Genetic Techniques

Southern Blotting

A Southern blot is a molecular biology method that is used to detect particular sequences of DNA in samples of DNA. It fuses, by probe hybridization, fragments of DNA that have been separated by electrophoresis and the membrane of a filter. The method of Southern blotting first entails endonucleases that are restricted, cutting strands of DNA that are of a high molecular weight into small fragments. Next, the fragments of DNA are size separated by the process of electrophoresis and a gel of agarose. After, the gel has the option to be treated with an acid to depurinate the fragments of DNA. The DNA will also be sliced into smaller segments which allows for the fragments of DNA to be efficiently transferred from the gel to the membrane filter. Then, the gel of DNA may be placed into a solution with alkaline properties which allows for the denaturation of the DNA that is double-stranded. A nitrocellulose sheet is placed over the gel with the use of pressure. A buffer solution is then used to seal the gel and to prevent the drying out of the gel. In a vacuum or an oven, the membrane is allowed to bake for a duration of 2 hours at 80◦C. A hybridization probe is used to expose the membrane. A detection label is attached to the DNA of the probe. Finally, hybridization has occurred and probe that has been produced in excess is cleaned from the membrane. On X-ray film, the scientist can now visualize the hybridization that has occurred by the use of autoradiography. Southern blotting can be applied to the identification, in specific genes, of sited of methylation. Southern blotting can also be used for cloning that is based on homology.

mini-prep (isolation of plasmid DNA)

A technique that utilizes extracts and purifies plasmid DNA is called plasmid preparation. There are various methods that involve plasmid preparation, yet each method involves the same three steps. These three steps include: bacterial culture growth, bacteria lysis and harvesting, and plasmid DNA purification. Liquid bacterial cultures are commonly utilized as a way to purify plasmids. Isolated and transformed E.coli is most commonly used as a liquid bacterial culture during the purification of plasmids. A selectable marker usually involves an antibiotic resistant gene for vectors of plasmids. An antibiotic resistant selectable marker allows the plasmid vector to grow unrestricted. Bacteria that have failed to pick up the plasmid vector are determined as lacking the gene of resistance and due to the selective marker differentiating between bacteria that have the antibiotic resistance and bacteria that do not exhibit bacterial resistance, scientists can determine transformations that are successful within colonies. Conditions that are favorable to the bacteria allow the bacteria to successfully grow. The denaturation of protein and DNA of chromosomes takes places under alkaline conditions. However, under alkaline conditions the plasmid DNA holds stable. Alkaline conditions are an important part of mini-prep isolation of plasmid DNA. Mini-prep is a type of plasmid isolation. It involves the isolation of bacterial plasmid DNA by that is both rapid and small-scale. Principally, the mini-prep method entails the use of the alkaline lysis technique in order to yield plasmid DNA. Alkaline lysis breaks bacterial cells open in order to isolate DNA of a plasmid. Bacteria comprised of the desired plasmid is first grown and then lysed using a buffer containing sodium dodecyl sulfate and sodium hydroxide. The cell membrane's phospholipid bilayer is cleaved by the detergent while the proteins are denatured by the alkali. The plasmid is eventually purified and isolated due to various processes which first include agitation and precipitation. Then the process allows for centrifugation, supernatant removal, and the removal of cellular debris. A performance of miniprep yields plasmid DNA that has been extracted; this extracted plasmid DNA is commonly referred to as "miniprep". Scientists apply the use of a miniprep in order to analyze clones of bacteria by molecular cloning. Depending on the strain of the cell, a miniprep can produce 50-100 micrograms of plasmid DNA.

YAC

A yeast artificial chromosome is engineered genetically and derived from yeast DNA. Into a plasmid of bacterial, the YAC is then ligated. Principally, by the insertion of large DNA fragments, the sequences that were inserted can now participate in cloning and also can be mapped physically. The mapping process is referred to as chromosome walking. Currently, YACS have been replaced with BACS (bacterial artificial chromosomes). The central components of YACS are the centromere, ARS, and yeast telomeres. In the selection of yeast cells that have been transformed, genes that are selectable markers have been used. The construction of YACS entails an initial plasmid that contains DNA that is circular. This plasmid is then sliced to create a molecule of DNA that is linear. The slicing of the DNA is due to the use of restriction enzymes. To ligate the sequence of DNA or desired gene into the DNA that is now linearized, a DNA ligase is utilized. The linearized DNA creates a singular piece of circular DNA that is large in size. The creation of linear YACS consists of six main steps. The steps include: (1) the creation of a plasmid vector by selective marker ligation, (2) mitotic stability by the sequences of centromeres being ligated, (3) ARS ligation which provides a replication origin for mitosis, (4) the creation of a piece of linear DNA by the conversion of plasmids that are circular after the ligation of telomeric artificial sequences, (5) amplification after the sequence of DNA being inserted, and (6) yeast colony transformation. Applications of YACS include the mapping and assemblage of an organisms entire genome and determining the location of various genetic and trait disorders. The Human Genome Project used YACS when they first started, but due to their loose stability, they were abandoned for BACs.

