Primary DNA Analysis Techniques That Have Been Used Since 1985

Genetics-based research is one of the more rapidly evolving scientific disciplines today. Early technology began with techniques using radioactive labels for DNA sequencing, identification of individual bases that make up DNA. DNA is the blueprint for every living organism, including viruses. It is formed from millions or billions of repeating units of four nitrogenous bases, designated as A for adenosine, G for guanosine, C for cytosine and T for thymine. Humans contain approximately 9 billion of these bases, repeating without a distinctive pattern. Three bases together symbolize an amino acid. A chain of amino acids determines a protein. The complement of different proteins determines the characteristics of a living organism called a phenotype. DNA analysis techniques are used to determine DNA sequences in order to understand how living organisms develop, and the mistakes in sequence that cause disease like cancer. Technology advanced rapidly after the development of PCR in 1985.

Polymerase Chain Reaction

• The polymerase chain reaction, or PCR, is perhaps the most important scientific breakthrough in genetics research. Kary Mullins invented PCR in 1985. The PCR process allows scientists to amplify specific areas of DNA, producing millions of copies within hours. PCR employs a heat-stable enzyme called TAQ polymerase, isolated from a bacteria species called Thermus aquaticus, found living in hot springs. In the presence of raw materials, TAQ polymerase synthesizes duplicate copies of DNA using the original DNA as a template. Researchers can determine the exact area of DNA to be amplified by including 20 base strands of DNA called primers. Primers initiate amplification by pairing, or annealing, to a matching set of bases on the DNA template. All new technologies developed since 1985 require some derivative of PCR amplification.

DNA Sequencing Techniques

• DNA sequencing determines the exact order of the nitrogenous bases. Early development of sequencing methods tagged each base with a radioactive label during PCR. The amplified DNA would be separated by an electrical current and move through a gel-like material called polyacrylamide. The technology was limited by the fact that each base composition was determine in separate lanes because radioactive labels appear the same when read by X-ray photography. One lane on the gel was used for each base. Development of fluorescent dyes automated the technology in the early 1990s, and each base was labeled with a different color. As the bases moved through the gel, a digital camera recorded the colors and sent the data to an attached computer system. Automated sequencing allowed up to 700 bases to be determined, versus the 200 base limitation for radioactive labels.

Development of Capillary Sequencing

• Around 1997, DNA sequencing techniques were further developed by replacing messy glass plates and polyacrylamide with glass capillaries. Researchers no longer needed to pour acrylamide between two glass plates and wait for formation of gel-like polyacrylamide before sequencing. Sequencing instead was performed using a thick syrup-like polymer derivative of polyacrylamide that was syringe- injected into hollow glass fibers. Samples are still amplified using PCR and fluorescent dyes, then automatically loaded into individual capillaries for sequencing. The result was more automation in techniques and the capacity to sequence greater number of samples in less time. The majority of the human genome, all 9 billion bases, was determined using capillary sequencing.

Real Time PCR

• After determining the DNA sequence for a specific organism, the next goal in research was to look for variations between organisms of the same and different species. One reason is to analyze differences and similarities in DNA sequence from different species; why humans are human and gorillas are not. Another reason is to use genetic techniques to determine mistakes, or mutations, that cause genetic diseases. Real time PCR employs technology similar to PCR that incorporates an additional fluorescent-labeled primer to mark a specific sequence. Any mistake in the DNA prevents the marker from annealing to the DNA strand. The ability for the marker to anneal is measured during PCR to determine whether a mutation is present.

Microchip Array Technology

• Microchip array analysis was developed soon after real time PCR and is primarily used for gene expression, or to determine what genes in a cell are active. Not every gene in the genome is active. The activation of specific genes determines the function of different types of cells in complex organisms; why skin cells are not liver cells, for example. Researchers isolate the product of active genes in the form of messenger RNA, or mRNA, and use PCR techniques to produce a DNA complement. The sample DNA is spotted on a plate with labeled probes that will fluoresce in the presence of DNA. The plates used today can concurrently test over 30,000 samples at one time.

Next Generation Sequencing

• The most recent development in DNA analysis technologies is next generation sequencers. The process is not unlike normal sequencing. However, the equipment is capable of determining entire genomic sequences isolated from bacteria, 2 million bases at one time. The process incorporates emulsion PCR, a technique that uses DNA encapsulated on a micro-bead or oil droplet. It greatly improves the efficiency of normal PCR techniques and allows multiple areas of DNA to be amplified simultaneously. The amplified DNA is then sequenced using specialized next generation sequencers. With the development of technology used in genetic research, genetics has become one of the most rapidly developing areas of scientific research.