Chapter 21 and 22
Palindromic sequences
A palindromic sequence is a sequence made up of nucleic acids within a double helix of DNA and/or RNA that is the same when read from 5' to 3' on one strand and 3' to 5' on the other, complementary, strand. It is also known as a palindrome or an inverted-reverse sequence. Many restriction endonucleases (restriction enzymes) recognize specific palindromic sequences and cut them. These enzymes predictably cut both strands because the sequences they recognize are palindromic.
Potential Uses of Stem Cells to Treat Diseases
The potential use of stem cells to help treat diseases has already come to fruition for a few cases. For example, patients diagnosed with certain cancers can get a bone marrow transfusion from a healthy donor. The bone marrow contains stem cells which will proliferate and differentiate in the patient's body thus creating a functioning immune system. The use of embryonic stem cells has great potential to treat a great array of diseases associated with tissues and cell damage because they are easy to identify, grow in a laboratory and are pluripotent. On the other hand, adult stem cells prove to be more challenging to use because they are difficult to locate in the body and hard to propagate. In order to control the fate of ES cells, researchers need to fully understand the internal/external factors that affect it. Stem cells can potentially be differentiated into cardiac cells to repair damage caused by heart attacks or neural cells to treat spinal cord injuries or Parkinson's disease.
Gene addition
Gene addition is a process that involves the insertion of a cloned gene into the genome. Additional copies of a gene that is already in the genome can be added. It is also possible to introduce a gene that is not already in the genome. To perform this process a researcher can insert the gene of interest into a particular site into the genome. An example of gene addition is the production of Glofish. A gene that encodes for a fluorescent protein is inserted into a zebrafish's genome, causing the zebrafish to glow.
Microinjection- gene transfer
A Microinjection is a genetic engineering technique involved in the transfer of DNA. In this process the foreign DNA is transferred to a cell by the use of a glass micropipette. The micropipette is heated until the glass is almost liquified and it is quickly stretched to give the pipette approximately a 0.5 mm diameter. Then the actual transfer of the foreign DNA is done under a strong microscope. The cells that are receiving the foreign DNA are placed into a container and a holding pipette holds the targeted cell in one place. Then the tip of the micropipette is injected through the membrane of the cell and the DNA is delivered into the cytoplasm of the living cell. This process is involved in the making of transgenic animals as well as in vitro fertilization which is the fertilization of egg and sperms in glassware. Electroporation- gene transfer: Electroporation is yet another form of gene transfer. In this DNA transfer process host cells and selected molecules are suspended in a conductive solution. The mixture is surrounded by an electrical circuit and then an electrical pulse at an optimal voltage is discharged for microseconds. This voltage disturbs the phospholipid bilayer of the membrane and forms temporary pores. These temporary pores allow the charged DNA molecules that are also in solution to cross the host cell's membrane.
cDNA library
A cDNA library is a DNA library whose recombinant vectors carry cDNA inserts. cDNA is first made using reverse transcriptase and linkers (short oligonucleotides) are attached to the cDNA using DNA ligase to insert the cDNA into vectors. The linkers contain DNA sequences with a unique site for a restriction enzyme. After the linkers are attached to the cDNAs, the cDNAs and the vectors are cut with restriction enzymes and then ligated to each other. This produces a cDNA library. So what is the template used in cDNA if the enzyme that is used is reverse transcriptase? A poly-dT primer binds to the 3' end of eukaryotic mRNAs. Reverse transcriptase catalyzes the synthesis of a complementary DNA strand (cDNA).
Genomic Library
A genomic library is a set of DNA clones that ideally contains the entire DNA content of a genome from which the library was derived. Contains thousands of different bacterial colonies and each one carrying a different piece of chromosomal DNA. A DNA clone is a DNA construct that is made by replication in a microorganism. The clone is composed of two parts that are fused into a single continuous DNA molecule. One part is the vector, which at a minimum contains genes coding for the proteins and other DNA elements necessary for the propagation and selection of the clone in the host microorganism. The other part of the clone is the insert DNA. This is the DNA that is isolated from the organism under study and inserted into the vector.
