Copying DNA

Today i hope to describe about Copying DNA. this is first post for Copying DNA..

• Polymerase Chain Reaction
• Also called PCR
• A method of making many copies of a piece of DNA

Steps in Copying DNA
• A DNA molecule is placed in a small test tube
• DNA polymerase that can work at high temps is added
• The DNA is heated to separate the two strands
• Primers, short pieces of DNA complementary to the ends of the molecule to be copied, are added
Copying DNA
• The tube is cooled, and DNA polymerase adds new bases to the separated strands

Human Genome Project

• Started in 1990
• Research effort to sequence all of our DNA (46 chromosomes)
• Over 3.3 billion nucleotides
• Mapping every gene location (loci)
• Conducted by scientists around the world
HGP Insights
• Only 2% of human genome codes for proteins (exons)
• Other 98% (introns) are non-coding
• Only about 20,000 to 25,000 genes (expected 100,000)
• Proteome – organism’s complete set of proteins
• About 8 million single nucleotide polymorphisms (SNP) – places where humans differ by a single nucleotide
• About ½ of genome comes from transposons (pieces of DNA that move to different locations on chromosomes)
Benefits of Human Genome Project
• Improvements in medical prevention of disease, gene therapies, diagnosis techniques …
• Production of useful protein products for use in medicine, agriculture, bioremediation and pharmaceutical industries.
• Improved bioinformatics – using computers to help in DNA sequencing

The Human Genome Project was started in 1989 with the goal of sequencing and identifying all three billion chemical units in the human genetic instruction set, finding the genetic roots of disease and then developing treatments. With the sequence in hand, the next step was to identify the genetic variants that increase the risk for common diseases like cancer and diabetes.
It was far too expensive at that time to think of sequencing patients’ whole genomes. So the National Institutes of Health embraced the idea for a "shortcut", which was to look just at sites on the genome where many people have a variant DNA unit. The theory behind the shortcut was that since the major diseases are common, so too would be the genetic variants that caused them. Natural selection keeps the human genome free of variants that damage health before children are grown, the theory held, but fails against variants that strike later in life, allowing them to become quite common. (In 2002 the National Institutes of Health started a $138 million project called the HapMap to catalog the common variants in European, East Asian and African genomes.)
The genome was broken into smaller pieces; approximately 150,000 base pairs in length. These pieces were then ligated into a type of vector known as "bacterial artificial chromosomes", or BACs, which are derived from bacterial chromosomes which have been genetically engineered. The vectors containing the genes can be inserted into bacteria where they are copied by the bacterial DNA replication machinery. Each of these pieces was then sequenced separately as a small "shotgun" project and then assembled. The larger, 150,000 base pairs go together to create chromosomes. This is known as the "hierarchical shotgun" approach, because the genome is first broken into relatively large chunks, which are then mapped to chromosomes before being selected for sequencing.
Funding came from the US government through the National Institutes of Health in the United States, and a UK charity organization, the Wellcome Trust, as well as numerous other groups from around the world. The funding supported a number of large sequencing centers including those at Whitehead Institute, the Sanger Centre, Washington University in St. Louis, and Baylor College of Medicine.
The Human Genome Project is considered a Mega Project because the human genome has approximately 3.3 billion base-pairs.
If the sequence obtained was to be stored in book form, and if each page contained 1000 base-pairs recorded and each book contained 1000 pages, then 3300 such books would be needed in order to store the complete genome. However, if expressed in units of computer data storage, 3.3 billion base-pairs recorded at 2 bits per pair would equal 786 megabytes of raw data. This is comparable to a fully data loaded CD.

Genetic Engineering and Fish

What is a genetically engineered fish?

Genetically engineered (also called transgenic) fish are those that carry and transmit
one or more copies of a recombinant DNA sequence (i.e., a DNA sequence produced
in a laboratory using in vitro techniques). Because genetic engineering is defined by
the technology that is used to create and transfer the DNA sequence, and not the
source species of the donor DNA, even fish that are engineered with DNA derived
entirely from fish species are considered to be genetically engineered. Currently, no
genetically engineered fish has been approved for food production in the United
States. To date only one company, AquaBounty, has publicly announced that it has
requested FDA approval to market a genetically engineered food animal, a growth-
enhanced Atlantic salmon that is capable of growing 4 to 6 times faster (but not larg-
er) than standard salmon grown under the same conditions.


What are the science-based concerns associated with genetically engineered fish?

