UNIT II. Techniques in Biotechnology PDF

**UNIT II. TECHNIQUES IN BIOTECHNOLOGY** There is a number of techniques used to genetically modify living organisms, including foods, as reported in the literature. The following sections discuss some of the techniques developed to modify living organisms, which are widely used today. ![](media/i...

**UNIT II. TECHNIQUES IN BIOTECHNOLOGY** There is a number of techniques used to genetically modify living organisms, including foods, as reported in the literature. The following sections discuss some of the techniques developed to modify living organisms, which are widely used today.  1. differentiate among the various techniques in biotechnology 2. outline the steps/procedure of the various techniques in biotechnology 3. perform some techniques in biotechnology such as tissue culture, DNA extraction, backcross breeding 4. discuss how women are affected by agricultural biotechnology  1. Tissue culture - Aguilar 2. DNA extraction - Amos 3. DNA cloning - Besana 4. Gene coding -- Caguan 5. Gene gun and alternative techniques - DeogrACIAS 6. Backcross breeding - Neuda 7. Cloning -- Delos Reyes 8. DNA sequencing - Sarsona **TISSUE CULTURE** Plants usually reproduce through sexual means -- they have flowers and seeds to create the next generation. Egg cells in the flowers are fertilized by pollen from the stamens (male part) of the flower of the same plant (self-pollination) or another plant (cross). Each of these sexual cells contains genetic material in the form of DNA. During sexual reproduction, DNA from both parents is combined creating offspring similar to the parents (in self-pollinated crops), or in new and unpredictable ways, creating unique organisms (in cross-pollinated crops). Some plants and trees on the other hand need several years before they flower and set seeds, making plant improvement difficult. Plant scientists have developed the science and art of tissue culture to assist breeders in this task. 6\. Plant tissue culture is a technique that has been around for more than 30 years. There are several types of tissue culture depending on the part of the plant (explant) used. Schematic presentation of the major areas of plant cell and tissue cultures, and some fields of application **The various branches of cell and tissue culture** ***Callus Culture*** In this approach, isolated pieces of a selected tissue, so-called explants (only some mg in weight), are obtained aseptically from a plant organ and cultured on, or in a suitable nutrient medium. For a primary callus culture, most convenient are tissues with high contents of parenchyma or meristematic cells. In such explants, mostly only a limited number of cell types occur, and so a higher histological homogeneity exists than in the entire organ. However, growth induced after transfer of the explants to the nutrient medium usually results in an unorganized mass or clump of cells---a callus---consisting largely of cells different from those in the original explant. *Cell suspensions* Whereas in a callus culture there remain connections among adjacent cells via plasmodesmata, ideally in a cell suspension all cells are isolated. Under practical conditions, however, also in these cell populations there is usually a high percentage of cells occurring as multicellular aggregates. A supplement of enzymes is able to break down the middle lamella connecting the cells in such clumps, or a mechanical maceration will yield single cells. Often, mechanical shearing of callus material in a stirred liquid medium produces cell suspensions. These cell suspensions generally consist of a great variety of cell types, and are less homogenous than callus cultures. A **cell suspension** or **suspension culture** is a type of [cell culture](https://en.wikipedia.org/wiki/Cell_culture) in which single [cells](https://en.wikipedia.org/wiki/Cell_(biology)) or small aggregates of cells are allowed to [function](https://en.wikipedia.org/wiki/Cell_physiology) and [multiply](https://en.wikipedia.org/wiki/Cell_division) in an agitated [growth medium](https://en.wikipedia.org/wiki/Growth_medium), thus forming a [suspension](https://en.wikipedia.org/wiki/Suspension_(chemistry)).  *Protoplast cultures* In this approach, initially the cell wall of isolated cells is enzymatically removed, i.e., "naked" cells are obtained, and the explant is transformed into a single-cell culture. To prevent cell lysis, this has to be done under hypertonic conditions. This method has been used to study processes related to the regeneration of the cell wall, and to better understand its structure. Also, protoplast cultures have served for investigations on nutrient transport through the plasma lemma, but without the confounding influence of the cell wall. The main aim in using this approach in the past, however, has been interspecies hybridizations, not possible by sexual crossing. Nowadays, protoplasts are still essential in many protocols of gene technology. From such protoplast cultures, ideally plants can be regenerated through somatic embryogenesis to be used in breeding programs. *Anther or microspore cultures* Culturing anthers, or isolated microspores from anthers under suitable conditions, haploid plants can be obtained