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
The concepts described in this website might be a review for some people, or it might be a new learning experience. Click in order to learn or review the basics of biology and genetics.
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!
DNA Sequencing and Analysis
DNA Sequencing- The Sanger Method
Through DNA sequencing, scientists can determine the complete sequence of bases, for a particular DNA. This could be used to "scan" for different diseases that are known to researchers.
This DNA sequencing method, called the Sanger Method or "chain termination method", was created by Fred Sanger in 1977.
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The goal of the Sanger Method, is to decode the sequence of A,T,C and G nucleotides in a piece of DNA. First, the template DNA must be copied repeatedly. The copying of the DNA is stopped when special "chain terminators" are added. The reaction can be started at either end of the DNA.
The two strands of DNA are first separated with a primer when complementary nucleotides to the template DNA sequence are added. The complement will bind with the DNA sequence.
DNA polymerase, an enzyme, binds to the primer, and creates a new strand of DNA using nucleotides complementary to the target DNA. The enzyme will continue to extend the DNA strand, until a chemically altered nucleotide is randomly inserted. This nucleotide will end the strand, and the enzyme will fall away.
By repeatedly generating fragments of DNA, and then ending it with the altered nucleotide, a large number of DNA strand can be created.
By now, the fragments of strands are freely floating, each in one of multiple "wells" in a plate.
The plate is loaded into a sequencing machine, where the fragments are sorted by size. The machine is also used to determine when each base had been added.
The samples are transferred into glass capillaries, where an electrical charge forces the DNA molecules to move through a gel matrix.
The longer the fragments, the slower they move compared to the shorter fragments. Therefore, the longest fragments are at the bottom, while the shortest are at the top.
The fragments can then be read from bottom to top, in order to determine the sequence of the DNA.
Genetic mapping (also called linkage mapping) provides clues about the location of a gene on a chromosome, making it the first step in isolating a gene. Genetic mapping is successfully used to find single gene responsible for inherited disorders like muscular dystrophy and cystic fibrosis.
To create a genetic map, researchers will use various laboratory techniques to isolate DNA from the blood or tissue samples of family members where a certain disease or trait is prominent. Scientists will then examine the isolated DNA and analyze the unique patterns of bases appearing only in family members with the trait or disease. These molecular patterns are called polymorphisms (markers). Before identifying the gene responsible for the disease or trait, researchers used DNA markers to find the rough location of a gene on the chromosome.
A genetic process called recombination causes the exchange of the 23 pairs of chromosomes within an egg or sperm. During this process, the closer two genes are, the more likely they will be together during recombination. So if a particular gene is close to a marker, the gene and marker will most likely stay together during the process of recombination. The gene and marker will also be passed on together from parent to child. If each family member with a disease/trait inherits a specific DNA marker, it is very likely that the gene responsible for the disease lie near that marker.
Markers are DNA that does not contain a gene, but can tell the identity of the person a DNA sample came from. The markers most used for genetic maps currently are called microsatellite maps. High resolution maps have also used single-nucleotide polymorphisms (SNPs).
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A genome-wide association study involves rapidly scanning markers across genomes of many people in order to find genetic variations associated with a particular disease. Once the variation is found, scientists can find strategies to detect, treat, and/or prevent the disease.
To carry out this study, researchers used two groups of participants. One of the group contains people with the disease, while the other group contains people without the disease.
Each person's genome is purified from collected blood samples or cells. The genomes are then scanned on machines that quickly survey each participant's genome for strategically selected markers of genetic variation. These genetic variations are called single nucleotide polymorphisms (SNPs).
The genetic variation is "associated" with the disease if it is found more frequently in people with the disease compared to people without the disease.
However, researchers must not assume that the variation directly cause the disease. The variation could simply be located near or "tagging" along with the actual variation responsible for the disease. Because of this, researchers often sequence the DNA base pairs in that particular region of the genome in order to identify the exact genetic change resulting in the disease.
Fluorescence In Situ Hybridization (FISH) is a versatile technique that allows researchers to visualize and essentially map the entire genetic material in the cell of an individual. It is extremely useful for finding the location of a specific gene within an individual's chromosomes.
Fluorescence In Situ Hybridization uses probes, short sequences of single-stranded DNA which matches a portion of a specific gene that scientists want to study. Scientists then label these probes by attaching them with fluorescent dye. The probes' single-stranded DNA will bind to complementary strands of DNA in a person's chromosomes. The fluorescent color allows scientists to see the location of the gene within a chromosome.
To find out where a gene is located on a chromosome, scientists use locus specific probes.
To find out whether an individual has the necessary number of chromosomes, scientists use alphoids, otherwise known as centromeric repeat probes. They are created from repeated sequences of DNA found in the middle of each chromosome. These probe can be used in conjunction with locus specific probes to determine whether an individual is missing any genetic material from specific chromosomes.
Whole chromosome probes are collections of smaller probes, which bind to different sequences along the length of a chromosome. These probes are used to create a full-color map called a spectral karyotype (explained below)
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Spectral karyotyping allows scientists to look at and visualize all of the human chromosomes without confusion. This laboratory technique paints each pair of chromosomes a different fluorescent color. SKY is used to analyze chromosomes and easily figure out abnormalities quickly and efficiently.
To use this technique, scientists first must prepare a large number of probes, short sequences of single-stranded DNA. Each probes are complementary to specific region of one chromosome. All of the probes in this "library" is complementary to all of the chromosomes in the human genome.
Each complementary probe of a chromosome is labeled in a distinct and different color than other probes. For example, all of the probes for chromosome one would be the same color, while all of the probes for chromosome two would be a different color.
The probes are mixed with chromosomes from a human cell, causing them to bind (hybridize) to the DNA of the chromosome. The probes will bind and paint the set of chromosomes in colors. Using computers, scientists can analyze the chromosomes and determine any structural abnormalities.