Transgenic Plants
A transgenic plant is a genetically modified plant that is considered a genetically modified organism (GMO). These plants are genetically modified using DNA recombinant technology to introduce new genes into the plant's genome. The modifying of the plant's genome involves the combining of DNA from other genomes or the insertion of foreign DNA into the genome of the plant using various DNA recombinant techniques. Transgenic plants can be made by introducing cloned genes into somatic tissue of the plant (ex: leaf). Once the leaf is transgenic then it can be treated with plant growth hormones which causes the transgenic plant to form roots and grow. Many scientists use the Ti plasmid of Agrobacterium tumefaciens to introduce new genes into the plants. The Ti plasmid contains T DNA which can be transferred from the bacterium to the infected plant and be integrated into the plant cell by recombination. The T DNA is used as a vector to introduce the cloned genes into plants and scientists genetically modify the Ti plasmid to contain desirable genes. Scientists and researchers have produced transgenic plants with desired characteristics such as resistance to herbicides, disease, and insects. Transgenic plants have been modified to have plant protection which includes resistance to herbicides, resistance to viral, bacterial and fungal pathogens, and resistance to insects. Genetic modification of plants is also used to improve the plant quality. Genes can be inserted into the plant to improve storage life by the expression of antisense RNA that silences fruit softening. Another example of altering the plant quality is changing the plant composition. Plants can also be modified to make new products. Transgenic plants have been made to synthesise biodegradable plastics. They can also be modified and used for vaccines and pharmaceuticals. Transgenic plants can also be modified to have human antibodies that can help battle various diseases.
Vectors-Plasmids
A vector is a small DNA molecule that replicates independently of the chromosomal DNA and can produce multiple copies of an inserted gene. Also, vectors are used in cloning experiments because they can carry a small segment of chromosomal genes, such as one gene. For instance, a host cell is a cell that has a replicating vector. Furthermore, a type of vector that is used in cloning experiments is a plasmid; plasmids are small circular pieces of DNA found in bacterial and some eukaryotic cells. Some plasmids are advantageous for bacteria providing them antibiotics or toxic substance resistance. When plasmids are in a host cell, the origin of replication of the plasmid is recognized by replication enzymes of the hosting cell resulting in plasmid replication. Ultimately, the origin of the replication sequence of the plasmid is vital for replication to occur; thus, some plasmids have a broad range of origin of replication. Conversely, some plasmids have a limiting origin of replication sequences that decrease host cell range, so these types of plasmids are used in a cloning experiment. Concurrently, the origin of replication determines the number of copies the plasmid will make once it replicates in the host cell. For example, there are plasmids with strong origins that have high copy numbers approximately 100 to 200 copies per cell, while weaker origin plasmid can make about 1 or two copies per cell .Lastly, many commercially available plasmids contain unique sites where geneticists inserted a piece of DNA. Subsequently, commercially available plasmids have a selectable marker that provides the host cell antibiotic or toxic product resistance.
Recombinant DNA technology
According to our text, recombinant DNA technology is the use of in vitro molecular technology to isolate and manipulate different pieces of DNA to produce new arrangements. The resulting recombinant DNA molecules are introduced to living cells and once inside the host, they are replicated by cellular activities, thus producing many identical copies of the genes, a process otherwise known as gene cloning.
Methods to Introduce Cloned Genes into Human Cells
An approach that human gene therapy takes is introducing a cloned gene into a person's somatic cells.A main concern with the approach is that it requires many cells to uptake the cloned gene and express it. Despite the concerns, there are two techniques used to transfer cloned genes: nonviral and viral gene transfer. For instance, a common nonviral technique involves the use of liposomes, which are a lipid vesicle. Initially, the liposomes are complexed with the DNA containing the gene of interest.Afterwards, the DNA-liposome complex is integrated into the cells via endocytosis.During endocytosis, the plasma membrane invaginates and creates endosome, which are intracellular vesicles. At the end of the process, the DNA is released into the cytosol and is integrated into the chromosome of the target cell by recombination. An advantage of this process is that the immune responses aren't activated by the liposomes, the efficiency of the transfer is low. A second way to transfer genes into humans is by using viruses. Some of the viruses used in this technique are retroviruses, adenoviruses, and parvoviruses. Furthermore, the reason why viruses can be used in transfer of genes is because gene therapy vectors were developed. When a gene vector is introduced into a virus, the virus can infect cells and tissues but can't replicate within the target cell. Similar to the first approach the genetically engineered viruses are taken up via endocytosis. After endocytosis, the viral coat is diasambled and the viral genome is released into the cytosol.Ultimately, the viral DNA that carries the gene of interest is integrated into a chromosome of the target cell by recombination. An advantage of using viruses to transfer gene is that it has a high efficiency to transfer the gene;however, this type of transfer can evoke an immune response that can be very strong or fatal.Immunosuppressive drugs and less immunogenetic viral vectors are used to prevent a strong or fatal reaction caused by viral transfer.