through somatic embryogenesis. Treating such plant material with, e.g., colchicines, it is possible to produce dihaploids, and if everything works out, within 1 year (this depends on the plant species) a fertile homozygous dihaploid plant can be produced from a heterozygous mother plant. This method is advantageous for hybrid breeding, by substantially reducing the time required to establish inbred lines. Often, however, initially a callus is produced from microspores, with separate formation of roots and shoots that subsequently join, and in due time haploid plants can be isolated. Here, the production of "ploidy chimeras" may be a problem. Another aim in using anther or microspore cultures is to provoke the expression of recessive genes in haploids to be selected for plant breeding or gene transfer purposes. **Micropopagation** is a tissue culture method developed for the production of disease-free, high quality planting material and for rapid production of many uniform plants. Actively-dividing young cells (meristem) are placed in a special medium and treated with plant hormones to produce many similar sister plantlets. Since the meristem divides faster than disease-causing virus, clean materials are propagated and hundreds of uniform plantlets are produced in a short time. Through micropropagation, it is now possible to provide clean and uniform planting materials in plantations -- oil palm, plantain, pine, banana, abaca, date, rubber tree; field crops -- eggplant, jojoba, pineapple, tomato; root crops -- cassava, yam, sweet potato; and many ornamental plants such as orchids and anthuriums. Micropropagated plants were found to establish more quickly, grow more vigorously and taller, have a shorter and more uniform production cycle, and produce higher yields than conventional propagules. A. **DNA Extraction** ***What is DNA Extraction?***\ \ Simply put, DNA Extraction is the removal of deoxyribonucleic acid (DNA) from the cells or viruses in which it normally resides. ***What is it used for?***\ \ Extraction of DNA is often an early step in many diagnostic processes used to detect bacteria and viruses in the environment as well as diagnosing disease and genetic disorders. These techniques include but are not limited to - - - - ***How does it work?***\ \ Outline of a basic DNA Extraction - 1. 2. 3. 4. 5. 6. ***Instrumentation used in DNA Extraction***  This ***bead beater*** is used in the breaking apart or \"lysing\" of cells in the early steps of extraction in order to make the DNA accessible. Glass beads are added to an eppendorph tube containing a sample of interest and the bead beater vigorously vibrates the solution causing the glass beads to physically break apart the cells. Other methods used for lysing cells include a french press and a sonication device. A ***centrifuge*** such as this can spin at up to 15,000 rpm to facilitate separation of the different phases of the extraction. It is also used to precipitate the DNA after the salts are washed away with ethanol and or isopropanol.  A ***gel box*** is used to separate DNA in an agarose gel with an electrical charge. When the red and black leads are plugged into a power supply the DNA migrates through the gel toward the positive charge due to the net negative charge of the molecule. Different sized pieces of DNA move at different rates, with the larger pieces moving more slowly through the porus medium, thereby creating a size separation that can be differentiated in a gel. B. **DNA Cloning** DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that "cut and paste" DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources. Diagram showing the construction of a recombinant DNA molecule. A circular piece of plasmid DNA has overhangs on its ends that match those of a gene fragment. The plasmid and gene fragment are joined together to produce a gene-containing plasmid. This gene-containing plasmid is an example of recombinant DNA, or a DNA molecule assembled from DNA from multiple sources. Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains. What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need many DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as "factories" to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics. Deoxyribonucleic acid (abbreviated DNA) is **the molecule that carries genetic information** for the development and functioning of an organism. [Steps of DNA cloning] DNA cloning is used for many purposes. As an example, let\'s see how DNA cloning can be used to synthesize a protein (such as human insulin) in bacteria. The basic steps are: - Cut open the plasmid and \"paste\" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA). - Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid. - Grow up lots of plasmid-carrying bacteria and use them as \"factories\" to make the protein. Harvest the protein from the bacteria and purify it. Let us take a closer look at each step. 1. **Cutting and pasting DNA** How can pieces of DNA from different sources be joined together? A common method uses two types of enzymes: restriction enzymes and DNA ligase. A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they will not combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.  