Finals- chapter 20 (DNA Tools and Biotechnology)

biotechnology

Biotechnology is the process of manipulating organisms or their components for the purpose of making useful products.

Using DNA technology for agricultural applications

Certain genes that produce desirable traits have been inserted into crop plants to increase their productivity or efficiency. An organism that has acquired by artificial means on or more genes from another species or variety is termed a genetically modified (GM) organism. Currently, a debate is in progress over the satfey of Gm organisms.

Using DNA technology for for forensic applications

DNA samples taken from the blood, skin cells, or hair of alleged criminal suspects can be compared to DNA collected from the crime scene. Genetic profiles can be compared and used to identify persons at the crime scene.

Uses for DNA technology

Diagnosis of disease, gene therapy, the production of pharmaceuticals, transgenic animals, forensic applications, environmental cleanup, agricultural applications

Embryonic stem cells

Embryonic stem cells are pluripotent, which means "capable of differentiating into many different cell types." The ultimate aim is to use them for the repair of damaged or diseased organs, such as insulin-producing pancreatic cells for people with diabetes or certain kinds of brain cells for people with Parkinson's disease.

how to find the gene of interest among many colonies present after transformation

For certain applications the next step in cloning a gene is finding the gene of interest among the many colonies present after transformation. A process known as nucleic acid hybridization can be used to find the gene. If we know at least part of the nucleotide sequence of the gene of interest, we can synthesize a probe complementary to it. For example, if the known sequence if G-G-C-T-A-A, then we would synthesize the complementary probe C-C-G-A-T-T. If we make the probe radioactive or fluorescent, the probe will be easy to track, taking us to the proper gene of interest.

gel electrophoresis

Gel electrophoresis is a lab technique used to separate macromolecules, primarily DNA and proteins. The principles of this separation of DNA include: 1. An electric current is applied to the field. DNA is negatively charged and migrates to the positive electrode. 2. A gel made of a polymer is used as a matrix to separate molecules by size. The gel allows smaller molecules to move more easily than larger fragments of DNA. 3. The DNA must be stained or tagged for visualization

genomic library

Gel electrophoresis leads to a genomic library. A genomic library is a set of thousands of recombinant plasmid clones, each of which has a piece of the original genome being studied. A cDNA library is made up of complementary DNA made from mRNA transcribed by reverse transcriptase. This technique rids the gene of introns buy may not contain every gene in the organism

Gene cloning

Gene cloning is the process by which scientists can produce multiple copies of specific segments of DNA that they can then work with in the lab. Many bacteria have DNA outside the main circular chromosome in plasmids.

Using DNA technology for production of pharmaceuticals

Gene splicing and cling can be used to produce large amounts of particular proteins in the lab (for example, human insulin and growth hormone.)

genetic engineering

Genetic engineering is the process of manipulating genes and genomes.

DNA microarray assays

Genome-wide studies of gene expression are made possible by the use of DNA microarray assays. DNA microarray chips work as follows: 1. Small amounts of single-stranded DNA (ssDNA) fragments representing different genes are fixed to a glass slide in a tight grid, termed a DNA chip 2. The mRNA molecules from the cells being tested are isolated and used to make cDNA using reverse transcriptase, then tagged with a fluorescent dye. 3. The cDNA bonds to the ssDNA on the chip, indicating which genes are "on" in the cell (actively producing mRNA). This enables researchers, for example, to see differences in gene expression between breast cancer tumors and noncancerous breast tissue.

animal cloning

In animal cloning the nucleus of an egg is removed and replaced with the diploid nucleus of a body cell, a process termed nuclear transplantation. The ability of a body cell to successfully form a clone decreases with embryonic development and cell differentiation.

Recombinant DNA

Recombinant DNA is DNA that has been artificial made, using DNA from different sources- and often different species. An example is the introduction of a human gene into an E. coli bacterium.

Restriction fragment length polymorphisms (RFLPs)

Restriction fragment length polymorphisms (RFLPs) result from small differences in DNA sequences and can be detected by electrophoresis. The difference in banding patterns after electrophoresis allows for diagnosis of disease or is used to answer paternity and identity questions.

Using DNA technology for environmental cleanup

Scientists engineer metabolic capabilities into microorganisms, which are then used to treat environmental problems, such as removing heavy metals from toxic mining sites.

stem cells

The major goal of most animal cloning is reproduction, but not for humans. In humans, the major goal is the production of stem cells. A stem cell can both reproduce itself indefinitely and, under the proper conditions, produce other specialized cells. Stem cells have enormous potential for medical applications.

Using DNA technology for gene therapy

This is alteration of an afflicted individual's genes. Gene therapy holds great potential for treating disorders traceable to a single defective gene, such as cystic fibrosis.

Using DNA technology for transgenic animals

Transgenic animals are created when eggs are fertilized in vitro and then a desired gene is cloned and inserted into the nucleus of the embryo. If successful, the transgenic animal will express the "foreign" gene, which might be for a human protein that can be produced in large quantities. For example, goats are used to express human antithrombin in milk.

what happens when a DNA molecule is cut

When a DNA molecule is cut by restriction enzymes, the result will always be a set of restriction fragments, which may have at least one single-stranded end, called a sticky end. Sticky ends can form hydrogen bonds with complementary single-stranded pieces of DNA. These unions can be sealed with the enzyme DNA ligase

PCR

pCR (polymerase chain reaction) is a method used to amplify a particular piece of DNA without the use of cells. PCR is used to amplify DNA when the source is impure or scanty (as it would be at a crime scene).

restriction enzymes

restriction enzymes are used to cut strands of DNA at specific locations (called restriction sites). They are mostly derived from bacteria where they serve the important function of protection against invading viruses.

basic steps of the PCR procedure

1. Denaturation: heat briefly to separate DNA strands 2. Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence. 3. Extension: DNA polymerase adds nucleotides to the 3' end of each primer 4. each cycle x^2 molecules are made

Steps to clone a gene

1. identify and isolate the gene of interest and a cloning vector (defined as a DNA molecule that can carry foreign DNA into a host cell and replicate there). The vector will carry the DNA sequence to be cloned and is often a bacterial plasmid. 2. Cut both the gene of interest and the vector with the same restriction enzyme. This gives the plasmid and the human gene matching sticky ends. 3. Join the two pieces of DNA. Form recombinant plasmids by mixing the plasmids with the DNA fragments. The human DNA fragments can be sealed into the plasmid using DNA ligase. 4. Get the vector carrying the gene of interest into a host cell. The plasmids are taken up by the bacterium by transformation. 5. Select for cells that have been transformed. The bacterial cells carrying the clones must be identified or selected. This can be done by linking the gene of interest to an antibiotic resistance gene or a reporter gene such as green fluorescent protein.

Using DNA technology for diagnosis of disease

A number of diseases can be detected by RFLP analysis (for example, cystic fibrosis, sickle-cell disease) or through amplification of blood samples to test for viruses (for example, HIV)

plasmid

A plasmid is a small, circular extra-chromosomal loop of DNA. Plasmids are often used in biotechnology.