency, though chances of error are not entirely ruled out.

• Applications of CRISPR-CAS9
  • The technology has had a revolutionary impact on life science.
  • Its applications include:
    • Curing diseases genetic in nature – i.e., the diseases are caused by unwanted changes or mutations in genes. These include common blood disorders like sickle cell Anaemia, eye diseases including color blindness etc.
    • Deformities arising out of abnormalities in gene sequences – like stunted or slow growth, speech disorders, or inability to stand or walk can also be treated by CRISPR.
    • Developing GM crops and animals.
      • For e.g., Japan has already approved the commercial cultivation of a tomato variety that has been improved using CRISPR-based intervention.
      • In India, several research groups are working on CRISPR-based enhancements for various crops including rice and banana.
• Limitation
  • Potential of misuse: (bioterrorism; designer babies)
  • Collateral Damage (Knock-on Effect):
  • Ethics of CRISPR – Should humans be allowed to modify how the nature works?

7) CRISPER TECHNOLOGY INSERTED GENES THAT ALLOW IMMUNE CELLS TO ATTACK CANCER CELLS (NOV 2022: SOURCE – THE HINDU)

• In the past, CRISPR gene editing tool has been used in humans to remove specific genes to allow the immune system to be more activated against cancer.
• For the first time, scientists have used CRISPR to insert gene that allow immune cells to attack cancer cells, potentially leaving normal cells unharmed and increasing the effectiveness of immunotherapy.
  • The research, published in the journal Nature, used CRISPR to not only take out specific genes, but also to insert new ones in immune cells efficiently redirecting them to recognize mutations in the patient’s own cancer cells.
• The human immune system has specific receptors on immune cells that can specifically recognize cancer cells and differentiate them from normal cells. These are different for every patient, so finding an efficient way to isolate them and insert them back into immune cells to generate a personalized cell therapy to treat cancer is key to making the approach feasible on a large scale.
• Significance
  • This is a leap forward in developing a personalized treatment for cancer, where the isolation of immune receptors that specifically recognize mutations in the patient’s own cancer are used to treat the cancer.
  • The generation of personalized cell treatment for cancer would not have been feasible without the newly developed ability to use the CRISPR technique to replace the immune receptors in clinical-grade cell preparation in single step.
• The researchers reported treating of 16 patients with a variety of solid cancers including colon, breast and lung cancers.

8) HOW GENE THERAPY USING CRISPR CAN CURE CANCER (DEC 2022: SOURCE THE HINDU)

• Why in news?
  • Scientists in the United Kingdom testing a new form of cancer therapy, reported success of teenage girl, Alyssia, with a form of cancer called T-cell acute lymphoblastic leukaemia (Dec 2022)
• What is T-cell acute lymphoblastic leukaemia (T-ALL)
  • It is a type of cancer where the T-cells, which are a class of white blood cells, equipped to hunt and neutralize threats to the body, turn against the body and end up destroying healthy cells that normally help with immunity. The disease is rapid and progressive and is usually treated by chemotherapy and radiation therapy.
• How gene therapy treated this?
  • Alyssia, a teenage girl, had tried several of the standard treatments including chemotherapy and radiation. But the treatment wasn’t successful.
  • Then she enrolled in an experimental trial conducted by doctors and scientists at the University College, London and Great Ormond Street Hospital. She was the first patient to receive experimental gene therapy that relied on a new technique called ‘base-editing’.
  • What is base editing?
    • When a misarrangement in the sequence of nitrogen bases (ATCG) is edited to arrange it properly, it is called base editing. David Liu, of the Broad Institute, Massachusetts has improvised on the CRISPR-cas9 to be able to directly change certain bases: thus, a C can be changed into G and T into an A. While still a nascent technology, base editing is reportedly more effective at treating blood disorders which were caused by so-called single point mutations, or when a change in a single base pair can cause terminal disease.
  • Alyssia’s case:
    • In Alyssia’s case, her T-cells – perhaps because of a misarrangement in the sequence of bases – had become cancerous. The objective of the gene therapy in thecaseofT-cellleukemia wastofix her immune system ina way that it stops making cancerous T-cells.
    • First, healthy T-cells were extracted from a donor and put through a series of edits.
    • The first base edit blocked the T-cells targeting mechanism so it would ceaseattacking Alyssa’s body.
    • The second removed a chemical marking, called CD7, which is on all T-cells.
    • Third prevented the T-cells from being killed by a chemotherapy drug.
    • Finally, the T-cells were programmed to destroy all cells – cancerous or protective – with CD7 marked on it.
    • After spending a month in remission,she was given a second donor transplant to regrow her immune system that would contain healthy T-cells.
    • How effective was the treatment?
      • Her cancer doesn’t seem to have re-surfaced.
    • More verification needed:
      • It has been 1.5 years since she was first diagnosed with the disease and whether the treatment has reliably and entirely fixed her immune system, remains to be established.

A) UNDERSTANDING T-CELLS IN MORE DETAILS

• T cells are a type of white blood cells. They are part of immune system and develop from hematopoietic stem cells (blood stem cells) present in bone marrow. They help protect body from infection and may help fight cancer. They are also called T Lymphocyte and thymocyte.
• After getting born from blood stem cells, they migrate to thymus gland to develop. T-cells derive their name from the thymus. In thymus, the precursor cells mature into several distinct type of T cells. This differentiation continues after they have left the thymus.
  • One of the important functions of T-cells is immune mediated cell death – it is carried out by two major subtypes – CD8+” Killer” and CD4+” helper” T cells. These are named for the presence of the cell surface proteins CD8 and CD4.
• T cells, also known as “Killer T-cells”, are cytotoxic – this means that they are able to directly kill virus-infected cells, as well as cancer cells.
• T-cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface.

9) SOMATIC CELL NUCLEAR TRANSFER

• In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory technique for creating an ovum with a donor nucleus.
  • In SCNT the nucleus, which contains the organism’s DNA, of a somatic cell (a body cell other than a sperm or egg cell) is removed and the rest of the cell discarded.
  • At the same time, the nucleus of an egg cell is removed.
  • The nucleus of the somatic cell is then inserted into the unnucleated egg cell.
  • After being inserted into the egg, the somatic cell nucleus is reprogrammed by the host cell.
  • The egg, now containing the nucleus of a somatic cell, is stimulated with a shock and will begin to divide.
  • After many mitotic divisions in culture, this single cell forms a blastocyst (an early stage embryo with about 100 cells) with almost identical DNA to the original organism

It can be used in embryonic stem cell research, or in regenerative medicine where it is sometimes referred to as “therapeutic cloning.” It can also be used as the first step in the process of reproductive cloning.