Our goal in cloning is to insert a target gene (e.g., for human insulin) into a plasmid. Using a carefully chosen restriction enzyme, we digest: - The plasmid, which has a single cut site - The target gene fragment, which has a cut site near each end Then, we combine the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene. 2. **Bacterial transformation and selection** 3. **Protein production**  [Uses of DNA cloning] - *Biopharmaceuticals* - *Gene therapy* - *Gene analysis* C. **Gene Coding** - At the 5' end of the chain, the phosphate group of the first nucleotide in the chain sticks out. The phosphate group is attached to the 5\' carbon of the sugar ring, which is why this is called the 5\' end. - At the other end, called the 3' end, the hydroxyl of the last nucleotide added to the chain is exposed. The hydroxyl group is attached to the 3\' carbon of the sugar ring, which is why this is called the 3\' end. - Many processes, such as DNA replication and transcription, can only take place in one particular direction relative the directionality of a DNA or RNA strand. - Polypeptides (chains of linked amino acids) have two distinct ends: - An N-terminus with an amino group exposed - A C-terminus with a carboxyl group exposed - During translation, polypeptide is built from N- to C-terminus. You can learn more about N- and C-termini in the article on proteins and amino acids. mRNA sequence: 5\'-UCAUGAUCUCGUAAGA-3\' Read in Frame 1: 5\'-UCA UGA UCU CGU AAG A-3\' Ser-STOP-Ser-Arg-Lys Read in Frame 2: 5\'-U CAU GAU CUC GUA AGA-3\' His-Asp-Leu-Val-Arg Read in Frame 3: 5\'-UC AUG AUC UCG UAA GA-3\' Met(Start)-Ile-Ser-STOP The start codon\'s position ensures that Frame 3 is chosen for translation of the mRNA. D. **Gene Gun and Alternative Techniques** E. **Backcross Breeding (Upgrading)** F. **Cloning** Cloning, the process of generating a genetically identical copy of a cell or an organism. Cloning happens often in nature---for example, when a cell replicates itself asexually without any genetic alteration or recombination. Prokaryotic organisms (organisms lacking a cell nucleus) such as bacteria create genetically identical duplicates of themselves using binary fission or budding. In eukaryotic organisms (organisms possessing a cell nucleus) such as humans, all the cells that undergo mitosis, such as skin cells and cells lining the gastrointestinal tract, are clones; the only exceptions are gametes (eggs and sperm), which undergo meiosis and genetic recombination.  In biomedical research, cloning is broadly defined to mean the duplication of any kind of biological material for scientific study, such as a piece of DNA or an individual cell. For example, segments of DNA are replicated exponentially by a process known as polymerase chain reaction, or PCR, a technique that is used widely in basic biological research. The type of cloning that is the focus of much ethical controversy involves the generation of cloned embryos, particularly those of humans, which are genetically identical to the organisms from which they are derived, and the subsequent use of these embryos for research, therapeutic, or reproductive purposes. [Early Cloning Experiments] Reproductive cloning was originally carried out by artificial "twinning," or embryo splitting, which was first performed on a salamander embryo in the early 1900s by German embryologist Hans Spemann. Later, Spemann, who was awarded the Nobel Prize for Physiology or Medicine (1935) for his research on embryonic development, theorized about another cloning procedure known as *nuclear transfer*. This procedure was performed in 1952 by American scientists [Robert W. Briggs] and [Thomas J. King], who used DNA from embryonic cells of the frog Rana pipiens to generate cloned tadpoles. In 1958 British biologist John Bertrand Gurdon successfully carried out nuclear transfer using DNA from adult intestinal cells of African clawed frogs (Xenopus laevis). Gurdon was awarded a share of the 2012 Nobel Prize in Physiology or Medicine for this breakthrough. Advancements in the field of molecular biology led to the development of techniques that allowed scientists to manipulate cells and to detect chemical markers that signal changes within cells. With the advent of recombinant DNA technology in the 1970s, it became possible for scientists to create transgenic clones---clones with genomes containing pieces of DNA from other organisms. Beginning in the 1980s mammals such as sheep were cloned from early and partially differentiated embryonic cells. In 1996 British developmental biologist [Ian Wilmut] generated a cloned sheep, named Dolly, by means of nuclear transfer involving an enucleated embryo and a differentiated cell nucleus. This technique, which was later refined and became known as somatic cell nuclear transfer (SCNT), represented an extraordinary advance in the science of cloning, because it resulted in the creation of a genetically identical clone of an already grown sheep. It also indicated that it was possible for the DNA in differentiated somatic (body) cells to revert to an undifferentiated embryonic stage, thereby reestablishing pluripotency---the potential of an embryonic cell to grow into any one of the numerous different types of mature body