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DNA sequencing methods
The area of biotechnology is of continuous change. Rapid development and growth of constant cutting-edge research is usually dependent on the creativity and innovation of scientists as well as their ability to foresee potential in a fundamental molecular technique in order to apply it into certain new processes (Janitz, 2011). Introduction of PCR opened up numerous doors in research of genetics, such as means of DNA identification and analysis of various genes based on DNA sequences. Furthermore, DNA sequencing is depended upon ability of scientists to use gel electrophoresis in order to separate DNA strands that differ in their size very slightly, by just less as one base pair (Graham & Hill, 2001).
DNA sequencing refers to the process of determining order of nucleotides contained ion a gene. Earliest methods of DNA sequencing were complex and time consuming. However, a major breakthrough appeared in 1975 when a process known as Sanger sequencing was developed (Hindley, 2000). Sanger sequencing is given this name after Fredrick Sanger, an English biochemist. Sanger sequencing is sometimes known as dideoxy sequencing or chain-termination sequencing. About 25 years after this sequencing was created, it was used to sequence genome of human beings, and, with addition of numerous technological modification and improvements, Sanger method today remains important in numerous laboratories in many parts across the globe (Janitz, 2011).
In late 1970’s, Sanger method was invented alongside another methods referred to as Maxam-Gilbert or chemical cleavage method. Maxam-Gilbert is based on specific cleavage by chemicals. This method is effectively used to various oligonucleotides, which are usually short nucleotide polymers (Hindley, 2000). Sanger method is mostly used mainly because it has been technically proven to be easier to apply. With automation of this technique and advent of PCR, Sanger method is easily applied especially to long DNA strands including certain entire genes. This method is mainly based on what is termed as chain termination during elongation reactions of PCR (Kieleczawa, 2006).
In Sanger method, strand of DNA to be analyzed is used together with DNA polymerase, in PCR reaction, in order to generate certain complimentary strands with use of primers. Four separate PCR reaction mixtures are then prepared, each having certain percentage level of dideoxynucleoside triphosphate analogs to just one of those four nucleotides (Graham & Hill, 2001). Synthesis of a new strand of DNA goes on until one of those analogs is included. At that time, strand of DNA is truncated prematurely. Each PCR reaction eventually end up encompassing mixture of various lengths of strands, all of them ending up with nucleotide, which was dideoxy labeled specially for that reaction (Janitz, 2011). In addition, electrophoresis is hence used so as to separate strands of those four reactions, in just four different lanes, in order to determine sequence of original template, on basis of what lengths of strands eventually end with what kind of nucleotide (Kornacki, 2010).
The diagram above shows polymerase chain reaction (Hindley, 2000).
Furthermore, in automated Sanger reaction, the primers that are used are normally labeled with four separate colored fluorescent tags. In presence of various dideoxy nucleotides, PCR reactions are conducted as described above. Eventually, those four reaction mixtures are applied and combined to just one lane of a gel (Janitz, 2011). Color of each separate fragment is detected with the use of a laser beam. Information is hence collected using a computer that generates chromatograms, displaying peak of each color, in which DNA sequence of the template can be determined. Generally, automated sequencing is only precise and accurate for sequences of up to a maximum of approximately 700 to 800 base-pairs in length (Graham & Hill, 2001). But it is possible to get full sequences of other larger genes and whole genomes, through the use of step-wise methods that include Shotgun and primer walking sequencing (Kornacki, 2010).
In the primer walking sequencing, a workable segment of a bigger gene is sequenced mainly using Sanger method. Other new primers are produced from a reliable portion of the sequence, and further used in order to continue sequencing the segment of gene which was out of range of the initial reactions (Janitz, 2011). Shotgun sequencing on the other hand entails cutting DNA portion of interest randomly into more manageable and appropriate sized fragments, sequencing of each fragment, as well as arranging the segments based on certain overlapping sequences. This method made effective and easier mainly by application of certain computer software that help in arranging those overlapping pieces (Graham & Hill, 2001).
Regardless of the method to the genome as whole, real process of sequencing DNA is the same. The technique that is used in sequencing is referred to as electrophoresis to different DNA pieces that usually differ in length (Janitz, 2011). In electrophoresis process, DNA which is being sequenced is normally placed on a gel. Electrodes are then placed at a gel and electrical current is then applied, making DNA molecules to go through the gel. Molecules which are smaller move through gel faster, so DNA molecules are hence separated into various bands in accordance to their size (Hindley, 2000).
