Medicine is transforming rapidly. During this era of human history, scientists are forever changing lives, because for the first time humans are not powerless against their genes! We now have the power to not only predict, but also alter our very future. The Genetic Revolution is here!
With the power of gene therapy, scientists now have a new tool that enables them to change a patient's DNA. Medical treatments usually counteract the symptoms of a disease. But with gene therapy, doctors can cure diseases. Click to learn more
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Medicine is transforming rapidly. During this era of human history, scientists are forever changing lives, because for the first time humans are not powerless against their genes! We now have the power to not only predict, but also alter our very future. The Genetic Revolution is here!
Tools, Techniques, and Technologies
The following is a list of tools, techniques, and technologies used by scientists in the field of genomics. They all have tremendous importances in DNA sequencing and analyzing, thus they are fundamental to the field of genomics.
Polymerase chain reaction (PCR) is an extremely fast and inexpensive technique used to basically copy or amplify small segments of DNA. In order to analyze a sample genetically, a large amount of DNA is needed, thus PCR allows studies of small isolated pieces of DNA.
It is now used in multiple laboratory procedures and clinical techniques such as DNA fingerprinting, genetic disorders screening, and detection of bacteria and/or viruses.
First the sample is heated to denature or separate the DNA into two single-stranded piece. Then polymerase, an enzyme, is used to build two new strands of DNA, using the original strands as "templates". This basically duplicates the original DNA. Using this technique over and over again will create two new copies from one old sample. Usually PCR is performed 30 to 40 times on a sample to create more then a billion copies of the original DNA segment.
A machine called a thermocycler direct the entire cycling process of PCR, usually completing it in just a few hours.
(157) PCR Machine
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(159) DNA often looks like this after isolation
Video on Total RNA Extraction
DNA Isolation is the process that extracts DNA from a cell in pure form. The DNA needs to be separated from proteins, RNA, lipids, and other part of the cells. The cells with the DNA are placed in a solution, referred to as a cocktail, which contains enzymes, chemicals, and salts. The enzymes chew the proteins, the chemicals incinerate any RNA, and the salt is used to pull the DNA out of the solution. To harvest the glob-like DNA, a centrifuge is used, which basically spin the tube of glob-like DNA. The DNA collects at the bottom, the solution is poured off, and the dissolved DNA is re- suspended in a second solution. The DNA is now concentrated in a better and easier to work form.
In order for researchers to work with "expressed DNA", or DNA that codes directly for the synthesis of a protein, scientists may sometimes isolate messenger RNA (mRNA). First a cell is ruptured so that the cellular contents can be exposed to synthetic beads coated with thymine nucleotides. Poly(A) mRNA is selectively retained on the beads, while the rest of the cellar components are washed away. The isolated mRNA is converted to single-stranded DNA with the help of the reverse transcriptase enzyme. DNA polymerase, an enzyme, is then used to make the mRNA into a stable double-stranded DNA. This DNA is more stable then the mRNA, and it only represents expressed DNA sequence.
DNA Microarray Technology allows scientists to examine how active thousands of genes are. Using this technology, scientists can classify dfferent diseases base on the level of gene activity.
Robot machines are used to create DNA microarrays. When a gene is activated the cell's machinery will copy only certain part of that gene, resulting in the creation of messenger RNA (mRNA). mRNA is complementary and will bind to the original DNA strand that it was copied from. Scientists collect this mRNA and label each one with a reverse transcriptase enzyme (RT), which generates a complementary cDNA to the mRNA. The cDNA is attached with fluorescent nucleotide, each with different dyes.
The researcher then placed the labeled cDNAs onto a DNA microarray slide. The cDNAs will hybridize (bind) with the synthetic complementary DNA attached on the microarray slide (just like a normal mRNA). This leaves a fluorescent tag on the DNA. A special scanner is then used to measure the fluorescent intensity of each spot/areas on the microarray slide.
The more active a gene is, the more molecule of messenger RNA is produced, resulting in more labeled cDNA hybridizing to the DNA microarray slide. A large number of cDNA hybridizing will result in a very bright fluorescent area. Genes that are not as active will produce less mRNA, resulting in a dimmer fluorescent spot. If there are no fluorescence, then the gene is inactive.
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(165) Reporter Gene Diagram
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How do scientists determine what a gene normally do?
They use a knockout mouse.
A knockout mouse is a laboratory mouse with an inactivated gene. The missing gene causes loss in gene activity, changing the phenotype of a mouse (appearance, physical characteristics, biochemical characteristics, behavior). Because humans share many genes with mice, scientists can gain clues to the effects of a gene. Knockout mice are used as models for cancer, heart diseases, anxiety, aging, arthritis, and much more.
Although knockout mice are an extremely valuable research tool, they are not perfect. Approximately 15 percent of gene knockouts are developmentally lethal, resulting in embryos that can not grow into adult mice. This deficiency in adult mice limits the studies exceptionally to embryonic development. Sometimes the gene will have a different function in adults than in developing embryos. A knockout mouse may also not produce an observable change or characteristics that are observable, or it might differ from characteristics observed in humans with the same deactivated gene.
To create a knockout mouse, scientists use two different strategies. Researchers used these two methods to insert artificial DNA into the chromosomes of embryonic stem cells harvested four days after fertilization. Because embryonic stem cells with a knockout gene will divide with the gene in every type of adult cell, the knockout gene can be observed in every tissue of the mouse.
The first method for inserting artificial DNA is called gene targeting or homologous recombination. Researchers specifically manipulate a gene in the nucleus of an embryonic stem cell by introducing an artificial piece of DNA with the same (homologous) sequence to the gene. The cell's nuclear machinery will recognize that the artificial DNA sequence is identical to the real one in the nucleus, and will swap the real DNA sequence with the artificial sequence. Because the artificial DNA is inactive, its knocks out the function of the gene. Usually the artificial DNA is tagged with a "reporter gene" used for tracking.
In the second method, called gene trapping, researchers randomly insert an artificial DNA bearing a reporter gene into an embryonic stem cell. This artificial DNA prevents the cell's RNA "splicing" machinery from working, preventing the existing gene from producing its proteins (effectively knocking out its function). Scientists can track the activity of the gene to figure out the existing gene's normal activities and patterns.
Because the baby mice have tissues with the altered embryonic stem cells, and tissues without the altered embryonic stem cells, it is necessary to crossbreed the mice to produce lines of mice with both copies of the gene. These mice are called homozygous knockouts.