The greatest science-based concerns associated with genetically engineered fish are
those related to their inadvertent release or escape. Concerns range from interbreed-
ing with native fish populations to ecosystem effects resulting from heightened com-
petition for food and prey species. There is, in principle, no difference between the
types of concerns associated with the escape of genetically engineered fish and those
related to the escape of fish that differ from native populations in some other way,
such as captively bred populations (Lynch and O’Hely 2001). Ecological risk assess-
ment requires an evaluation of the fitness of the genetically engineered fish relative
to non–genetically engineered fish in the receiving population in order to determine
the probability that the transgene will spread into the native population. Ecological
impacts are the result of the characteristics of the organism, regardless of whether the
organism acquired those characteristics through natural selection, artificial selection,
or genetic engineering. The presence of genetically engineered fish does not a priori
have a negative effect on native populations. If genetically engineered fish are ill-suit-
ed to an environment or are physically unable to survive outside of containment, they
may pose little risk to the native ecosystems. Regulators apply a scientifically derived,
risk-based framework to assess the ecological risks involved with each transgene, spe-
cies, and receiving ecosystem combination on a case-by-case basis. Risks will be quite
specific to the gene, species, and site in question, and simple generalizations concerning
the risks (and benefits) of genetically engineered fish are not scientifically meaningful.



Commercialization of genetically engineered fish will likely depend on the development
of effective containment strategies. If genetically engineered fish are adequately con-
tained, they pose little risk to native populations. The NRC recommended the simulta-
neous use of multiple containment strategies for genetically engineered fish (National
Research Council 2004). Physical containment is an obvious first line of defense to pre-
vent the escape of genetically engineered fish. Examples of such measures may include
building facilities on land or in locations removed from native populations, or ensuring
that water chemistry (temperature, pH, salinity, and concentrations of certain chemi-
cals) is lethal to one or more life stages of the genetically engineered fish, such as treat-
ing effluent water to prevent the release of viable gametes or fry. Biological containment
or bioconfinement approaches such as sterilization are also being developed.

Cell Experiments & Activities

Cells are the smallest, most basic functional units that comprise living organisms. They are made up of smaller structures called organelles, which carry out different cell functions (for example, the mitochondria are responsible for respiration). Some ideas for cell science projects and activities include experimenting with salt and cell cytoplasm, examining cell nucleus and genome size, and extracting DNA from plant and animal cells.

SALT & CELL CYTOPLASM

All living cells have cytoplasm, which is a fluid-like substance (sometimes referred to as protoplasm) that cell bodies---such as mitochondria and ribosomes---float around in. In plant cells, the cytoplasm is contained both by an inner cell membrane, and an outer cell wall. As an experiment, you can observe the effect salt has on plant cell cytoplasm, assuming---of course---that it can penetrate the cell wall and membrane. According to crystal-clear-science-projects.com, you will need to take a small tissue sample from an onion and examine it under a compound microscope (100x magnification will be sufficient). You can then saturate your sample with a five percent solution of salt in water, observe, and do the same with a 10 percent solution. You may discover that the salt dissolves the cytoplasm of the cells in your onion, allowing you to have a better glimpse at the inner-workings of the plant cell.

CELL NUCLEUS & GENOME SIZE

Every species of animal stores DNA in the nuclei of its cells. However, some store more DNA than others, which correlates to a larger genome size. According to sciencebuddies.org, genome size is determined by weighing DNA, and is measured in picograms (one picogram equals one trillionth of a gram). For this experiment, you will determine if there is a correlation between the size of animal genomes and the size of their cell nuclei. To do this, you will need to utilize the Cell Size Database, which lists the genome sizes and nuclei sizes (measured in μm2, or in millionths of a square meter) of different animals. You will likely want to organize your results according to animal kingdom (for example, have a section for amphibians, mammals, etc.) and make a chart and/or graph to display them.


EXTRACTING DNA FROM PLANT & ANIMAL CELLS

This experiment will require equipment and chemicals that you may only be able to access at a high school or---perhaps more likely---university or college laboratory. According to usc.edu, your goal will be to determine if it is easier to extract DNA from an animal cell as opposed to a plant cell (which, as mentioned earlier, features a sturdy cell wall). You will need to take samples of animals and plants, such as small bits of chicken liver or onion, and prep them for extraction. This requires adding a buffering solution, a detergent and a neutralizing solution to each sample, and then subjecting each to a centrifuge (which rotates fluids at extremely high speeds). Finally, you need to add isopropanol to isolate and extract the DNA. For an additional twist on the experiment, treat plant samples with cellulase---which will breakdown cell walls---and see what effect this has on extraction.