Antisense Technology

Antisense technology involves the method of silencing of the disease-causing gene sequence by interfering with RNA translation. Using specific gene sequences, antisense oligonucleotides, are able to inhibit messenger RNA engaging in the process of translation. By inhibiting the translation of the messenger RNA, the disease-causing gene is rendered inactive. Another way to stop the translation of the messenger RNA is by the use of steric blocking or by utilizing the RNase-H enzyme, to destroy the attached mRNA. Antisense technology is used as a form of therapy to treat infections and disorders dealing with the genes of a patient. It is possible to treat a disease by the inactivation of the disease-causing gene, if a particular gene's genetic sequence is known. Antisense technology has developed an essential branch of therapy, antisense therapy, to treat cancer, HIV/AIDS, Hemorrhagic fever viruses, and Familial hypercholesterolemia. Antisense Technology is able to treat diseases and infections due to its ability to halt the expression of genes. The use of antisense oligonucleotides is essential for the effective ness of antisense technology and antisense therapy. There are two types of antisense oligonucleotides that both aid in inhibiting a disease-causing gene. One class of antisense oligopeptides involves the destruction of mRNA by the induction of oligonucleotides that are RNase H-dependent. The destruction of mRNA inhibits translation, which then leads to protein not being formed. The other class of oligonucleotides involves steric blocking which prevents slicing by acting as a physical inhibitor. The most common form of antisense oligonucleotides are the ones that involve RNase H due to its efficiency. Reduction by H-dependent RNase results in expression of mRNA being down-regulated to 80-95%. Plus, any mRNA region that is actively involved in protein expression can be targeted and inhibited by H-dependent RNase. Steric blockers are viewed as less efficient than H-dependent RNase because it is region and codon specific since oligonucleotides engaging in steric blocking only work upon the initiation codon region of AUG or the 5' region of a RNA strand.

DNA fingerprinting (RFLP)

DNA fingerprinting involves taking a sample and then extracting the DNA from it. Once DNA extraction has occurred, the DNA is segmented by using unique restriction enzymes that focus on cutting segments that hold bases of repeated DNA. The technique of electrophoresis is then used to separate the segments by length. After separation has occurred, an autoradiograph (a visual pattern), also referred to as a "DNA fingerprint" is created by radioactively tagging the separated DNA segments onto an X-ray film. DNA fingerprinting involves a variety of applications, one it being utilized at criminal investigations as an essential forensics tool. DNA fingerprinting is often used to compare crime scene evidence to a suspect's blood or bodily fluids. DNA fingerprinting is also used in paleontology research, archeological research, diagnostics in the medical field, and in a variety of biology fields. In research regarding evolution, DNA fingerprinting is known to be used as a ways to observe change through time at both the evolutionary and molecular level. DNA fingerprinting involves studying the unique sequences of nucleotides within certain regions of DNA of each individual. No two people have the same DNA fingerprint. Principally, DNA fingerprinting relies on that fact that a very small number of base pairs contribute to each person's genetic individuality. Plus, mutations causing different expression of genes in regions containing repetitive, noncoding DNA, accumulate and essentially lead to DNA polymorphism. The foundation of the mapping of genes and DNA fingerprinting is DNA polymorphism. Below is an example of a DNA fingerprint:

epigenetic technology

Epigenetics involves an organism's phenotype undergoing heritable changes that occur without the influence of the organisms' genotype. Epigenetic technology refers to the two common uses of epigenetics which involve the process of DNA methylation and modifications of the histones of a protein. Currently, researchers have been analyzing epigenetics as a key player in the progression cancer. Epigenetics, as a type of novel drug therapy, is being researched in the pharmaceutical world due to epigenetics having the potential to repress transcription by manipulating the structure of chromatin in a specific gene sequence. Epigenetics is being monumentally researched by scientists interested in stem cell research, specifically in epigenetics having the potential to aid in cell pluripotency. Another important application of epigenetics involves the potential of epigenetics being essential biomarkers of disease. Epigenetics involves the analysis of gene expression in regards to changes in the pattern of a DNA underlying sequence. It is often referred to when talking about DNA methylation. In epigenetics, these changes are due to workings not dealing with mutations. Principally, modifications due to epigenetics occur due to the structure of chromatin being altered due to changes in the histone proteins and bases of DNA that are within chromatin. Once altered, modifications involving epigenetics, are passed down to daughter cells within the process of cell division.

Golden Gate Assembly

Golden Gate cloning is molecular biology method that lets a scientist, using restriction enzymes that are type II and DNA ligase that is T4, put together multiple fragments of DNA both directionally and simultaneously into a single piece. The assemblage is done in vitro. The most common enzymes that are type IIs are Bbsl, Bsal, and BsmBl. These three enzymes slice DNA on the exterior of their sites of recognition which allows the creation of overhangs that are non-palindromic. By using numerous combination sequences that overhang, the assemblage of multiple DNA fragments is possible. The method of Golden Gate cloning entails, at one time, the assemblage of nine fragments in a plasmid that is a recipient. Cloning then occurs by taking one tube and pipetting all plasmid donors, a restriction enzyme and ligase that are both type IIS, and a vector that is a recipient. The mixture is then incubated in a thermal cycler. The desired product, that is assembled, accumulates over time. A Golden Gate assembly is applied when a scientists wants to clone single inserts.

