Comprehensive Notes on Nucleic Acids and Biotechnology
Nucleic Acids and Biotechnology
Introduction to Biotechnology
Biotechnology involves manipulating DNA, which has led to:
Recombinant DNA molecules.
Cloning of organisms.
Production of glowing mice.
We consume genetically modified foods and use DNA analysis in forensics.
Human DNA manipulation has resulted in:
Bacteria protecting plants from pests.
Ecosystem restoration.
Production of insulin, hormones, antibiotics, and clot-dissolving medicines.
Comparative genomics provides insights into species relationships, and DNA sequencing reveals personal genetic make-up.
Ethical questions arise regarding the appropriate uses of DNA manipulation.
Basic Techniques and Processes
Isolating Nucleic Acids:
Nucleic acids are isolated by lysing cell membranes and enzymatically destroying other macromolecules.
Gel Electrophoresis:
Fragments or whole chromosomes are separated by size (base pair length) using gel electrophoresis.
Polymerase Chain Reaction (PCR):
Short DNA or RNA sequences are amplified using PCR.
Recombinant DNA Technology:
Combines DNA from different sources using bacterial plasmids or viruses as vectors to carry foreign genes into host cells, creating genetically modified organisms (GMOs).
Transgenic Organisms:
Transgenic bacteria, agricultural plants (corn, rice), and farm animals produce protein products like hormones and vaccines for human benefit.
Recombinant technology relies on the universality of the genetic code and the fundamental similarity of transcription and translation across all organisms.
Cloning:
Cloning produces genetically identical copies of DNA, cells, or entire organisms (reproductive cloning).
Genetic Testing and Gene Therapy:
Genetic testing identifies disease-causing genes, and gene therapy treats or cures inheritable diseases.
Ethical and Practical Considerations
Questions about the safety of GMOs and privacy issues arise from these technologies.
Living systems store, retrieve, transmit, and respond to information essential to life processes.
Big Idea 3: Living systems store, retrieve, transmit, and respond to information essential to life processes.
Enduring Understanding 3.A: Heritable information provides for continuity of life.
Essential Knowledge 3.A.1: DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 6.4: The student can make claims and predictions about natural phenomena based on scientific theories and models.
Learning Objective 3.5: The student can justify the claim that humans can manipulate heritable information by identifying an example of a commonly used technology.
Enduring Understanding 3.C: The processing of genetic information is imperfect and is a source of genetic variation.
Essential Knowledge 3.C.1: Changes in genotype can result in changes in phenotype.
Science Practice 7.2: The student can connect concepts in and across domain(s) to generalize or extrapolate in and/or across enduring understandings and/or big ideas.
Learning Objective 3.24: The student is able to predict how a change in genotype, when expressed as a phenotype, provides a variation that can be subject to natural selection.
Biotechnology and Its Applications
Biotechnology is the use of biological agents for technological advancement.
It has been used in breeding livestock and crops long before the scientific basis was understood.
Since the discovery of DNA structure in 1953, biotechnology has grown rapidly.
Primary applications are in medicine (vaccines, antibiotics) and agriculture (genetic modification of crops).
Industrial applications include fermentation, oil spill treatment, and biofuel production.
Basic Techniques to Manipulate Genetic Material
Nucleic acids are macromolecules made of nucleotides (sugar, phosphate, and nitrogenous base) linked by phosphodiester bonds.
Phosphate groups have a net negative charge.
The entire set of DNA molecules in the nucleus is called the genome.
DNA has two complementary strands linked by hydrogen bonds between paired bases.
DNA denaturation occurs at high temperatures, strands reanneal upon cooling.
DNA is replicated by DNA polymerase enzyme.
RNA molecules, unlike DNA, leave the nucleus.
mRNA (messenger RNA) is commonly analyzed because they represent actively expressed, protein-coding genes.
RNA molecules are often less stable than DNA.
DNA and RNA Extraction
DNA or RNA must be isolated or extracted from cells to study or manipulate nucleic acids.
