Genotyping Tools

Genetic researchers have combined efforts to determine the entire genetic sequence, 9 billion bases, found in the human genome. The genome is the DNA content found in every human cell consisting of repeating nitrogenous bases labeled as "A," "G," "C" and "T." Genetics research continues to look for variation in different human genomes as well as different species for evolutionary studies. Medical genetic studies are interested in locating genetic abnormalities or genetic variation that could cause disease. Genotyping is a method for determining genetic variation without sequencing an entire genome. Researchers isolate small regions of a genome to look for size differences, the result of different number of nitrogenous bases. Different base number can be caused by a genetic abnormality that results in diseases such as diabetes, Alzheimer's and cancer. The tools used to perform genotyping applications have evolved and developed to rapidly and accurately determine an individual genotype.

The Polymerase Chain Reaction

• The evolution of genetic technology originated from development of the Polymerase Chain Reaction, or PCR. This method allows researchers to amplify or make copies of small regions of a genome within hours. The region of interest is determined by the addition of short DNA molecules --- primers that match the beginning and end of the region of interest.

  PCR has become an invaluable tool in genetic research. Amplifying the same region from different individuals provides a method of comparison. Forensics identification currently uses 15 separate regions for comparison. One region can match in different people. However, no two people should match all 15 regions.

Agarose Gel Electrophoresis

• PCR is the method for amplifying specified regions of DNA. However, it does not provide a method to compare actual size of the fragments. Products of PCR are separated by size using a gel-like material called "agarose." Smaller fragments migrate more quickly across agarose in response to an electrical current in a process called "agarose gel electrophoresis."

  Following PCR, fragments are loaded on the agarose and migrate along the gel when an electrical current is applied. Ethidium bromide allows fragments to be seen when under ultraviolet light. Agarose gel electrophoresis provides a method for quick determination of successful PCR as well as approximates fragment size. However, the disadvantage of agarose as a tool for genotyping is that fragments must be substantially different in size for accurate results. Agarose does not provide a good medium for comparing fragments different by a single base.

Automated Sequencers and Genetic Analyzers

• Fluorescent labeled DNA fragments recorded by an automated sequencer.

  Automated sequencers and genetic analyzers have become the main tool used in genotyping. These allow fragments, different by a single nitrogenous base, to be determined quickly and inexpensively. As with agarose gel electrophoresis, DNA fragments are first amplified by PCR. However, one primer is tagged with a fluorescent dye adding color to the fragment. Samples are separated according to size by migrating through a gel-like polymer in response to an electrical current. Smaller fragments move more rapidly than larger fragments. As the fragments migrate towards the end of the polymer, a digital camera records the color and sends the information to a computer for analysis. Automated sequencing equipment is currently used in forensic identification and paternity testing.

Next Generation Sequencers also Determine Genotypes

• Next generation sequencing instruments have rapidly emerged during the since 2006. This technology combines PCR amplification and sequencing together in order to determine millions of bases at one time. The process begins with PCR; however, different primers are bound to beads mixed in one reaction allowing thousands of regions to be amplified at one time.

  Next generation sequencers then separate the fragments by size and allow a multitude of DNA regions to be analyzed. The method is disadvantageous as a genotyping tool when researchers are interested in comparing a single region of the genome. The advantage is that the genome isolated from a single cell provides enough material for PCR amplification. Comparing the genotypes of normal and abnormal cells isolated from a single individual can help identify cancerous cells and potential treatments.