agarose gel electrophoresis

Has a standard procedure, but the experiment could potentially vary due to alternative methods. The first step of the procedure involves the casting of the gel. Using an appropriate buffer (TAE or TBE), the powder of the agarose is dissolved to prepare the gel. The gel is prepared to later be used in electrophoresis. The agarose-containing gel is then melted till right before it reaches it melting point. Once the scientist has agarose that is melted, the agarose is cooled sufficiently until the scientist is able to pour the solution into a cast. The next phase of the method of agarose gel electrophoresis involves the sample being loaded into the newly created wells of the agarose gel. The wells were created by the comb residing on the set gel being removed. A mixture of the loading buffer and the sample of DNA is placed into the wells. The third phase of the method of agarose gel electrophoresis is electrophoresis. The most common method of agarose gel electrophoresis is completely submerging the slab gel into a buffer in a horizontal, submarine mode. A less popular method of agarose gel electrophoresis is performing electrophoresis in a vertical manner. Another method is utilizing the legs of agarose to raise the gel on an apparatus. The process of electrophoresis involves the use of colored dyes and voltage. The common colored dyes involve Xylene cyanol and Bromophenol. The fourth phase involves staining and visualization. After electrophoresis, the gel may be stained. Ethidium bromide is a stain that is commonly used to view the DNA under an ultraviolet transilluminator. The wavelengths of 302/312-nm is the standard wavelength used by the ultraviolet transilluminator. Further procedures may be used after staining, such as downstream procedures. A band of DNA may be separated from a purified and dissolved gel slice to later be analyzed. Picture of the method (procedure of agarose gel electrophoresis) Agarose Gel Electrophoresis is used in a variety of ways. For example, it is used to determine a molecule of DNA's size after undergoing digestion involving restriction enzymes. It is also used in diagnosis of molecular genetics and fingerprinting of genes by the analyzation of the products of PCR. Extracted and purified DNA fragment separation is also accomplished by using agarose gel electrophoresis. The last application of agarose gel electrophoresis involves DNA genomic restriction separation. The process of Agarose Gel Electrophoresis is able to occur due to the backbone, consisting of phosphate, possessing a negative charge which allows the DNA to progress in the direction of the positively charged anode within the process of electrophoresis. DNA separation, by the determination of size, is able to occur due to the gel matrix during the process of electrophoresis.

BAC

In the human genome, BAC clones' DNA location is determined by the "mapping" of every BAC clone. This ensures that the exact location and the spatial relation of every BAC clones' sequenced letters of DNA are pinpointed. Sequencing occurs when small 2,000 base fragments of each BAC clone are spliced. These small fragments of DNA are referred to as "subclones". The subclones engage in a "reaction of sequencing" to later be placed in a machine that sequences the subclones. Each sequencing reaction results in the production of C,G,A,T base pairs that are estimated to be sequenced to times once the process is finished. It is estimated that about 500-800 A,T,C, and G base pairs are created. In the BAC clone, a representation of human DNA is processed by the synthesizing of short sequences into long, continuous strands, utilizing computer software. BAC clones are often used as a way to show genetic diseases by partnering with an organism-the transgenic mice. Alzheimer's disease and Down syndrome are two disorders that are analyzed with the use of BAC clones. Sceintists have also studied the genes often associated with the development of cancer-oncogenes. Recently, BACs have been used as a component of mapping when sizable gene sequences are of scientific interest. Principally, scientists can refer to BACs as an example of a plasmid. BACs carry within them the observed human or mice DNA which are then injected into a host, usually a bacterium, while in the BAC plasmid. The BAC plasmid replicates, along with the human or mice DNA now within the host bacterium. Due to the easy replication of large sequences of human DNA by using BACs, BACs are an essential tool to the project of the Human Genome.

microprojectile bombardment (gene gun)

Microprojectile bombardment (gene gun) is a device that is used as a way to insert genetic information into cells. Principally, microprojectile bombardment deals with taking a heavy metal elemental particle and then using the DNA of a plasmid to coat the elemental particle. The method of Microprojectile bombardment (gene gun) involves obtaining culture cells of tissue that are placed into a vacuum chamber. Once placed into a vacuum chamber, a gas of high pressure propels he particles of metal in spontaneous bursts that resemble a burst of a balloon that has been popped. Pictured below is the process of the gene gun. In this example, particles of gold are being transformed with the use of a gene gun: Gold microscopic particles are utilized to deliver DNA, as bullets, into callus cells The desired genes are then used to coat the particles of gold Microprojectile bombardment (Gene gun) can be applied to plants, animals, and even humans. The most common use of gene guns involves plant cells. Gene guns target plant cells that are undifferentiated and growing in a petri dish on a gel medium. Currently, gene guns are being used in DNA vaccine delivery. This is done by rat neuron receiving a plasmid delivery with the use of a gene gun. The rat neurons usually being analyzed are DRG neurons. Studies dealing with Alzheimer's disease and other diseases dealing with the degradation of neurons effects, use gene guns as pharmacological precursors.

Phage Display Technology

Phage display technology is a common technique, done in the laboratory that involves the analyzation of interactions that involve either protein-DNA, protein-peptide, or protein-protein. These three interactions are only analyzed if they utilize bacteriophages to bind proteins with the information of the genes that encodes the proteins. The method of phage display technology first entails the insertion of a gene of a desired protein into a protein gene of a phage coat. This results in the phage displaying the desired protein on its exterior while enclosing the protein gene on its interior. This leads to the bonding of the phenotype and genotype. The now displayed phages result in interaction detection between the protein that is displayed and the other molecules. This detected interaction is due to sequences of DNA, proteins, and peptides being screened against the displayed phages. In vitro selection, uses phage display technology to screen and amplify proteins of huge libraries. M13 and a filamentous phage are the most commonly utilized bacteriophages when one is using phage display technology. Principally, phage display is used to screen interactions of proteins by high-throughput. Ligation, numerous sites of cloning, the insertion of a DNA hybrid and phage gene, the immobilization of desired DNA to a microtiter plate's surface, and panning are the principles of phage display technology. Phage display technology is applied when a scientists wants to determine the partner interaction of a protein, finding interactions of protein-DNA, engineering of protein, the discovery of drugs, searching for current ligands, and to pinpoint antigens of tumors.

