The Polymerase Chain Reaction (or PCR) is a laboratory technique which greatly amplifies the amount of a short segment of DNA. It is based on the process by which DNA is copied in cells. Since replicated DNA is itself available for later replication, the PCR process can feed upon itself, leading to an accelerating increase to millions of copies over the original DNA sample.Polymerase Chain Reaction Polymerase Chain Reaction
PCR solves two of the major problems encountered in the analysis of DNA in an organism. The first problem is specificity - an area of interest may be as little as one unit of DNA among billions. Since PCR only amplifies a small segment (leaving the rest of the DNA largely unaltered), the required specificity of a later genetic assay might be reduced to just one part among hundreds. The second problem is sensitivity - a sample of DNA might contain just a few copies of an area of interest. By increasing that to millions (or billions) of copies, PCR greatly speeds or simplifies later genetic analysis.
PCR has become a widely used tool for research in molecular genetics. It is also the basis for medical genetic testing, where individuals are screened for the presence of disease mutations. DNA Fingerprinting, the unique identification of individuals by patterns of variation in their DNA, is also based on PCR, and is used in criminal forensics and parental testing. The technique had such widespread impact that its inventor was awarded the Nobel Prize in 1993, just eight years after it was first reported
Wednesday, April 30, 2008
DNA
Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints or a recipe, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.
Chemically, DNA is a long polymer of simple units called nucleotides, with a backbone made of sugars and phosphate groups joined by ester bonds. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.
Within cells, DNA is organized into structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms (animals, plants, and fungi) store their DNA inside the cell nucleus, while in prokaryotes (bacteria and archae) it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.
Chemically, DNA is a long polymer of simple units called nucleotides, with a backbone made of sugars and phosphate groups joined by ester bonds. Attached to each sugar is one of four types of molecules called bases. It is the sequence of these four bases along the backbone that encodes information. This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.
Within cells, DNA is organized into structures called chromosomes. These chromosomes are duplicated before cells divide, in a process called DNA replication. Eukaryotic organisms (animals, plants, and fungi) store their DNA inside the cell nucleus, while in prokaryotes (bacteria and archae) it is found in the cell's cytoplasm. Within the chromosomes, chromatin proteins such as histones compact and organize DNA. These compact structures guide the interactions between DNA and other proteins, helping control which parts of the DNA are transcribed.
Genetic disorder
A genetic disorder is a condition caused by abnormalities in genes or chromosomes. While some diseases, such as cancer, are due to genetic abnormalities acquired in a few cells during life, the term "genetic disease" most commonly refers to diseases present in all cells of the body and present since conception. Some genetic disorders are caused by chromosomal abnormalities due to errors in meiosis, the process which produces reproductive cells such as sperm and eggs. Examples include Down syndrome (extra chromosome 21), Turner Syndrome (45X0) and Klinefelter's syndrome (a male with 2 X chromosomes). Other genetic changes may occur during the production of germ cells by the parent. One example is the triplet expansion repeat mutations which can cause fragile X syndrome or Huntington's disease. Defective genes may also be inherited intact from the parents. In this case, the genetic disorder is known as a hereditary disease. This can often happen unexpectedly when two healthy carriers of a defective recessive gene reproduce, but can also happen when the defective gene is dominant. Currently about 4,000 genetic disorders are known, with more being discovered. Most disorders are quite rare and affect one person in every several thousands or millions. Cystic fibrosis is one of the most common genetic disorders; around 5% of the population of the United States carry at least one copy of the defective gene. Some types of recessive gene disorder confer an advantage in the heterozygous state in certain environments.
Genetic diseases are typically diagnosed and treated by geneticists. Genetic counselors assist the physicians and directly counsel patients. The study of genetic diseases is a scientific discipline whose theoretical underpinning is based on population genetics.
Genetic diseases are typically diagnosed and treated by geneticists. Genetic counselors assist the physicians and directly counsel patients. The study of genetic diseases is a scientific discipline whose theoretical underpinning is based on population genetics.
Cloning DNA in Bacteria
The word cloning for this type of amplification entails making multiple identical copies of a sequence of DNA. The target DNA sequence is then inserted into a [cloning vector]. Because this vector originates from a self-replicating virus, plasmid, or higher organism cell when the appropriate size DNA is inserted the “target and vector DNA fragments are then ligated and create a recombinant DNA molecule. The recombinant DNA molecules are then put into a bacteria strain (usually E. coli) which produces several identical copies by transformation. Transformation is the DNA uptake mechanism possessed by bacteria. However, only one recombinant DNA molecule can be cloned within a single bacteria cell, so each clone is of just one DNA insert.
Separation and Detection
In separation and detection DNA and mRNA are isolated from cells (the separation) and then detected simply by the isolation. Cell cultures are also grown to provide a constant supply of cells ready for isolation.