cells that make up a complete organism. The realization that the DNA of somatic cells could be reprogrammed to a pluripotent state significantly impacted research into therapeutic cloning and the development of stem cell therapies. Soon after the generation of Dolly, a number of other animals were cloned by SCNT, including pigs, goats, rats, mice, dogs, horses, and mules. Despite those successes, the birth of a viable SCNT primate clone would not come to fruition until 2018, and scientists used other cloning processes in the meantime. In 2001 a team of scientists cloned a rhesus monkey through a process called embryonic cell nuclear transfer, which is similar to SCNT except that it uses DNA from an undifferentiated embryo. In 2007 macaque monkey embryos were cloned by SCNT, but those clones lived only to the blastocyst stage of embryonic development. It was more than 10 years later, after improvements to SCNT had been made, that scientists announced the live birth of two clones of the crab-eating macaque (Macaca fascicularis), the first primate clones using the SCNT process. (SCNT has been carried out with very limited success in humans, in part because of problems with human egg cells resulting from the mother's age and environmental factors.) **Reproductive Cloning** Reproductive cloning involves the implantation of a cloned embryo into a real or an artificial uterus. The embryo develops into a fetus that is then carried to term. Reproductive cloning experiments were performed for more than 40 years through the process of embryo splitting, in which a single early-stage two-cell embryo is manually divided into two individual cells and then grows as two identical embryos. Reproductive cloning techniques underwent significant change in the 1990s, following the birth of Dolly, who was generated through the process of SCNT. This process entails the removal of the entire nucleus from a somatic (body) cell of an organism, followed by insertion of the nucleus into an egg cell that has had its own nucleus removed (enucleation). Once the somatic nucleus is inside the egg, the egg is stimulated with a mild electrical current and begins dividing. Thus, a cloned embryo, essentially an embryo of an identical twin of the original organism, is created. The SCNT process has undergone significant refinement since the 1990s, and procedures have been developed to prevent damage to eggs during nuclear extraction and somatic cell nuclear insertion. For example, the use of polarized light to visualize an egg cell's nucleus facilitates the extraction of the nucleus from the egg, resulting in a healthy, viable egg and thereby increasing the success rate of SCNT. Reproductive cloning using SCNT is considered very harmful since the fetuses of embryos cloned through SCNT rarely survive gestation and usually are born with birth defects. Wilmut's team of scientists needed 277 tries to create Dolly. Likewise, attempts to produce a macaque monkey clone in 2007 involved 100 cloned embryos, implanted into 50 female macaque monkeys, none of which gave rise to a viable pregnancy. In January 2008, scientists at Stemagen, a stem cell research and development company in California, announced that they had cloned five human embryos by means of SCNT and that the embryos had matured to the stage at which they could have been implanted in a womb. However, the scientists destroyed the embryos after five days, in the interest of performing molecular analyses on them. **Therapeutic Cloning** Therapeutic cloning is intended to use cloned embryos for the purpose of extracting stem cells from them, without ever implanting the embryos in a womb. Therapeutic cloning enables the cultivation of stem cells that are genetically identical to a patient. The stem cells could be stimulated to differentiate into any of the more than 200 cell types in the human body. The differentiated cells then could be transplanted into the patient to replace diseased or damaged cells without the risk of rejection by the immune system. These cells could be used to treat a variety of conditions, including Alzheimer disease, Parkinson disease, diabetes mellitus, stroke, and spinal cord injury. In addition, stem cells could be used for in vitro (laboratory) studies of normal and abnormal embryo development or for testing drugs to see if they are toxic or cause birth defects. Although stem cells have been derived from the cloned embryos of animals such as mice, the generation of stem cells from cloned primate embryos has proved exceptionally difficult. For example, in 2007 stem cells successfully derived from cloned macaque embryos were able to differentiate into mature heart cells and brain neurons. However, the experiment started with 304 egg cells and resulted in the development of only two lines of stem cells, one of which had an abnormal Y chromosome. Likewise, the production of stem cells from human embryos has been fraught with the challenge of maintaining embryo viability. In 2001 scientists at Advanced Cell Technology, a research company in Massachusetts, successfully transferred DNA from human cumulus cells, which are cells that cling to and nourish human eggs, into eight enucleated eggs. Of these eight eggs, three developed into early-stage