PCR

Polymerase chain reaction is a type of technology that is commonly used in molecular biology when scientists want to produce numerous specific DNA sequences. This is done by amplifying one copy of a DNA piece or several DNA pieces, over orders of various magnitudes. Application of a polymerase chain reaction technique include cloning DNA for sequencing, analysis of functional genes, genetic fingerprint identification, phylogeny based on DNA, diagnosing diseases that are hereditary, and the diagnosis and discovery of diseases that are infectious. Principally, the polymerase chain reaction primarily involves thermal cycling. Thermal cycling involves the reactions for the melting of DNA and the replication of the enzymes of the DNA being regularly heated and cooled by numerous cycles. Primers consisting of complementary sequences to the desired region and a DNA polymerase are two essential components that allow for amplification that is both repeated and selective. Deoxynucletotide triphosphates, a solution containing buffer, ions (manganese, magnesium, and bivalent cations can be used), and reaction tubes that are small in size, are used in a PCR. The eventual numerously repeated amplification of the template of DNA occurs due to the chain reaction created by PCR progression and the use of the DNA itself as the template. Polymerase chain reaction derived its name from the fact that a DNA polymerase, that is heat-stable, is used during the PCR process. The procedure of the PCR process consists of six major steps: initialization, denaturation, annealing, elongation, elongation that is final, and final hold. The initialization step involves the reaction being heated by 94-96◦C for a duration of 1-9 minutes. The denaturation step is the start of the cycling process and, for a duration of 20-30 seconds, the reaction is heated to 94-98◦C. During this step, DNA molecules that are single-stranded are produced by the hydrogen bonds being disrupted between bases that are complementary. The disruption of the hydrogen bonds occurs by the template of DNA being melted by DNA. The annealing step involves the DNA template containing single strands, being annealed with the primers for a duration of 20-40 seconds at a temperature of 50-65◦C. The elongation step involves, in a direction of 5' to 3', the addition of dNTPS by the DNA polymerase allows for the creation of a new strand of DNA that is complementary to the template of DNA. The final elongation step involves the assurance that the DNA, that is single-stranded, is completely extended. This occurs for a duration of ranging from 5-15 minutes at a temperature of 70-74◦C. The final step involves reaction storage for a short duration at the temperature range of 4-15◦C.

metagenomics technique

Principally, metagenomics technique is based upon the utilization of techniques of the sequencing of DNA in order to analyze extracted DNA from samples within the environment. Metagenomics involves new genes being found with biological activity that is desired (bioprospecting) and the analyzation of microbes located within the environment without the process of cultivation. In nature, microorganisms' function and diversity are studied by first crafting the metagenomics library. This is done by first obtaining an environmental sample in which its prokaryotic cells can be extracted. This environmental sample could consist of seawater, sediment, and soil. Extraction and purification of the DNA content, as a whole, is sheared. A cloning vector is then utilized to clone appropriately sized fragments of DNA. These clones are then placed into a host cell to be transformed. This will result in a metagenome fragment of DNA being carried in thousands of cells. These thousands of cells constitutes the metagenomic library. In the case of projects that deal with the sequencing of DNA on a massive scale, shotgun sequencing of the whole genome is utilized. Shotgun sequencing involves constructing libraries that are small-insert based on 2-5 kb plasmids and the omission of cloning (pyrosequencing). Metagenomics can be applied to ecology, medicine, engineering, sustainability, and agriculture. Currently, metagenomics sequencing is being used to analyze and categorize communities containing microbial growth from an estimated 250 individuals from 15-18 body sites. This is being done to determine if there is a core of the human microbiome that affects human health. In relation to biofuel, metagenomics technology is used to observe communities of complex microbial growth and target screen enzymes that have the potential to aid in the production of biofuel. Another application of metagenomics technology is in the remediation of the environment. For instance, improvement in strategies of the way pollutants impact ecosystems and how contaminated environments are cleaned are two ways in which the technique of metagenomics is utilized. The sector of agriculture uses metagenomics technology to analyze plant and microbe interaction while the ecology sector uses metagenomics to provide an insight into environmental communities' ecology.

RNA interference technology-RNAi

RNA interference technology is an emerging technology that is reforming the mammalian sector of research, applied particularly in the expression of genes of mammalians. RNAi is revolutionizing the way, the quickness, and easiness in which the function of gene loss analysis is being carried out in the cells of mammals and within the models of animals. It is now being used as a way to determine and assess the numerous genes of the genome that may play a role in the phenotypes of diseases. It can also be used as a method to block a specific gene's expression and observing its response to compounds that are chemical. Signal pathway changes are also identified by RNA interference technology. RNA interference technology works by utilizing the natural machinery of the cell aided by molecules of RNA that are short and interfering. By taking advantage of the molecules of RNA, RNAi technology dismantles the desired gene's expression. There is more than one way to start RNAi, molecules that are synthetic, vectors of RNAi, and dicing that is in vitro. Particular degradation of desired cellular mRNA, in the cells of mammals, is initiated by siRNA and short strands of dsRNA. Then, the siRNA duplex's antisense strand bonds with a complex of multiple proteins (silencing complex that is RNA-induced), which leads to the identification of the corresponding mRNA. The mRNA is cleaved at a particular site. After, targeted degradation occurs at the cleaved message. This results in the expression of protein loss.