Cell (biology)
The cell is the structural and functional unit of all known living organisms. It is the smallest unit of an organism that is classified as living, and is sometimes called the building block of life.Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans, are multicellular. (Humans have an estimated 100 trillion or 1014 cells; a typical cell size is 10 µm; a typical cell mass is 1 nanogram.) The largest known cell is an ostrich egg. In 1837 before the final cell theory was developed, a Czech Jan Evangelista Purkyně observed small "granules" while looking at the plant tissue through a microscope. The cell theory, first developed in 1839 by Matthias Jakob Schleiden and Theodor Schwann, states that all organisms are composed of one or more cells. All cells come from preexisting cells. Vital functions of an organism occur within cells, and all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells.
The word cell comes from the Latin cellula, meaning, a small room. The descriptive name for the smallest living biological structure was chosen by Robert Hooke in a book he published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in.
The word cell comes from the Latin cellula, meaning, a small room. The descriptive name for the smallest living biological structure was chosen by Robert Hooke in a book he published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in.
Tissue culture
Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth media, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue culture is used for plantIn modern usage, "tissue culture" generally refers to the growth of eukaryotic cells in vitro. It is often used interchangeably with cell culture to specifically describe the in vitro culturing of sperm donor cells.
However, "tissue culture" can also be used to refer to the culturing of tissue pieces, i.e. explant culture or whole organs, i.e. organ culture.s.
However, "tissue culture" can also be used to refer to the culturing of tissue pieces, i.e. explant culture or whole organs, i.e. organ culture.s.
DNA Isolation
DNA isolation extracts DNA from a cell in a pure form. First, the DNA is separated from cellular components such as proteins, RNA, and lipids. This is done by placing the chosen cells in a tube with a solution that mechanically, chemically, breaks the cells open. This solution contains enzymes, chemicals, and salts that breaks down the cells except for the DNA. It contains enzymes to dissolve proteins, chemicals to destroy all RNA present, and salts to help pull DNA out of the solution.
Next, the DNA is separated from the solution by being spun in a centrifuge, which allows the DNA to collect in the bottom of the tube. After this cycle in the centrifuge the solution is poured off and the DNA is resuspended in a second solution that makes the DNA easy to work with in the future.
This results in a concentrated DNA sample that contains thousands of copies of each gene. For large scale projects such as sequencing the human genome, all this work is done by robots.
Next, the DNA is separated from the solution by being spun in a centrifuge, which allows the DNA to collect in the bottom of the tube. After this cycle in the centrifuge the solution is poured off and the DNA is resuspended in a second solution that makes the DNA easy to work with in the future.
This results in a concentrated DNA sample that contains thousands of copies of each gene. For large scale projects such as sequencing the human genome, all this work is done by robots.
Thymine
Thymine is one of the four bases in the nucleic acid of DNA that make up the letters ATGC. The others are adenine, guanine, and cytosine. Thymine (T) always pairs with adenine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at the 5th carbon. In RNA, thymine is replaced with uracil in most cases. In DNA, thymine(T) binds to adenine (A) via two hydrogen bonds to assist in stabilizing the nucleic acid structures.
Thymine combined with deoxyribose creates the nucleoside deoxythymidine, which is synonymous with the term thymidine. Thymidine can be phosphorylated with one, two, or three phosphoric acid groups, creating, respectively, TMP, TDP, or TTP .One of the common mutations of DNA involves two adjacent thymines or cytosine, which, in presence of ultraviolet light, may form thymine dimers, causing "kinks" in the DNA molecule that inhibit normal function.
Thymine could also be a target for actions of 5-fluorouracil (5-FU) in cancer treatment. 5-FU can be a metabolic analog of thymine (in DNA synthesis) or uracil (in RNA synthesis). Substitution of this analog inhibits DNA synthesis in actively-dividing cells.
Thymine bases are frequently oxidized to hydantoins over time after the death of an organism
Thymine combined with deoxyribose creates the nucleoside deoxythymidine, which is synonymous with the term thymidine. Thymidine can be phosphorylated with one, two, or three phosphoric acid groups, creating, respectively, TMP, TDP, or TTP .One of the common mutations of DNA involves two adjacent thymines or cytosine, which, in presence of ultraviolet light, may form thymine dimers, causing "kinks" in the DNA molecule that inhibit normal function.
Thymine could also be a target for actions of 5-fluorouracil (5-FU) in cancer treatment. 5-FU can be a metabolic analog of thymine (in DNA synthesis) or uracil (in RNA synthesis). Substitution of this analog inhibits DNA synthesis in actively-dividing cells.
Thymine bases are frequently oxidized to hydantoins over time after the death of an organism
Complementary DNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes. cDNA is also produced by retroviruses (such as HIV-1, HIV-2, Simian Immunodeficiency Virus, etc.) which is integrated into its host to create a provirus.The central dogma of molecular biology outlines that in synthesizing proteins, DNA is transcribed into mRNA, which is translated into protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes can contain introns (intervening sequences), which are not coding sequences, and must be spliced out of the RNA primary transcript before it becomes mRNA and can be translated into protein. Prokaryotic genes have no introns, so their RNA is not subject to splicing.