embryos (containing four to six cells); however, the embryos survived only long enough to divide once or twice. In 2004 South Korean researcher Hwang Woo Suk claimed to have cloned human embryos using SCNT and to have extracted stem cells from the embryos. However, this later proved to be a fraud; Hwang had fabricated evidence and had actually carried out the process of parthenogenesis, in which an unfertilized egg begins to divide with only half a genome. The following year a team of researchers from the University of Newcastle upon Tyne was able to grow a cloned human embryo to the 100-cell blastocyst stage using DNA from embryonic stem cells, though they did not generate a line of stem cells from the blastocyst. Scientists have since successfully derived embryonic stem cells from SCNT human embryos. Progress in research on therapeutic cloning in humans has been slow relative to the advances made in reproductive cloning in animals. This is primarily because of the technical challenges and ethical controversy arising from the procuring of human eggs solely for research purposes. In addition, the development of induced pluripotent stem cells, which are derived from somatic cells that have been reprogrammed to an embryonic state through the introduction of specific genetic factors into the cell nuclei, has challenged the use of cloning methods and of human eggs. **Ethical Controversy** Human reproductive cloning remains universally condemned, primarily for the psychological, social, and physiological risks associated with cloning. A cloned embryo intended for implantation into a womb requires thorough molecular testing to fully determine whether an embryo is healthy and whether the cloning process is complete. In addition, as demonstrated by 100 failed attempts to generate a cloned macaque in 2007, a viable pregnancy is not guaranteed. Because the risks associated with reproductive cloning in humans introduce a very high likelihood of loss of life, the process is considered unethical. There are other philosophical issues that also have been raised concerning the nature of reproduction and human identity that reproductive cloning might violate. Concerns about eugenics, the once popular notion that the human species could be improved through the selection of individuals possessing desired traits, also have surfaced, since cloning could be used to breed "better" humans, thus violating principles of human dignity, freedom, and equality. There also exists controversy over the ethics of therapeutic and research cloning. Some individuals and groups have an objection to therapeutic cloning, because it is considered the manufacture and destruction of a human life, even though that life has not developed past the embryonic stage. Those who are opposed to therapeutic cloning believe that the technique supports and encourages acceptance of the idea that human life can be created and expended for any purpose. However, those who support therapeutic cloning believe that there is a moral imperative to heal the sick and to seek greater scientific knowledge. Many of these supporters believe that therapeutic and research cloning should be not only allowed but also publicly funded, similar to other types of disease and therapeutics research. Most supporters also argue that the embryo demands special moral consideration, requiring regulation and oversight by funding agencies. In addition, it is important to many philosophers and policy makers that women and couples not be exploited for the purpose of obtaining their embryos or eggs. There are laws and international conventions that attempt to uphold certain ethical principles and regulations concerning cloning. In 2005 the United Nations passed a nonbinding Declaration on Human Cloning that calls upon member states "to adopt all measures necessary to prohibit all forms of human cloning inasmuch as they are incompatible with human dignity and the protection of human life." This does provide leeway for member countries to pursue therapeutic cloning. The United Kingdom, through its Human Fertilization and Embryology Authority, issues licenses for creating human embryonic stem cells through nuclear transfer. These licenses ensure that human embryos are cloned for legitimate therapeutic and research purposes aimed at obtaining scientific knowledge about disease and human development. The licenses require the destruction of embryos by the 14th day of development, since this is when embryos begin to develop the primitive streak, the first indicator of an organism's nervous system. The United States federal government has not passed any laws regarding human cloning due to disagreement within the legislative branch about whether to ban all cloning or to ban only reproductive cloning. The Dickey-Wicker amendment, attached to U.S. appropriations bills since 1995, has prevented the use of federal dollars to fund the harm or destruction of human embryos for research. It is presumed that nuclear transfer and any other form of cloning is subject to this restriction. G. **DNA Sequencing** The ability to determine the sequence of bases in DNA is a central part of modern molecular biology and provides what might be considered the ultimate structural information. Rapid methods for sequence analysis were developed in the late 1970s, and the technique is now used in laboratories worldwide. In recent years the basic techniques have been revolutionized by automation, which has improved the efficiency of sequencing to the point where genome sequencing is possible. **What is sequencing?