RNAseq

RNA-seq, a technology that is also referred to as whole transcriptome shotgun sequencing, utilizes the capacity of sequencing that is next-generation to uncover a presence of RNA snapshot and genome quantity at any given instance in time. RNA-seq principally involves a RNA population being transformed into a library consisting of fragments of cDNA with attached adaptors to a single or multiple ends. The most common method of RNA-seq entails each observed molecule, in a manner of high-throughput, being sequenced, with or without amplification, to capture sequences that are short in length. These short sequences are obtained from sequencing of single ends or sequencing of both ends. 30-400 base pairs are typically read depending on the type of technology that is being used in the DNA-sequencing process. Other RNA-seq methods of RNA-seq include a library of RNA'Poly(A)', sequencing of small RNA/non-coding RNA, assembly of the Transcriptome, and sequencing that is RNA direct. RNA-seq can be applied as a tool that is accurate in expression measuring over the entire span of a transcriptome. This provides an image of changes previously undetected in occurring states of disease. RNA-seq also allows researchers, in a single assay, to pinpoint novel features, both known and unknown. This allows for the detection of variants of single nucleotides, fusions of genes, isoforms of transcripts, gene expression that is specific to an allele, and many other features.

stem cell techniques

Stem cells are biological cells that are undifferentiated, but have the ability to differentiate into cells, that are specialized, and, by mitosis, can generate more stem cells. They are only found in organisms that are multicellular. In mammals, two types of stem cells exist: stem cells that are embryonic and adult stem cells. In organisms that are adults, stem cells act as a system of repair within the body by regenerating the tissues of adult mammals. There are more than one type of stem cells of adults; they are: marrow of the bone, adipose tissue, and blood. The umbilical cord of a baby can also provide stem cells. There are also numerous ways to extract stem cells from an organism. The extraction of stem cells by the technique of autologous harvesting provides the least risk. Currently, stem cells can be grown artificially and differentiated into specific and unique types of cells such as nerve and muscle cells. Stem cells are commonly used as a type of medical therapy such as transplantation of bone marrow in humans. Another medical therapy technique, using stem cells, include transfer of nuclear somatic cells. Embryonic stem cells are developed in the laboratory by first transporting cells into a culture medium from an embryo in the stage of pre-implantation. While on the dish, division and separation of the cells commences. Skin cells of embryonic mouse skin cells are used in the culture dish's inner surface so that the cells located there will not divide. This coating of mouse cells is referred to as the feeder layer. A sticky surface for the cells to attach is provided by mouse cells being placed at the dish's bottom. If the cells survive and multiply in the dish, they are gently removed and placed into dishes with fresh cultures. Cell re-plating occurs many times during the technique of extracting embryonic stem cells and each time the re-plating process is done it is referred to as a passage. The cells may then be extracted and frozen for later use. Just like the method of embryonic stem cell extraction, the adult stem cell method involves isolation and growing the stem cells in a culture until a large enough amount of stem cells has been reached. Stem cells have numerous applications, including transplants of bone marrow, replace cells that are damaged, to study diseases, and to study the development of an organism that is complex.

maxam-gilbert dna sequence method

The Maxam-Gilbert DNA sequence method involves partial DNA chemical modification that is specific to each nucleobase. Maxam-Gilbert sequencing also entails the successive cleavage of the backbone of DNA located at adjacent regions to modified nucleotides. For a successful Maxam-Gilbert sequencing to occur, a fragment of DNA must be radioactively labeled at its 5' end and the DNA must be purified. A treatment utilizing chemicals must then be used to divide small quantities of maybe one or two of the four bases of nucleotides, within each of these reactions: C+T, C, G, A+G. Once the chemical treatment has occurred, hot piperidine is used to cleave the modified DNAs. A modification will then occur that is estimated to be a single modification per molecule of DNA. This results in the generation of a sequence of labeled fragments, starting from the radioactively-labeled end to the site of the first "cut" within every molecule. The four reactions' fragments are then processed by electrophoresis using gels of acrylamide that aid in separation based on size. In order to view the fragments, autoradiography must be utilized along with the exposure of the gels to X-ray film. This results in observable locations of the molecules of DNA containing radioactive labels due to the presence of dark bands. Pictured below is an example of Maxam-Sequencing and the use of hot piperidine to cleave the modified DNAs: Maxam Sequencing allows for the direct reading of DNA that has been purified. It also allows for the efficient sequencing of runs of DNA that are homopolymeric and for the analysis of interactions that deal with DNA and protein (footprinting). Another application of Maxam-Gilbert Sequencing involves the analysis of the structure of nucleic acid and DNA that are modified epigenetically. Principally, Maxam-Gilbert DNA sequencing involves the use of four cleavage chemical reactions that are specific to bases to break fragments of DNA that are not only double-stranded, but are also 32P labeled. The fragments of DNA are denatured, electrophoresized, viewed by autoradiography, and observed.

electroporation

The method of electroporation is fairly simple. A conducive solution is used to suspend selected molecules and host cells while a circuit containing electricity, encloses the mixture. The circuit's electricity is dispersed throughout the suspension of the cell for only less than a millisecond. This results in a disturbance of the cell membrane's phospholipid bilayer which leads to the creation of impermanent pores. In a similar process to electrophoresis, the temporary pores and rising electric potential allow for DNA, and other molecules possessing a charge, to cross the cell membrane. Another use of electroporation involves the delivery of drugs and even genes. Electroporation is used in gene and drug delivery by the cell membrane being transiently permeable by intensely short pulses of electricity. Electroporation has many medical applications, one being in the treatment of malignant tumors that cause cancer. Researchers have discovered that irreversible electroporation can target and destroy small ranges of target cells while leaving healthy cells virtually unharmed. This discovery has led to better cancer treatments and to improved treatment of any disease that deals with tissue removal to rid a person of the disease. Electroporation allows for transfection of both stable and transient of every type of cell. It also allows for a large quantity of cells to be easily transfected after the determination of electroporation optimum conditions. Principally, electroporation deals with the use of fields of electricity that are applied to cell membranes in order to raise the permeability of cell membranes. Once the permeability of a cell membrane is increased, charged molecules are allowed to be driven across the cell membrane. Electroporation is essential in plant, yeast, and bacteria protoplast transformation due new coding DNA being introduced. Plus, foreign genes are often introduced into cells of tissue culture by the process of electroporation.