Often it is desirable to express eukaryotic genes in prokaryotic cells. A simplified method of doing so would include the addition of eukaryotic DNA to a prokaryotic host, which would transcribe the DNA to mRNA and then translate it to protein. However, as eukaryotic DNA has introns, and since prokaryotes lack the machinery to splice them, the splicing of eukaryotic DNA must be done prior to adding the eukaryotic DNA into the host. This DNA which was made as a complementary to the RNA is called complementary DNA (cDNA). To obtain expression of the protein encoded by the eukaryotic cDNA, prokaryotic regulatory sequences would also be required .
Often it is desirable to express eukaryotic genes in prokaryotic cells. A simplified method of doing so would include the addition of eukaryotic DNA to a prokaryotic host, which would transcribe the DNA to mRNA and then translate it to protein. However, as eukaryotic DNA has introns, and since prokaryotes lack the machinery to splice them, the splicing of eukaryotic DNA must be done prior to adding the eukaryotic DNA into the host. This DNA which was made as a complementary to the RNA is called complementary DNA (cDNA). To obtain expression of the protein encoded by the eukaryotic cDNA, prokaryotic regulatory sequences would also be required .
DNA polymerase
A DNA polymerase is an enzyme that assists in DNA replication. Such enzymes catalyze the polymerization of deoxyribonucleotides alongside a DNA strand, which they "read" and use as a template. The newly-polymerized molecule is complementary to the template strand and identical to the template's partner strand.
DNA polymerase is considered to be a holoenzyme since it requires a magnesium ion as a co-factor to function properly. In the absence of the magnesium ion, it is referred to as an apoenzyme.
DNA-Polymerase initiates DNA replication by binding to a piece of single-stranded DNA.
DNA polymerase is considered to be a holoenzyme since it requires a magnesium ion as a co-factor to function properly. In the absence of the magnesium ion, it is referred to as an apoenzyme.
DNA-Polymerase initiates DNA replication by binding to a piece of single-stranded DNA.
mRNA Isolation
Expressed DNA that codes for the synthesis of a protein is the final goal for scientists and this expressed DNA is obtained by isolation mRNA (Messenger RNA). First, laboratories use a normal cellular modification of mRNA that adds up to 200 adenine nucleotides to the end of the molecule (poly(A) tail). Once this has been added, the cell is ruptured and its cell contents are exposed to synthetic beads that are coated with thymine string nucleotides. Because Adenine and Thymine pair together in DNA, the poly(A) tail and synthetic beads are attracted to one another, and once they bind in this process the cell components can be washed away without removing the mRNA. Once the mRNA has been isolated, reverse transcriptase is employed to convert it to single-stranded DNA, from which a stable double-stranded DNA is produced using DNA polymerase. Complementary DNA (cDNA) is much more stable than mRNA and so, once the double-stranded DNA has been produced it represents the expressed DNA sequence scientists look for.
Human Genome Project
The Human Genome Project (HGP) was an international scientific research project with a primary goal to determine the sequence of chemical base pairs which make up DNA and to identify the approximately 25,000 genes of the human genome from both a physical and functional standpoint.
The project began in 1990 initially headed by James D. Watson at the U.S. National Institutes of Health. A working draft of the genome was released in 2000 and a complete one in 2003, with further analysis still being published. A parallel project was conducted by the private company Celera Genomics. Most of the sequencing was performed in universities and research centers from the United States, Canada and Great Britain. The mapping of human genes is an important step in the development of medicines and other aspects of health care.
While the objective of the Human Genome Project is to understand the genetic makeup of the human species, the project also has focused on several other nonhuman organisms such as E. coli, the fruit fly, and the laboratory mouse. It remains one of the largest single investigational projects in modern science.
The HGP originally aimed to map the nucleotides contained in a haploid reference human genome (more than three billion). Several groups have announced efforts to extend this to diploid human genomes including the International HapMap Project, Applied Biosystems, Perlegen, Illumina, JCVI, Personal Genome Project, and Roche-454.
The "genome" of any given individual (except for identical twins and cloned animals) is unique; mapping "the human genome" involves sequencing multiple variations of each gene. The project did not study the entire DNA found in human cells; some heterochromatic areas (about 8% of the total) remain un-sequenced.
The project began in 1990 initially headed by James D. Watson at the U.S. National Institutes of Health. A working draft of the genome was released in 2000 and a complete one in 2003, with further analysis still being published. A parallel project was conducted by the private company Celera Genomics. Most of the sequencing was performed in universities and research centers from the United States, Canada and Great Britain. The mapping of human genes is an important step in the development of medicines and other aspects of health care.
While the objective of the Human Genome Project is to understand the genetic makeup of the human species, the project also has focused on several other nonhuman organisms such as E. coli, the fruit fly, and the laboratory mouse. It remains one of the largest single investigational projects in modern science.
The HGP originally aimed to map the nucleotides contained in a haploid reference human genome (more than three billion). Several groups have announced efforts to extend this to diploid human genomes including the International HapMap Project, Applied Biosystems, Perlegen, Illumina, JCVI, Personal Genome Project, and Roche-454.
The "genome" of any given individual (except for identical twins and cloned animals) is unique; mapping "the human genome" involves sequencing multiple variations of each gene. The project did not study the entire DNA found in human cells; some heterochromatic areas (about 8% of the total) remain un-sequenced.
Subscribe to:
Posts (Atom)