Until late 1980s, gels were usually read by an individual. Each piece of DNA used to be attached to radioactive label. An X-ray picture could be made of a gel in order to make positions of the bands to be visible (Kieleczawa, 2006). Painstakingly analyzing columns and rows of band on gel, an individual could determine DNA sequence. However, this process was tedious, slow as well as fraught with error. Large-scale sequencing projects of today could be impossible without various automated sequencing machines that became available in 1980s, and has made sequencing process to be more reliable and much quicker (Graham & Hill, 2001).
Most sequencing machines have been designed mainly based on initial, manual sequencing process. So as to run the machine, technician usually pours gel into a certain space that located between two plates and set apart in less than half millimeter. After gel sets, the DNA is hence loaded into various lanes (Hindley, 2000). As pieces of DNA go through the gel, sequencing machines usually reads order of bases as well as stores that information in computer memory. However, in certain newer machines referred to as capillary sequencers, DNA moves through array of gel-filled capillaries rather than through slab of the gel. But just as slab-gel machines, these newer machines read sequence of the base as DNA goes through the gel (Kieleczawa, 2006).
The above picture shows a lab with sequencing machines (Janitz, 2011).
Capillary sequencers have potential of sequencing each DNA piece about twice as fast as compared to slab-gel machines (Graham & Hill, 2001). In addition, these machines are fully automated. It has a robotic arm that places DNA into top capillaries. It automatically fills capillaries with the gel as well as cleans gel between runs. These machines require less attention of a person (Pierce, 2008). However, these sequencing machines are very expensive and they are new such that some labs normally have trouble obtaining them in order to work as utmost efficiency (Kornacki, 2010). But most large-scale sequencing projects usually use combination of capillary and slab-gel machines.
In current years, technology of DNA sequencing has advanced numerous areas of science. For instance, the area of functional genomics is focusing on figuring out what various DNA sequences do, and which DNA pieces code for proteins as well as which have significant regulatory functions. An invaluable step in making such determination is studying nucleotide sequences of various DNA segments (Hindley, 2000). Another field of science that mainly rely on DNA sequencing is what is referred to as comparative genomics, whereby researchers normally compare genetic material of various organisms so as to learn more about their history of evolution as well as degree of relatedness (Janitz, 2011). Furthermore, DNA sequencing has helped in the research of complex disease by allowing researchers to catalogue some genetic variations among individuals which can influence their susceptibility to certain different conditions (Kieleczawa, 2006).
At individual level, the ongoing biomedical research into course and cause of human diseases and illnesses is primed to improve healthcare greatly (Janitz, 2011). Application of sequencing to identification of various disease-causing genetic factors will result in expansion and improvement of genetic testing, and also to development of more personalized, targeted drug therapies in future (Pierce, 2008). Currently, advantages of DNA sequencing are noticeable in agriculture. Thanks to development of disease-resistant animals and plants. Furthermore, microbial genome sequencing projects might someday result in development of certain new biofuel as well as pollutant-monitoring systems (Kornacki, 2010).
In addition, DNA sequencing technology is used in forensic science, and has helped greatly in providing important evidence in many criminal cases (Kieleczawa, 2006). For example, in United States, Federal Bureau of Investigation operates and funds a national database, encompassing genetic profiles of various known offenders which may be searched whenever evidence of DNA is obtained in any crime scene. According to FBI, as of the year 2008, this national database had profiles of more than 6.5million offenders and had further helped in about 81,000 investigations (Janitz, 2011).
Therefore, DNA sequencing helps scientists to determine sequence of genome. Human genome project can be said to be the greatest example of what is called DNA sequencing. When human genome was initiated in 2001, numerous issues rose. However, today people can see impacts of Human genome project on pharmaceutical and medical research. Scientists are currently able to identify various genes responsible for causing certain diseases such as Cystic fibrosis, Alzheimer’s disease, myotonic dystrophy as well as other many diseases caused by genes disability to function properly (Hindley, 2000). Furthermore, many forms of acquired diseases such as cancer can be detected by examining certain genes.
DNA sequencing is applied in various industries and field. The first area where this technology is applied is in forensics. These methods for a long time have been used in forensics science to help crime detectives to identify individuals because each person has a unique and different sequence of his or her DNA (Graham & Hill, 2001). DNA sequencing is mainly used to identify criminals by finding some evidence from crime scene through form of nail, skin, hair or blood samples among others. In addition, this method is also used to determine paternity of a child. It also identifies various protected and endangered species (Kornacki, 2010).