Extraction techniques involve breaking open the cell and using enzymatic reactions to destroy unwanted macromolecules.
Cells are broken using a lysis buffer (detergent solution).
Lysis means "to split."
Enzymes break apart lipid molecules in cell and nuclear membranes.
Macromolecules are inactivated using enzymes like proteases (break down proteins) and RNAses (break down RNA).
DNA is then precipitated using alcohol.
Human genomic DNA is visible as a gelatinous, white mass.
DNA samples can be stored frozen at for several years.
RNA analysis studies gene expression patterns in cells.
RNA is naturally very unstable because RNAses are common and difficult to inactivate.
RNA extraction uses buffers and enzymes to inactivate macromolecules and preserve the RNA.
Gel Electrophoresis
Nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field.
Gel electrophoresis separates molecules based on size using this charge.
Nucleic acids can be separated as whole chromosomes or fragments.
Nucleic acids are loaded into a slot near the negative electrode of a semisolid, porous gel matrix and pulled toward the positive electrode.
Smaller molecules move faster through the pores than larger molecules, separating fragments by size.
Molecular weight standard samples provide a size comparison.
Nucleic acids in a gel matrix are observed using fluorescent or colored dyes.
Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (negative electrode end) based on their size.
A mixture of genomic DNA fragments of varying sizes appear as a long smear, while uncut genomic DNA is usually too large to run through the gel, forming a single large band at the top.
Amplification of Nucleic Acid Fragments by Polymerase Chain Reaction
Genomic DNA is visible to the naked eye when extracted in bulk, DNA analysis often requires focusing on specific regions of the genome.
Polymerase chain reaction (PCR) amplifies specific regions of DNA for further analysis.
PCR is used for cloning gene fragments, analyzing genetic diseases, identifying contaminant foreign DNA, and amplifying DNA for sequencing.
Practical applications include detecting genetic diseases.
Primers—short pieces of DNA complementary to each end of the target sequence—are combined with genomic DNA, Taq polymerase, and deoxynucleotides.
Taq polymerase is a DNA polymerase isolated from Thermus aquaticus (a thermostable bacterium) that can withstand high temperatures used in PCR.
Thermus aquaticus grows in Yellowstone National Park's Lower Geyser Basin.
Reverse transcriptase PCR (RT-PCR) is similar to PCR, but cDNA is made from an RNA template before PCR begins.
DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR).
The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA.
This process is called reverse transcription.
This requires an enzyme called reverse transcriptase.
After cDNA is made, regular PCR can be used to amplify it.
Hybridization, Southern Blotting, and Northern Blotting
Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences.
Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection.
Gel electrophoresis separates nucleic acid fragments according to their size.
The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting.
The nucleic acid fragments bound to the surface of the membrane can then be probed with specific radioactively or fluorescently labeled probe sequences.
When DNA is transferred to a nylon membrane, the technique is called Southern blotting, and when RNA is transferred to a nylon membrane, it is called northern blotting.
Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression.
In western blotting, proteins are run on a gel and detected using antibodies.
Molecular Cloning
"Cloning" means the creation of a perfect replica; in biology, the re-creation of a whole organism is referred to as "reproductive cloning."
Researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.
Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation.
A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA.
In cloning, the plasmid molecules can be used to provide a "folder" in which to insert a desired DNA fragment.
Plasmids are usually introduced into a bacterial host for proliferation.
The fragment of DNA from the human genome (or the genome of another organism being studied) is referred to as foreign DNA, or a transgene, to differentiate it from the DNA of the bacterium, which is called the host DNA.
Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics).
Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone.
An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS).
The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly available restriction endonucleases.
Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner; they are naturally produced by bacteria as a defense mechanism against foreign DNA.
Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang.
Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.”
Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds.
Any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease.
Recombinant DNA Molecules
Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature.
They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis.
Proteins that are expressed from recombinant DNA molecules are called recombinant proteins.
Not all recombinant plasmids are capable of expressing genes.
The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression.
Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.