Didexoy Sequencing

The process of dideoxy sequencing occurs by first creating DNA containing a single strand from a DNA that is double stranded. This process is done by utilizing NaOH to denature the double stranded DNA. Once single stranded DNA is formed, DNA primers allows for the process of DNA synthesis to start. A specific ddNTP and a dNTP form a mixture that is then utilized in stopping DNA strand elongation. Once a ddNTP, that is modified and a phosphodiester bond is created due to the absence of a 3' hydroxyl group, is incorporated into DNA synthesis, the action of the DNA polymerase is ceased. Once this termination has occurred, a division of the sequencing reactions occur until there are four separate DNA sequences each containing dTTP, dCTP, dATP, and dGTP along with the DNA polymerase. Heat is then used to denature the fragments of DNA and by using gel electrophoresis, separation of the DNA fragments by size occurs. UV light is used to actually view the DNA bands once electrophoresis has taken place. The figure below shows the proponents of a Sanger reaction (dideoxy sequencing). Note that the proponents of a Sanger reaction are: a DNA primer, a sequenced strand, and a specific ddNTP and dNTP mixture. Dideoxy sequencing is primarily used as an application of DNA and RNA sequencing. It is now used as a mutation detector for various research projects that involve cancer, genetic disease, biology of agriculture, and human pathogens. It is also used as a supporter for numerous DNA sequencing applications. For instance, it is used in De novo sequencing, microbial sequencing, and sequencing dealing with targeted DNA. Often referred to as the Sanger sequencing due to the creator of this sequencing's name quencing represents a DNA sequencing method. Principally, this method involves utilizing 2'3'-dideoxynucletotide tripohosphates to terminate elongation of the chain of DNA Termination by 2'3'-dideoxynucletotide tripohosphates occurs because a bond containing phosphodiester cannot attach with its adjacent deoxynucleotide.

northern blotting

The technique of Northern blotting is commonly used in research of molecular biology in order to analyze the expression of genes. The analyzation of gene expression, by the technique of northern blotting) involves RNA detection (or mRNA that is isolated) contained within a sample. Northern blotting allows scientists to examine the function and structure of a cell under cellular control by pinpointing the exact expression of genes rates during the two processes of morphogenesis and cell differentiation. Northern blotting can also analyze conditions that are diseased or unique within the cell and an mRNA's molecular weight. Principally, northern blotting concerns the techniques of electrophoresis-that divides samples of RNA by size-and, using a complementary probe of hybridization, allows for the detection of whole targeted sequences. The transfer of RNA, with the use of capillaries, from the gel used in electrophoresis to the blotting membrane is what comprises the name "Northern blot". The technique of northern blotting is done by, first, using gel electrophoresis to separate RNA (mRNA or total RNA). The result is rather than discrete bands, a smear is shown. The transfer of RNA to nitrocellulose (a type of blotting paper) then occurs with the molecules of RNA displaying the same pattern separation. Then, single-stranded DNA probe is used to incubate the blot. A molecule of RNA-DNA, that is double-stranded, is eventually formed by the bonding of pairs of bases to a complementary sequence of RNA. The probe can be radioactive or bound with an enzyme. Finally, a colorless substrate, with an enzyme attached to it, is used to incubate the probe. The incubation of the probe reveals its location by the substrate's attached enzyme being converted to a product that is colored which exposes X-ray film due to light emission. Northern blotting can be applied to show oncogene overexpression and the downregulation of genes that suppress tumors cells containing cancer. Another important application includes analyzing organs that have been transplanted into patients, for rejection, by observing the expression of genes.

knockout mice technology

There are multiple ways to create knockout mice. The most common way involves extracting the gene that is to be knocked out. A newly created DNA sequence is formulated that shows similarities to both the parent gene and the DNA sequence that immediately neighbors it. The fact that the newly created DNA sequence's genes are rendered inoperable due to sufficient change upon the gene. A marker gene is placed upon the new DNA sequence, in order to be able to observe possible toxic agent resistance, and any notable change due to the fluorescent nature of the marker. In order to create a successful selection that is complete, a second gene may be added as well; an example of a second gene that could be added is herpes tK+. After selection, the blastocyst of the mouse is obtained in order to obtain stem cells. The stem cells are then grown in vitro. Once cells have grown, the sequence of new DNA and the obtained stem cells are synthesized together by electroporation. The stem cells that have been processed by electroporation will combine with the sequence containing the knockout gene and replace their original gene with the knockout gene in their chromosomes. The marker gene allows for the isolation of the new sequence containing a combination of the stem cells and the knocked-out gene from cells that haven unaltered. Once the stem cells containing the knocked-out gene are isolated, they are then placed inside the blastocyst of the mouse. The original cells and the cells containing the knocked-out gene are now the two types of stem cells within the mouse blastocyst. Implantation of the blastocysts then occurs by the blastocysts being inserted into the female mice's uterus. Offspring can both be heterozygous, and contain the knockout gene, or mice with a wildtype genotype when a mice containing the knocked-out gene are crossed with mice with a wildtype genotype. Knockout mice technology is often used to provide scientists information on what a normal gene does. The use of mice to analyze the functions of genes is due to the close similarity mice genes have to human genes. Mice have become instrumental in scientists knowing how certain genes cause diseases. Specific examples of how knocked-out genes in mice have been a powerful gene research tool is in the areas of aging, arthritis, substance abuse, cancer, diabetes, obesity, heart disease, and Parkinson's disease. Knockout mice technology principally involves the use of the process of transfection. Overall, gene knockout technology focuses on the genetic transference of genes from individual cells into a construct of DNA. This often leads to the goal of gene knockout technology: an altered gene within a transgenic animal that is then able to produce offspring, allowing for the altered gene to be passed on down to generations in the future. The gene is passed down through future generations due to stem cells in the embryo of the animal being transformed genetically and then are placed into early developed embryos. The recommended gene knockout method when dealing with mice involves protoplast infection.