** You may have heard of genomes being sequenced. For instance, the human genome was completed in 2003, after a many-year, international effort. But what does it mean to sequence a genome, or even a small fragment of DNA? **DNA sequencing** is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today, with the right equipment and materials, sequencing a short piece of DNA is relatively straightforward. Sequencing an entire genome (all of an organism's DNA) remains a complex task. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences into a single long \"consensus.\" However, thanks to new methods that have been developed over the past two decades, genome sequencing is now much faster and less expensive than it was during the Human Genome Project. **Women in Biotechnology** [Some of the leading women in biotechnology] **June Almeida** (1930 - 2007) Born: Glasgow. Almeida has recently attracted a great deal of media attention in the midst of the COVID-19 crisis as the person responsible for the first visualization of a human coronavirus. Yet, this barely scratches the surface of her achievements. Leaving school at the age of 16, Almeida managed to forge a major career based on her outstanding skills in electron microscopy. This she did in the midst of bearing a daughter and raising her as a single parent following divorce. Almeida\'s pioneering advances in immune electron microscopy put her at the forefront of many key breakthroughs in virology in the 1960s and 1970s.  **Brigitte Askonas** (1923 - 2013) Born: Vienna, Austria. Askonas co-developed one of the first systems for the cloning of antibody-forming B cells in vivo, some of the earliest monoclonal antibodies. She was also one of the first scientists to isolate and clone virus specific T lymphocytes, laying the foundation for defining different influenza sub-sets and improving vaccines. (Photo credit: Anne-Katrin Purkiss, Wellcome Images B0007461). **Sally Davies** (1949) Born: Birmingham, United Kingdom. Sally Davies was named the sixth most powerful woman in the UK by Woman's Hour, a BBC radio programme, in 2013. She is the first woman to hold the post of Chief Medical Officer for England. In 2006 she set up the National Institute for Health Research, a body that has revolutionized the approach to clinical and applied research in the UK. She is also at the forefront of spearheading efforts to combat antimicrobial resistance around the world. All this she has achieved in the midst of dealing with a likely variant of dyslexia, being widowed young and becoming a mother in her forties. Much of her career has been shaped by serendipity and her strong desire to make the world a better place.  **Margaret Dayhoff** (1925 - 1983) Born: Philadelphia, Pennsylvania, United States. Dayhoff is known as the founder of bioinformatics. This she did by pioneering the application of mathematics and computational techniques to the sequencing of proteins and nucleic acids and establishing the first publicly available database for research in the area. (Photo credit: Ruth E Dayhoff, National Library of Medicine). **Jennifer Doudna** (1964) Born: Washington DC, United States. Doudna first made her name uncovering the basic structure and function of the first ribozyme, a type of catalytic ribonucleic acid (RNA) that helps catalyse chemical reactions. This work helped lay the foundation for her later helping to pioneer CRISPR-Cas 9, a tool that has provided the means to edit genes on an unprecedented scale and at minimal cost. In addition to her scientific contributions to CRISPR, Doudna is known for spearheading the public debate to consider the ethical implications of using CRISPR-Cas9 to edit human embryos. **Rosalind Franklin** (1920 - 1958) Born: London, United Kingdom. Rosalind Franklin was an x-ray crystallographer whose work helped uncover the double-helix structure of DNA. (Photo credit: Vittorio Luzzati). **Carolyn Green** (1965 - 2017) Born: Pittsburgh, PA, USA. Carolyn E Green was a serial entrepreneur in the life sciences community and a leading voice for women in the sector. In her short life she managed to found and head up numerous companies and held many leadership and sales positions in the biopharmaceutical industry. Her crowning achievement was to be hired by Pfizer to head up its new strategic research and development investments initiative. This entailed working with the corporation's venture fund to establish partnerships with early-stage companies. Green's enduring patience and strong mentoring skills, together with her passion to develop products to help patients, set her apart from many others in the biotechnology world. **Beverly Griffin** (1930 - 2016) Born: Delhi, Louisiana. Griffin earned two doctorates in chemistry in an era when it was rare for women to pursue a scientific career. She is best known for her pioneering work on the molecular biology of two viruses that cause cancer - the polyomavirus and Epstein Barr Virus (EBV). From the 1980s she was devoted to understanding how in one setting EBV could cause glandular fever, a largely harmless disease, and yet in another Burkitt\'s Lymphoma, a major killer of children in Central Africa. She also spearheaded efforts to improve the diagnosis and treatment of the cancer and was a tireless campaigner for raising awareness of the plight of children with the disease in Africa. (Photo credit: Tomas Lindahl). **Esther Lederberg** (1922 - 2006) Born: Bronx, New York, United States. Esther Lederberg was a major pioneer of bacterial genetics. She discovered the lambda phage, a bacterial virus which is widely used as a tool to study gene regulation and genetic recombination. She also invented the replica plating technique, which is used to isolate and analyse bacterial mutants and track antibiotic resistance. (Photo credit: The Esther Lederberg Memorial Trust). **Rita Levi-Montalcini** (1909 - 2012) Born: Turin, Italy. An Italian scientist, Rita Levi-Montalcini helped discover the chemical tools the body uses to direct cell growth and build nerves. This knowledge underpins current investigation into how these processes go wrong in diseases like dementia and cancer. (Photo credit: Bernard Becker Medical Library). **Janet Mertz** (1949) Born: The Bronx, New York, USA. Mertz was pivotal to the discovery of the first enzyme for easily joining together DNA from different species and designing the protocol that underpinned the development of the first recombinant DNA cloned in bacteria. Her work not only helped lay the foundation for the development of genetic engineering, but also spurred on the establishment of the first safety guidelines for laboratories involved in genetic manipulation. She has also made key contributions to our understanding about how the human tumour viruses SV40, hepatitis B virus, and Epstein-Barr virus regulate expression of their genes and identified roles oestrogen-related receptors play in breast cancer and responses to therapies. (Photo credit: Janet Mertz).  **Christiane Nusslein-Volhard** (1942) Born: Magdeburg, Germany. Christiane Nüsslein-Volhard won the Nobel Prize in 1995, the sixth woman to do so. She was awarded the Prize on the basis of her groundbreaking research that showed how genes regulate the early development of fruit fly embryos. Her discoveries helped create the new discipline of developmental genetics and laid the foundation for understanding genetic defects in human embryos. **Padmanee Sharma** (1970) Born: Georgetown, Guyana. Padmanee Sharma is a leading figure in oncology, specialising in renal, bladder and prostate cancer. Her prime focus is to understand the mechanisms and pathways within the immune system responsible for tumour rejection. Since 2005 she has been a principal investigator for several clinical trials launched to improve the efficacy of cancer immunotherapies. Driven by the desperation of her cancer patients, much of Sharma's high-flying career is down to the strong determination she developed to overcome the poverty and hardships she faced as the child of Indo-Guyanese immigrants who settled in New York when she was 10 years old. **Rosemary Versteegen** (1948) Born: Glasgow, Scotland. Rosemary J Versteegen worked for over twenty years with Life Technologies Inc, which in the 1990s was one of largest suppliers of culture cell products and other scientific reagents to the biotechnology industry. She was pivotal to the company's success in winning FDA approval for the first diagnostic test using synthetic nucleic acid probes for detecting infection with the human papillomavirus, one of the most common causes of cervical cancer. In addition, Versteegen is one of the co-founders and the Chief Executive Officer of the International Serum Industry Association, an organisation that works to promote standards of excellence and ethics in the animal serum and animal derived products industry. **Francoise Barre-Sinoussi** (1947) Born: Paris, France. Barre-Sinoussi shared the 2008 Nobel Prize in Physiology or Medicine for helping to identify the human immunodeficiency virus (HIV) as the cause of AIDS in 1983. Over the years she has made substantial contributions to understanding the role of innate immune defences in the host in controlling HIV/AIDS and how HIV is transmitted between the mother and child. She has also studied the characteristics that allow some HIV-positive individuals gain resistance to HIV without antiretrioviral drugs. (Photo credit: Karolinska Institute, Press conference, 2008). **Elizabeth Blackburn** (1948) Born: Hobart, Tasmania, Australia. Blackburn is a molecular biologist who was awarded the Nobel Prize in Physiology or Medicine in 2009. She is best known for having discovered a particular repetative sequence of DNA on the telomere, a particular region found at the end of a chromosome that prevents the chromosome ends from fraying and sticking to each other. She also helped identify telomerase, an enzyme that helps replenish telomeres which get shorter every time a cell divides. Such shortening is associated with aging and cancer. (Photo credit: Chemical Heritage Foundation). **Gertrude Elion** (1918 - 1999) Born: New York City, United