Cellular Cloning
Unicellular organisms, such as bacteria and yeast, naturally produce clones of themselves when they replicate asexually by binary fission, known as cellular cloning.
The nuclear DNA duplicates by mitosis, which creates an exact replica of the genetic material.
Reproductive Cloning
Reproductive cloning is a method used to make a clone or an identical copy of an entire multicellular organism.
Most multicellular organisms undergo reproduction by sexual means, which involves genetic hybridization of two individuals (parents), making it impossible for generation of an identical copy or a clone of either parent.
Recent advances in biotechnology have made it possible to artificially induce asexual reproduction of mammals in the laboratory.
Parthenogenesis, or “virgin birth,” occurs when an embryo grows and develops without the fertilization of the egg occurring; this is a form of asexual reproduction.
An example of parthenogenesis occurs in species in which the female lays an egg and if the egg is fertilized, it is a diploid egg and the individual develops into a female; if the egg is not fertilized, it remains a haploid egg and develops into a male.
The unfertilized egg is called a parthenogenic, or virgin, egg.
Some insects and reptiles lay parthenogenic eggs that can develop into adults.
Sexual reproduction requires two cells; when the haploid egg and sperm cells fuse, a diploid zygote results.
The zygote nucleus contains the genetic information to produce a new individual.
However, early embryonic development requires the cytoplasmic material contained in the egg cell.
If the haploid nucleus of an egg cell is replaced with a diploid nucleus from the cell of any individual of the same species (called a donor), it will become a zygote that is genetically identical to the donor.
Somatic cell nuclear transfer is the technique of transferring a diploid nucleus into an enucleated egg. It can be used for either therapeutic cloning or reproductive cloning.
Dolly the sheep was the first mammal to be cloned in 1996, but the success rate was very low.
Dolly lived for seven years and died of respiratory complications.
There is speculation that because the cell DNA belongs to an older individual, the age of the DNA may affect the life expectancy of a cloned individual.
Since Dolly, several animals such as horses, bulls, and goats have been successfully cloned, although these individuals often exhibit facial, limb, and cardiac abnormalities.
There have been attempts at producing cloned human embryos as sources of embryonic stem cells, sometimes referred to as cloning for therapeutic purposes.
Therapeutic cloning produces stem cells to attempt to remedy detrimental diseases or defects (unlike reproductive cloning, which aims to reproduce an organism).
Therapeutic cloning efforts have met with resistance because of bioethical considerations.
Genetic Engineering
Genetic engineering is the alteration of an organism’s genotype using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits.
The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering.
The organism that receives the recombinant DNA is called a genetically modified organism (GMO).
If the foreign DNA that is introduced comes from a different species, the host organism is called transgenic.
Bacteria, plants, and animals have been genetically modified since the early 1970s for academic, medical, agricultural, and industrial purposes.
In the US, GMOs such as herbicide-resistant soybeans and borer-resistant corn are part of many common processed foods.
Gene Targeting
Classical methods of studying gene function start with a phenotype and determine the genetic basis.
Modern techniques allow researchers to start at the DNA sequence level and ask: "What does this gene or DNA element do?"
This technique, called reverse genetics, has resulted in reversing the classic genetic methodology.
This method involves mutating or deleting genes to understand their function.
Gene targeting involves using recombinant DNA vectors to alter the expression of a particular gene, either by introducing mutations or by eliminating the expression of a certain gene by deleting part or all of the gene sequence.
Biotechnology in Medicine and Agriculture
Biotechnology is used for medicinal purposes, leveraging knowledge of genetic makeup, heritable diseases, and technology to manipulate and fix mutant genes.
In agriculture, biotechnology enhances resistance to disease, pests, and environmental stress, and improves crop yield and quality.
Genetic Diagnosis and Gene Therapy
Genetic diagnosis involves testing for suspected genetic defects before administering treatment.
Family members may be advised to undergo genetic testing based on the inheritance patterns of a disease-causing gene.
For example, women diagnosed with breast cancer are usually advised to have a biopsy to determine the genetic basis of cancer development, and treatment plans are based on these findings.
Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.
Gene therapy is a genetic engineering technique used to cure disease by introducing a good gene at a random location in the genome to aid the cure of a disease caused by a mutated gene.
The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA.
More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID).
Production of Vaccines, Antibiotics, and Hormones
Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response.
Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen.
The antigen is then introduced into the body to stimulate the primary immune response and trigger immune memory.
Genes cloned from the influenza virus have been used to combat the constantly changing strains of this virus.
Antibiotics are a biotechnological product naturally produced by microorganisms, such as fungi, to attain an advantage over bacterial populations.
Antibiotics are produced on a large scale by cultivating and manipulating fungal cells.
Recombinant DNA technology was used to produce large-scale quantities of human insulin in E. coli as early as 1978.
Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product.
Human growth hormone (HGH) is used to treat growth disorders in children.
The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector.
Transgenic Animals
Several recombinant proteins used in medicine are successfully produced in bacteria, some proteins require a eukaryotic animal host for proper processing.
For this reason, the desired genes are cloned and expressed in animals, such as sheep, goats, chickens, and mice.
Animals that have been modified to express recombinant DNA are called transgenic animals.
Several human proteins are expressed in the milk of transgenic sheep and goats, and some are expressed in the eggs of chickens.
Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.
Transgenic Plants
Manipulating the DNA of plants (i.e., creating GMOs) has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life.
Plants are the most important source of food for the human population.
Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established.
Plants that have received recombinant DNA from other species are called transgenic plants.
Because they are not natural, transgenic plants and other GMOs are closely monitored by government agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life.
Foreign genes can spread to other species in the environment, extensive testing is required to ensure ecological stability.
Staples like corn, potatoes, and tomatoes were the first crop plants to be genetically engineered.
Transformation of Plants Using Agrobacterium tumefaciens
Gene transfer occurs naturally between species in microbial populations.
Many viruses that cause human diseases act by incorporating their DNA into the human genome.
In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by transfer of DNA from the bacterium to the plant.
The tumors do not kill the plants, they make the plants stunted and more susceptible to harsh environmental conditions.
Many plants, such as walnuts, grapes, nut trees, and beets, are affected by A. tumefaciens.
The artificial introduction of DNA into plant cells is more challenging than in animal cells because of the thick plant cell wall.
Researchers used the natural transfer of DNA from Agrobacterium to a plant host to introduce DNA fragments of their choice into plant hosts.
In nature, the disease-causing A. tumefaciens have a set of plasmids, called the Ti plasmids (tumor-inducing plasmids), that contain genes for the production of tumors in plants.
DNA from the Ti plasmid integrates into the infected plant cell’s genome.
Researchers manipulate the Ti plasmids to remove the tumor-causing genes and insert the desired DNA fragment for transfer into the plant genome.
The Ti plasmids carry antibiotic resistance genes to aid selection and can be propagated in E. coli cells as well.
The Organic Insecticide Bacillus thuringiensis
Bacillus thuringiensis (Bt) is a bacterium that produces protein crystals during sporulation that are toxic to many insect species that affect plants.
Bt toxin has to be ingested by insects for the toxin to be activated.
Insects that have eaten Bt toxin stop feeding on the plants within a few hours.
After the toxin is activated in the intestines of the insects, death occurs within a couple of days.
Modern biotechnology has allowed plants to encode their own crystal Bt toxin that acts against insects.
The crystal toxin genes have been cloned from Bt and introduced into plants.
Bt toxin has been found to be safe for the environment, non-toxic to humans and other mammals, and is approved for use by organic farmers as a natural insecticide.
Flavr Savr Tomato
The first GM crop was in 1994, tomato that resisted rotting and maintained flavor for longer periods of time.
Antisense RNA technology was used to slow down the process of softening and rotting caused by fungal infections, which led to increased shelf life of the GM tomatoes.
Additional genetic modification improved the flavor of this tomato.
This GM tomato did not successfully stay in the market because of problems maintaining and shipping the crop.