western blotting

Western blotting, also referred to as the protein immunoblot, is a commonly used technique of analysis that is used to identify particular proteins in a homogenate tissue sample or extract. Principally, it uses gel electrophoresis to divide proteins that are native. The separation is caused by using the proteins 3-D structure or the proteins are denatured by the polypeptide length. The proteins are then relocated to a nitrocellulose or even a PVDF membrane. After relocation, using specific antibodies to the protein being targeted, the proteins are stained and incubated. There are two steps in incubation: primary antibody incubation and secondary antibody incubation. The visualization of the proteins and antibodies is allowed by the scientists examining the bands of proteins that appear after staining. There are various types of detection/visualization which includes: colorimetric detection, chemiluminescent detection, radioactive detection, and fluorescent detection. The inclusion of gel electrophoresis is due to the issue of resolving antibody cross-reactivity. Western blotting has many applications in molecular biology and immunogenetics. Specifically, western blotting is applied in the detection of HIV (during the HIV test), bovine spongiform encephalopathy, some types of lyme disease, infection of Hepatitis B, and in the confirmation of FIV+ in animals.

Synthetic Biology

a branch of interdisciplinary biology that fuses disciplines such as biology of evolution, molecular biology, biotechnology, biology of systems, biophysics, engineering of computers, and engineering of genetics. A method of synthetic biology entails obtaining the desired genes of the experiment and then transferring the desired genes into a targeted cell. In this case, once the genes are transferred, the programmed cells become cellular factories that lead to the production of products of high value. Synthetic biology has been presently defined as, "the artificial design and engineering of biological systems and living organisms for purposes of improving applications for industry or biological research as it has expanded to many interdisciplinary fields." There are various applications of synthetic biology such as, the creation of artificial life from biomolecules while in vitro, transformation of living cells, storage of digital information, the synthesis of genetic pathways, nucleotides that are unnatural being inserted into proteins and nucleic acids, the production of unnatural amino acids, and amino-acid reduced libraries. Other applications include, the creation of designed proteins and biosensors, production of materials (nanofibers) and industrial enzymes, and aiding in the exploration of space.

Gilson assembly

a method of DNA assembly in which numerous fragments of DNA are joined by an isothermal reaction. The process involves few manipulations and a relatively small quantity of components. Knowing the identity of the sequence, fragments of DNA can combine simultaneously based on the identity of their sequence. A requirement of the Gibson Assembly method is for the fragments of DNA to possess an overlap of base 20-40 base pairs with adjacent fragments of DNA. Three enzymes, the fragments of DNA, and components of buffers are then mixed to make a sort of chemical cocktail. Another requirement in a successful Gibson assembly process is three enzymatic activities consisting of a DNA ligase, exonuclease, and a DNA polymerase. The role of the enzyme, exonuclease, is breaking the DNA from the end containing 5' which allows fragments of adjacent DNA to anneal. The role of the DNA polymerase is to fill gaps by including nucleotides using a DNA polymerase. The role of the DNA ligase is to join DNA segments that are adjacent to one another; this also results in the removal of any nicks contained within the DNA. Once the three enzyme activities are completed, the mixture is allowed to incubate at a temperature of 50◦C for a duration of one hour. This should result in different fragments of DNA being joined together as one. The Gibson assembly is primarily used in the community of synthetic biology due to the Gibson assembly being suitable, flexible, and easy-to-use when trying to create constructs of DNA. It is also used as a cloning method for DNA. Principally, the Gibson method uses three enzymatic activities, differing in functions, to create a molecule of DNA that is both double-stranded and sealed fully. This results in a functioning PCR template.