Biolistic gene transfer
Biolistic gene transfer is a process in which a target cell is hit with microprojectiles coated with DNA. The microprojectiles are fired from a device called a "gene gun", then they penetrate the cell membrane and cell wall if in a plant cell. The cells that received the DNA can be identified and regenerated into a new plant, creating a transgenic plant.
Bioremediation
Biotransformation is the chemical change to toxins structure by an enzyme produced by a microorganism. This can then lead the toxin to go on to the process of biodegradation. Biotransformation can be done on many different substances such as hydrocarbons,pharmaceutical compounds and metals. Some ways The biotransformation process can be done are oxidation,hydrolysis,condensation. It can convert a material into a less active one like taking DDT(pesticide) and breaking it into DDE & DDA
CRISPR
CRISPR, which stands for Clustered, Regularly Interspaced, Short, Palindromic Repeats, is a gene editing tool in current modern genetics. CRISPER allows scientists to precisely snip out and replace genes. The CRISPER-Cas system requires an enzyme such as CAS9, which will cut the genome at a site targeted by an RNA (in some research) or DNA guide molecule. In the natural type II system found in bacteria, there are two different non-coding RNAs (tracrRNA and crRNA) which play a key role. The tracrRNA will bind to the CAS9 protein and also to the crRNA. The crRNA will bind to. Target DNA such as a DNA segment within a bacteriophage. These binding interactions guide the CAS9 protein to the bacteriophage DNA and the CAS9 makes a double strand. Researchers have also made modifications to this system to make it efficient for gene mutations. They create a single RNA in which the tracrRNA and crRNA are linked to each other which is called a single guide RNA (sgRNA). The spacer region of the sgRNA is designed to be complementary to one of the strands of the genes that is to be mutated. The sgRNA binds to CAS9 and guides it to the gene of interest. CAS9 then makes a double-strand break in this gene. Following the repair there are 2 different repair events. It can be repaired by nonhomologous end joining and has caused a small deletion to create a gene inactivation. It can be repaired by homologous recombination repair which uses a double stranded DNA known as DNA donor which now carries the desired mutation.
Outline of Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a method in which small amounts of DNA sequences are amplified. To do this, you need a template DNA, two oligonucleotide primers, deoxyribonucleoside triphosphates (dNTPs), a heat resistant polymerase, and an ion (cofactor) and buffer solution. The steps for completing PCR are as follows: The dsDNA strands are separated by heating of the DNA to 95℃ The now single stranded DNA is cooled to 55℃ and annealed so the primers and DNA polymerase can attach The strands are heated again to 72℃ and extended back into dsDNA The process is repeated continuously for amplification of the original DNA sequence. Very Good!
Restriction endonucleases or restriction enzymes
Cloning experiments use restriction enzymes or restriction endonuclease. In cloning experiments, restriction enzymes cut the backbone at two defined locations and one cut on each strand. An example of a restriction enzyme is EcoRI, and its job is to make recombinant DNA. A distinct characteristic of the EcoRI enzyme is that it cuts G and A bases only. Furthermore, the EcoRI enzyme is useful in cloning experiments because it forms sticky ends when it cuts DNA into fragments. If two different DNA strands have sticky ends, they can create a hydrogen bond because their sticky ends are complementary to each other. Despite the bond formed, the bond between the different DNA strands is unstable because the sugar-phosphate backbones within the DNA must be linked. In an experiment, DNA ligase is used to stabilize the bond from the two different DNA strands. There are several commercialized restriction enzymes, and the proteins often recognize palindromic sequences. Subsequently, a palindromic sequence is the same as that in the complementary strand when reading it in the opposite direction. Aside from their usage in cloning experiments, restriction enzymes are naturally found in bacteria and protect the prokaryotic cell from foreign DNA.