The Difference of the Genomic DNA Extraction Between Animal & Plant

The structure of double-stranded DNA is universal in all living cells, but differences occur in the methods for extracting genomic DNA from animal and plant cells. Genomic DNA is found in the nucleus of cells. The amount and purity of extracted DNA depends on the type and size of the cell, as certain cells contain more DNA and impurities than others.




GENERAL DNA EXTRACTION
Plant and animal cells treated with a soapy substance will degrade the lipids in the cell and nuclear membranes. The DNA mixture will then separate from the cell membranes and proteins. The DNA in solution can be precipitated using alcohol. Depending on the amount in the sample, DNA may be visible by the naked eye. Such a simple procedure does not necessarily produce DNA of high purity.


PLANT AND ANIMAL CELLS
Plant cells are distinguishable from animal cells by their rigid cell wall and organelles like the chloroplast. They also contain proteins and enzymes that play a role in photosynthesis. Some plant cells are polyploidy, meaning they have more than one copy of each chromosome per cell. Cellular processes occurring in plants such as photosynthesis produce a range of secondary metabolites. Animal cells do not have a cell wall, but still need to be treated with chemicals like sodium dodecyl sulphate (SDS) to disrupt the cell membrane to release genomic DNA.


PLANT DNA EXTRACTION
Plant genomic DNA is more difficult to extract because of the plant's cell wall, which is removed by homogenization, or by adding cellulase to degrade the cellulose that makes up the cell wall. Also, the metabolites present in the plant cell may interfere with genomic DNA extraction by contaminating the DNA sample during the precipitation process.


ANIMAL DNA EXTRACTION
Peripheral blood leukocytes are a main source of animal genomic DNA, but sample collection is difficult as blood must be withdrawn from the animal. Blood contains a range of compounds like proteins, lipids, white blood cells, red blood cells, platelets, and plasma, which can contaminate the DNA sample. The primary contaminant of animal DNA extracted from blood samples is heme, the non-protein component of hemoglobin.


DIFFERENCES
The differences between plant and animal DNA lie in the sequence of bases in the helix. Compounds found in plant cells are absent in animal cells, and DNA base sequences reflect this, as the genomic plant DNA is often larger than animal DNA. These differences affect extraction methods, as it impacts on yield and purity of DNA.

Cloning and relation to plasmids

The use of cloning is interrelated with recombinant DNA in classical biology, as the term "clone" refers to a cell or organism derived from a parental organism, with modern biology referring to the term as a collection of cells derived from the same cell that remain identical. In the classical instance, the use of recombinant DNA provides the initial cell from which the host organism is then expected to recapitulate when it undergoes further cell division, with bacteria remaining a prime example due to the use of viral vectors in medicine that contain recombinant DNA inserted into a structure known as a plasmid. Plasmids are extrachromosomal self-replicating circular forms of DNA present in most bacteria, such as Escherichia coli (E. Coli), containing genes related to catabolism and metabolic activity, and allowing the carrier bacterium to survive and reproduce in conditions present within other species and environments. These genes represent characteristics of resistance to bacteriophages and antibiotics and some heavy metals, but can also be fairly easily removed or separated from the plasmid by restriction endonucleases, which regularly produce "sticky ends" and allow the attachment of a selected segment of DNA, which codes for more "reparative" substances, such as peptide hormone medications including insulin, growth hormone, and oxytocin. In the introduction of useful genes into the plasmid, the bacteria are then used as a viral vector, which are encouraged to reproduce so as to recapitulate the altered DNA within other cells it infects, and increase the amount of cells with the recombinant DNA present within them.The use of plasmids is also key within gene therapy, where their related viruses are used as cloning vectors or carriers, which are means of transporting and passing on genes in recombinant DNA through viral reproduction throughout an organism. Plasmids contain three common features—a replicator, selectable marker and a cloning site. The replicator or "ori" refers to the origin of replication with regard to location and bacteria where replication begins. The marker refers to a particular gene that usually contains resistance to an antibiotic, but may also refer to a gene that is attached alongside the desired one, such as that which confers luminescence to allow identification of successfully recombined DNA. The cloning site is a sequence of nucleotides representing one or more positions where cleavage by restriction endonucleases occurs.[1] Most eukaryotes do not maintain canonical plasmids; yeast is a notable exception. In addition, the Ti plasmid of the bacterium Agrobacterium tumefaciens can be used to integrate foreign DNA into the genomes of many plants. Other methods of introducing or creating recombinant DNA in eukaryotes include homologous recombination and transfection with modified viruses.