DNA footprinting

a way of analyzing the specificity of a sequence involving in vitro proteins containing proteins that are involved in the binding of DNA. The DNA footprinting process beings with cloning a DNA segment in which a repressor will bind to the site of the operator. The DNA is then digested by DNase I. DNase I digests the DNA by randomly cutting the DNA molecules. This results in a mixture containing various lengths of radioactive fragments. The smallest fragment is observed to only be one nucletotide. Once digestion has occurred, the technique of electrophoresis is used to separate the DNA fragments. Electrophoresis will result in separated fragments with the fragments containing the repressor being absent from the autoradiogram. The gaps that are formed to the absence of the lengths being covered by the repressor, results in the DNA "footprint" of the analyzed piece of DNA. The technique of DNA fingerprinting is used in a variety of ways. One being In vivo footprinting which involves observing and analyzing certain protein-DNA interactions. A second use of DNA footprinting involves the assessment of the ability of a protein to bind to a DNA region. A third use of DNA footprinting is detecting fragments of DNA by capillary electrophoresis. Principally, DNA footprinting involves the use of DNase I-a type of nuclease. The nuclease, DNase I will participate in the degradation of the molecule of DNA. Degradation will not occur if there is a protein surrounding the DNA. The region that is protected from nucleases by the surrounding protein, is called the DNA "footprint".

subtractive hybridization

an essential technique that allows scientists to study the expression of genes in particular tissues, a particular stage, or types of cells. Subtracted cDNA probes can be produced by using Dynabeads Oligo(dT)25. There are two methods that can be used for subtractive hybridization. The first method involves the hybridization of the target material's mRNA, with cDNA of the first strand, from immobilized subtractor material. After separation, that is magnetic, of subtractor cDNA that is bound to beads, the mRNA that has been subtracted remains in the supernatant. After the step, involving final hybridization has occurred, the particular mRNA, that has been subtracted, is transcribed in the reverse to cDNA that is radio-labelled. The second method involves a different approach in the creation of cDNA libraries that are immobilized from mRNA that is both subtractor and target. The cDNA that is second-strand is then formed by priming, that is random, of desired cDNA. The fragments are then mixed and eluted with subtractor cDNA that is in excess and immobilized. Annealation and removal of fragments then occurs while fragments that are unique, remain in the supernatant and are utilized to screen libraries of cDNA. Subtractive hybridization has applications in the differentiation between RNA of cells, organisms, tissues, and/or sexes after treatments. These treatments can include application of hormones and heat shock. It can also differentiate between mutant and wild-type cells. Organisms can be differentiated, using subtractive hybridization, by the expression of their sequences. Principally, subtractive hybridization involves the use of two types of nucleic acids. These two types of nucleic acids are referred to as the driver and the tester. The tester, also known as the tracer, consists of the nucleic acid that is to be targeted whereas, the driver does not contain the sequences to be targeted during experimentation. During subtractive hybridization, the tester and the driver are hybridized and then any drivers that are double-stranded and any driver molecules that are single-stranded, are removed. This results in sequences of nucleic acids that are specific to the tester.

DNA microarrays technology

first involves taking a DNA blood sample possessing a specific disease and a sample that will act as the control sample during the experiment. The DNA contained within the samples is denatured by the separation of two strands that are complementary to each other, into single-stranded molecules. After the DNA is separated into single-stranded molecules, the long-stranded DNA is cut into smaller segments. Fluorescent dye is then utilized to label every fragment. Green dye is used to label the patient's DNA and red dye is used to label the control DNA. Once labeling had occurred, the DNA labeled sets are placed into the chip to allow for hybridization of the chip and synthetic DNA. This hybridization results in the determination of a mutated or non-mutated gene. If the patient's DNA contains a mutated gene, then the patient's DNA will improperly bind to the "normal" sequence chip, but will properly bind to the chip that holds the mutated DNA. If the patient's DNA does not possess a mutated gene, then both the DNA samples containing red or green dye, will bind to the non-mutated chip. DNA microarray technology has various applications. One application involves the discovery and identification of genes. Another application of DNA microarray technology is the diagnosis of diseases by observing the activity of genes within a cell. Diseases that are analyzed with the use of DNA microarrays technology are various forms of cancer, disease spread through infection, and mental illness. A third application is the discovery of drugs, specifically pharmacogenomics. A fourth application is research of toxins in cells and genes. DNA mircoarrays involve the main principle of taking two strands of DNA and hybridizing them. Principally, the DNA microarrays technology also involves taking the base pairing rules and matching known and unknown DNA samples. Microplates and membranes that are able to blot are used an array experiments. A microplate can involves numerous spots (possibly a thousand spots) that have a diameter that is less than 200 micros. Analyzed samples of spots can contain oligonucletotides, cDNA, and DNA. On a hard and stationary support, these spotted samples are immobility placed to analyze the discovery of genes and their expression. This analyzation occurs by the determination of the binding of complementary unknown sequences. There are more than one type of microarray technology, one being DNA microarray technology. Other types include analysis of the expression of microarrays, analysis of mutations of microarrays, and hybridization of comparative genomics.

CRISPR/Cas systems for genome sequencing

stands for clustered regularly interspaced short palindromic repeats. CRISPR are segments of DNA that are prokaryotic and consist of short repetitions of sequences of bases. After every short sequence, "spacer DNA" follows. "Spacer DNA" comes from past exposures to a virus from a bacteria or plasmid. The CRISPR/Cas system is an immune system of prokaryotes that allows resistance to elements that are foreign and genetic, such as bacteriophages and plasmids. The CRISPR/cas system provides immunity that is acquired. The genetic elements, that are exogenous, are cut out by CRISPR spacers. CRISPRs can be found in an estimated 40% of bacterial genomes, that are sequenced, and 90% of archaea that are sequenced. The CRISPR/cas system has been applied to the editing of genes, treatments for genetic diseases, infection fighting, and expediting food crop yields. There are three types of mechanisms of CRISPR, but the most studied mechanism is type II. Type II involves viral and plasmid invading DNA being sliced into fragments of a small quantity. The short segments are then fused into a locus of CRISPR among sequences of short repeats. Then, loci transcription occurs with the transcripts being processed in order to produce RNAs. The small RNAs are used to facilitate endonucleases that are effectors, which target DNA that is invading based on complementarity of sequences.