DNA sequencing
DNA sequencing is the process of determining the order of nucleotides. This means determining the order in which the bases: cytosine, adenine, thymine, and guanine are placed. This allows scientists to determine what each part of the gene is responsible for. The first step to DNA sequencing is to use PCR to break up the DNA into many different fragments. Then dideoxynucleotides are added to the fragments, which stop the elongation of the fragments, due to the lack of an oxygen. Then gel electrophoresis is performed on the fragments. Then a computer is able to read the terminating dideoxynucleotides and depending on the length it can determine the sequence of the nucleotides in the DNA.
Gel electrophoresis
Gel Electrophoresis: Is a technique that is used to separate high molecular weight molecules, such as protein and nucleic acids. The molecules are separated based on size, electrical charge and other properties such as shape using an electrical field. To begin, agarose gel, a molecular sieve that separates the different size molecules, is prepared by increasing the temperature to 90°C and then the gel is allowed to cool to below 55°C. Agar, a polymer that is made from algae, becomes hard when it cools down. This process creates pores within the gel. The size of the pores depends on the concentration of the gel. The lower the concentration, the larger the pores. Therefore, the separation of molecules is influenced by the size of the pores in the gel and as a result smaller fragments thread their way through the pores easier and faster. Heavier molecules will stay on top and lighter molecules will pass through the pores. Movement also depends on their electrical charge. There are two electrophoresis chambers. A positive node called anode that attracts the ions that have a negative charge such DNA due to the presence of the negatively charged phosphate groups in the backbone of DNA. There is a negative node called the cathode which attracts molecules with a positive charge. The cathode chamber is on top and the anode chamber is on the bottom. Electrical charge is applied to the electrophoresis chamber causing the DNA to move towards the anode, which is at the bottom, separating itself from other molecules. There are also control lanes containing DNA of known sizes and genotypes as a reference.
The Steps in Gene Cloning
Gene cloning begins with a plasmid DNA (origin of replication, ampR gene, lacZ gene and a unique restriction site) and segment of chromosomal DNA from human cells containing the gene of interest. Next, cut the DNA with the same restriction enzyme. Mix the DNAs together. Allow time for sticky ends to base-pair. Add DNA ligase to covalently link the DNA backbone. The desired result is a recombinant vector with the gene of interest. Mix the DNA with many E. coli cells that don't already have a plasmid. Treat cells with agents that make them permeable to DNA. Plate the cells on media containing X-gal, IPTG, and ampicillin. Incubate overnight. Each bacterial colony comes from a single cell that has taken up the plasmid. Cells that do not take up a plasmid cannot grow because they are not resistant to ampicillin. Steps provided by Robert J. Brooker, textbook page 516 Very Good!
Gene cloning
Gene cloning is the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. The DNA containing the target gene is split into fragments using restriction enzymes. In 1973, Stanley Cohen and Herbert Boyer developed the techniques that make recombinant DNA which is a form of artificial DNA. Recombinant DNA is engineered through the combination of 2 or more DNA strands, which is combining DNA sequences which would not normally occur together. The following steps involved in gene cloning are Restriction enzyme digestion and ligation, Isolation of DNA, Ligation, Transfection and Selection, and Gel electrophoresis.
Gene Knockouts
Gene knockout is the total removal or permanent deactivation of a gene through genetic engineering. Gene knockout strategies are also known as gene replacement. This approach can be used to study either gain of function or loss of function phenotypes. It is based on the concept that a piece of DNA, when introduced into a nucleus, is able to find its matching sequence in the host genome and trade places through a mechanism called homologous recombination. Investigators can replace a specific target gene with a completely inactive copy or a mutated version of the piece, and study the resulting phenotype. Once a genomic target has been identified, a gene replacement transcript is constructed and transfected into embryonic stem cells by electroporation or lipofection. Following selection, the genomic DNA of the cells is tested by PCR to verify that the correct homologous recombination has occurred. Correctly targeted embryonic stem cells are microinjected into normal donor mouse blastocysts, where they mix with the population of normal embryonic stem cells. The injected blastocysts are then implanted into surrogate females, and the subsequent procedure is similar to that of the transgene approach.