In medicine field, DNA sequencing may be used to detect genes that are linked with some acquired and heredity diseases. Scientists normally used various methods of genetic engineering such as gene therapy in order to identify defected genes so as to replace such genes with healthy ones (Graham & Hill, 2001). In field of agriculture, DNA sequencing methods have played important role. The sequencing and mapping of entire microorganism’s genome has allowed agriculturists to make that genome useful for food plants and other crops (Kieleczawa, 2006). For instance, specific genes of certain bacteria have been effectively used in certain food plants in order to increase their tolerance and resistance against pests and insects and as a result, nutritional value and productivity of some plants have increased. Such plants can now fulfill need of food sufficiency in the poor nations (Janitz, 2011). Furthermore, DNA sequencing has been useful in development of livestock with improved milk and meat.
While such technologies provide a glimpse at future of sequencing, observers in various industries question how they will be able to deliver their promises. Hopefully, these new technologies will be able to make DNA sequencing cheaper (Kornacki, 2010). Companies that started commercial DNA sequencing are focusing on the future of this technology. For example, Applied Biosystems group is currently working on new usage and application of its capillary electrophoresis-based instruments, with special interest in pharmacogenics (Pierce, 2008). This company is focusing on making instruments to be more economical for sequencing.
Introduction of internet accessible databases such as MLST database will enhance global and large-scale tracking and surveillance of bacterial food pathogens and large-scale evolution studies (Janitz, 2011). Sequencing of various housekeeping genes as well provide a chance to probe evolutionary linkages of bacteria without confounding impacts of adaptive selection normally occurring among certain virulence genes (Kornacki, 2010). However, sequencing of such virulence genes may provide important information on evolutionary characteristics among groups of various bacterial pathogens. As DNA sequencing is continually becoming more broad accessible and less expensive, as well as DNA chip-based sequencing methods are being developed, use of sequencing data for evolutionary studies and bacterial sub-typing will further continue to expand (Pierce, 2008).
Current advances in genomic technologies and genomics have led to development of various new sub-typing methods likely to be effectively applied in food industry (Kornacki, 2010). Technologies which most likely may translate into application in agriculture and food industry include, full genome DNA sequencing methods, microarrays, as well as high-through put sole nucleotide polymorphism detection approaches (Janitz, 2011). Such technologies allow reproducible and rapid subtype characterization mainly by detecting large numbers of polymorphism such as DNA sequencing usually on a chip, and detection of gene absence or presence polymorphism, through use of various genomic microarrays. Furthermore, full genome DNA sequencing data will also enhance development of numerous other discriminatory sub-typing methods like multilocus variable tandem repeat analysis, a technique that is currently used in investigation of peanut butter outbreak (Pierce, 2008).
In conclusion, DNA sequencing methods have played a great role and have resulted in improvement of medical, forensics and agricultural industries. After analyzing DNA data with a computer, scientists normally use that information for numerous experiments. DNA sequencing methods can help to solve various murder mysteries, determine if and how species are related, as well as sequence human genome (Hindley, 2000). DNA sequencing for a long time have been used in forensics science to help crime detectives to identify individuals because each person has a unique and different sequence of his or her DNA. DNA sequencing is mainly used to identify criminals by finding some evidence from crime scene through form of nail, skin, hair or blood samples among others. In medicine field, DNA sequencing may be used to detect genes that are linked with some acquired and heredity diseases (Kieleczawa, 2006). Scientists normally used various methods of genetic engineering such as gene therapy in order to identify defected genes so as to replace such genes with healthy ones. In field of agriculture, DNA sequencing methods have played important role. The sequencing and mapping of entire microorganism’s genome has allowed agriculturists to make that genome useful for food plants and other crops (Graham & Hill, 2001). Therefore, DNA sequencing methods are very important in many fields and industries, as well has contributed much in improving welfare of many people across the globe.
Graham, C. A. & Hill, A. M. (2001). DNA Sequencing Protocols. London: Springer.
Hindley, J. (2000). DNA Sequencing. Amsterdam: Elsevier.
Janitz, M. (2011). Next-Generation Genome Sequencing: Towards Personalized Medicine. Hoboken: John Wiley & Sons.
Kieleczawa, J. (2006). DNA Sequencing II: Optimizing Preparation and Cleanup. Burlington: Jones & Bartlett Learning.
Kornacki, J. L. (2010). Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. London: Springer.
Pierce, L. T. (2008). Computational Modeling of DNA Sequence Effects on the Nucleosome Core Particle. Ann Arbor: ProQuest.