States. Elion shared the 1988 Nobel Prize in Physiology or Medicine for her contributions to the development of a multitude of new drugs. This included drugs for herpes, leukemia, malaria, gout, immune disorders, and AIDS, and immune suppressants to overcome rejection of donated organs in transplant surgery. Her work earned 45 patents. (Photo credit: Wellcome Images). **Carol Greider** (1961) Born: San Diego, California, United States. Greider shared the Nobel Prize for Physiology or Medicine in 2009 for helping to elucidate the structure of telomeres, a particular region found at the end of a chromosome that prevents the chromosome ends from fraying and sticking to each other, and to identify telomerase, an enzyme that helps replenish telomeres which get shorter every time a cell divides. Such shortening is associated with aging and cancer. She also collaborated in the development of the first telomerase knockout mouse which helped demonstrate how premature aging is linked to increasingly short telomeres. (Photo credit: Keith Weller). **Ingeborg Hochmair-Desoyer** (1953) Born: Vienna, Austria. Hochmair-Desoyer is an electrical engineer who helped create the world\'s first micro-electric multi-channel cochlear implant. Developed in 1977 the implant enables the user to not only hear sounds but also to understand speech. Since 2000 she has co-founded a number of medical device companies working to help with hearing loss. In 2013 she was awarded the Lasker-DeBakey Clinical Medical Research Award. (Photo credit: Ingeborg J Hochmair-Desoyer). **Dorothy Hodgkin** (1910 - 1994) Born: Cairo, Egypt. Dorothy Hodgkin, was a British biochemist who developed protein crystallography and X-ray crystallography which was used to confirm the structure of penicillin, for which she won the Nobel Prize in Chemistry in 1964. (Photo credit: Peter Lofts Photography, National Portrait Gallery, London Peter Lofts Photography, National Portrait Gallery, London ). **Mary-Claire King** (1946) Born: Illinois, United States. King is a human geneticist who studies the interplay between genetics and the environment on human disease. She is best known for having identified BRCA1, a single gene responsible for many breast and ovarian cancers. Her technique for identifying the BRCA1 gene is now used for studying many other diseases. She was also responsible for the development of a technique, using mitrochondial DNA and human leukocyte antigen, for genetically identifying the remains of missing people. (Photo credit: Mary-Claire King).  **Barbara McClintock** (1902 - 1992) Born: Hartford, Connecticut, United States. Through her work on maize, McClintock demonstrated the ability of genes to change position on the chromosome. (Photo credit: American Philosophical Society). **Sherie Morrison** Born: United States. A key pioneer in the development of antibody engineering techniques, Morrison helped develop some of the first chimeric monoclonal antibodies. This work paved the way to the creation of safer and more effective monoclonal antibody drugs. (Photo credit: Sherie Morrison). **May-Britt Moser** (1963) Born: Fosnavag, Norway. Moser shared the 2014 Nobel Prize in Physiology or Medicine for helping to discover cells located in the centre of the brain that are important for determining spacial position. Her work has helped scientists gain new understanding into the cognitive processes and spacial deficits linked to neurological conditions like Alzheimer\'s disease. (Photo credit: NBC News). **Evelyn Witkin** (1921) Born: New York City, United States. Witkin is an American geneticist who is best known for her work on DNA mutagenesis and DNA repair. She helped elucidate the first co-ordinated stress response. This she did studying the response of bacteria to UV radiation. Witkins was one of the first few women to be elected to the US National Academy of Sciences, in 1977 and in 2002 was awarded the National Medal of Science. (Photo credit: YouTube). **Rosalyn Yalow** (1921 - 2011) Born: New York City, United States. The second American woman to ever be awarded the Nobel Prize for Physiology or Medicine, Yalow is best known for having co-developed a diagnostic technique, known as a radioimmunoassay, for measuring tiny quantities of various biological samples in blood and other bodily fluids. The test\'s primary detection mechanism is an antibody combined with a radioisotope. First devised for determining insulin levels in diabetes patients, the technique is now used for hundreds of other substances previously difficult to detect because they were too small. Among the substances it can quantify are hormones, vitamins, enzymes. It is also used to measure the effectiveness of dose levels of antibiotics and other drugs. (Photo credit: US Information Agency). **Tu Youyou** (1930) Born: Zhejiang, China. Tu Youyou is a Chinese chemist who discovered artemisinin and dihydroartemisinin, used to treat malaria. YouYou received the 2015 Nobel Prize in Physiology or Medicine jointly with William Campbell and Satoshi Omura. Youyou is the first Chinese Nobel laureate in physiology or medicine and the first female citizen of the People\'s Republic of China to receive a Nobel Prize in any category. (Photo credit: Bengt Nyman).