Human Gene Therapy
Gene therapy is when cloned genes are introduced into somatic cells or genes are altered to cure a disease. The reason why Human gene therapy is important is because there are over 7000 human genetic diseases known to be caused by a mutated gene. Some diseases that human gene therapy could help combat are cystic fibrosis,sickle cell disease,hemophilia,cancer, and AIDS. Unfortunately, relatively few patients have successfully been treated, but by more research the outcome could change.
Genetically Modified Animals
Genetically modified animals are animals whose DNA has been altered using genetic engineering techniques. Biotechnological developments have allowed scientists to alter the genetic make-up of bacteria, plants, and animals. Initially, these modifications have served the purpose of basic research (the study of gene function and genetic mechanisms), but these techniques quickly became promising tools from agricultural point of view since they allow the addition of novel traits to organisms which may increase their suitability for use such as enhancing yields, increase resistance to disease, etc
Use of LacZ to Identify Recombinant Plasmid
Lac Z is part of the lac operon of E. coli and encodes the enzyme beta galactosidase. This enzyme catalyzes the hydrolysis of lactose into glucose and galactose, allowing the bacteria to use lactose as an energy source. Beta galactosidase can also break down an artificial substrate called X-gal to produce a compound that is blue in color. X-gal can thus be used to test for the presence of active beta galactosidase. For instance, the gene for human growth hormone (hGH) was inserted into a plasmid. The plasmid, as well as the hGH gene are cut with restriction endonucleases to create compatible DNA ends that can be ligated. While the ends of the hGH gene are capable of being ligated to the ends of the plasmid, the two ends of the plasmid could also rejoin. In fact, given that the two ends of the plasmid are on the same molecule, the chances of their finding each other are much higher than of a plasmid end finding an hGH gene. This would mean that many of the ligated molecules would not be recombinants, but simply recircularized plasmids. Five percent of the plasmids having inserts of the hGH gene would be very good. That would mean that 95% of the bacterial colonies arising from transformation would contain the original plasmid rather than the recombinant. How do we use the LacZ to identify the cells that have the gene of interest? The LacZ enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate X-gal. This is referred to as the blue-white screen. The main steps of blue white screening are: Ligation: ligation of foreign DNA into the plasmid vector Transformation: introduction of plasmid vector with foreign DNA insert into competent E. coli Screening: blue-white screening to identify recombinant bacterial colonies. Okay so ... the colonies that have the gene of interest will appear...What color? White would be containing recombinant and Blue would be without.
Multipotent cells and example
Multipotent cells are stem cells that are unspecialized and possess the ability to self-renew by dividing and becoming multiple different cell types. Multipotent stem cells give rise to cells with a specific purpose and function. Multipotent cells can develop into more than one cell type, but are more limited than pluripotent cells which can develop into all body cell types. Brain cells are an example of multipotent cells since they can produce different neural cells, glia or haematopoietic cells, which can give rise to different blood cell types, but they can't create brain cells, which shows they are limited. Bone marrow also has multipotent cells that are able to divide and produce all blood cell types but not other cells. Multipotent cells are also found in the tissues of adult animals and are thought to replace and replenish diseased or aging cells.
Northern Blotting
Northern blotting is a technique used to find a specific RNA sequence in blood or tissue samples. Similar to Southern blotting, restriction enzymes cut an RNA sample into segments. Next, the RNA segments are separated using gel electrophoresis. Using an absorbent paper, the RNA segments in the gel are transferred to a porous membrane. Then, the membrane is exposed to a DNA probe. If the probe binds to the membrane, then there is a complementary RNA sequence in the sample.
Pluripotent cells and example
Pluripotent stem cells are stem cells that can self-renew and differentiate into all or nearly all the types of body cells. An example of a pluripotent cell is an Embryonic stem cell (ES cell) which is located in the inner cell mass of a blastocyst during early embryonic mammalian development.
Reverse transcriptase
Reverse transcriptase is an enzyme that transcribes a complementary DNA strand from an RNA template. The process involves a single strand of mRNA, the enzyme, a promoter, and dNTPs to produce cDNA. The mRNA must be heated to denature the structure, and then chilled to allow the promoter to bind to the RNA. Then the reverse transcriptase and the dNTPs must be added and after the complementary DNA strand is completed it is heated to deactivate the enzyme. What is the origin of this enzyme? Is this a human enzyme?Reverse Transcriptase was discovered by Howard Temin at University of Wisconsin. Temin discovered it in Rous Sarcoma virions. Reverse Transcriptase was isolated by David Baltimore from murine leukemia virus and Rous Sarcoma Virus. This enzyme can be found in humans with immune diseases such as AIDS. Good! It is found in humans because it is part of the virus. This enzyme is NOT a human enzyme. Dr. I
Southern Blotting
Southern blotting is a technique used to detect a specific DNA sequence in blood or tissue samples. First, restriction enzymes cut a DNA sample into segments. Next, the DNA segments are separated using gel electrophoresis. Using an absorbent paper, the DNA segments in the gel are transferred to a porous membrane. Finally, the specific DNA sequences are detected by hybridization using DNA probes. DNA probes are complementary sequences that are fluorescently or radioactively labeled. The DNA probes bind to the DNA sequence via base pairing, and thus the specific DNA sequence can be found.
Stem cells and example
Stem cells have the ability to divide and differentiate into one or more specialized cell types. They give rise to the cells that make up the human body from a fertilized egg and also replace damaged or worn out cells. When a stem cell divides, one daughter cell remains an undifferentiated stem cell while the other differentiates into a specialized cell. This allows the stem cell population to remain constant while still producing specialized cells. Cells such as erythrocytes and skin epithelial cells depend on this mechanism because they have a finite life span. The developmental stage and ability to differentiate categorizes each stem cell for mammals. An example of a stem cell is an Embryonic stem cell (ES cell) which is located in the inner cell mass of a blastocyst during early embryonic mammalian development. ES cells are pluripotent, meaning they can differentiate into majority if not all body cells
Unipotent cells and example
Stem cells that can differentiate into only a single type of cell. Found in the adults. For example, primordial germ cells in the testis differentiate nly into a single cell type, the sperm.
Biodegradation
The breakdown of a larger molecule into a smaller molecule via cellular enzymes. Toxic pollutants are degraded ,yielding less complex, nontoxic metabolites. For example, toxic heavy metals can be rendered less toxic by oxidation or reduction reactions carried out by microorganisms. Another way to alter the toxicity of organic pollutants is by promoting polymerization. In many cases polymerized toxic compounds are less likely to leach from the soil and therefore, are less environmentally toxic than parent compounds. How is biodegradation different from bioremediation?? Biodegradation is a natural process that takes place without human intervention. Bioremediation is an engineered process of application of biological need to degrade material. Biodegradation is a slow process while bioremediation is a faster process. Human intervention is used to control the rate of bioremediation, by controlling temperature, availability of food or nutrients, etc. Biodegradation, on the other hand, is controlled by nature. Biodegradation takes place anywhere and everywhere, while bioremediation is planned at a contaminated site. Biodegradation can be both beneficial or harmful (such as degradation of metals in biofouling), while bioremediation is designed to be beneficial to us. Good way to differentiate them. Very Good! You can think of sewage treatment where the bacteria does the degradation with minimal human intervention. While Bioremediation will need more intervention since the microorganisms are usually modified to do a specific task such as degrading plastic. Dr. I
Totipotent cells and example
Totipotent stem cells are cells that can divide and give rise to any cell type in the organism. This includes becoming a cell in any of the three germ layers (ectoderm, mesoderm, and endoderm) or a placental cell. This type of cell is only present in the first few cell divisions of the zygote after fertilization. Thus, an example of a totipotent cell is a placental cell.
Western Blotting
Western blotting is a laboratory method which is used to detect specific protein molecules from among a mixture of proteins. They also can be used to evaluate the size of a protein of interest, and measure the amount of protein expression. This method is closely related to Southern Blot. First, to prepare the protein by mixing it with detergent which makes the protein unfold into linear chains and coats. Next, the protein molecules are separated according to their sizes using gel electrophoresis. After the separation, the proteins are transferred from the gel into a blotting membrane. The membrane is then incubated with an antibody,primary antibody, which specifically binds to protein of interest. Following incubation any unbound antibody is washed away, then incubated again. The secondary antibody is linked to a reporter enzyme that produces light or color, easily detected and images. The steps allow a specific protein to be detected among a mixture of proteins.
