How is sequencing performed

Electrodes are placed at either end of the gel and an electrical current is applied, causing the DNA molecules to move through the gel. Smaller molecules move through the gel more rapidly, so the DNA molecules become separated into different bands according to their size. Until the late s, electrophoresis gels were always read by a person. Each piece of DNA was attached to a radioactive label, and an X-ray picture was made of the gel to make the positions of the DNA bands visible. Painstakingly analyzing the rows and columns of bands on the gel, a person could determine the sequence of the DNA.

But this process was slow, tedious, and fraught with error. Today's large-scale sequencing projects would be impossible without automatic sequencing machines, which became commercially available in the late s and have made DNA sequencing much quicker and more reliable.

In one year, a person can produce a finished sequence of 20, to 50, bases; a machine can produce a rough draft of a sequence that long in just a few hours. Most automatic sequencing machines have a design based closely on the original, manual sequencing process. To run the machine, a technician pours gel into the space between two glass plates set less than half a millimeter two-hundredths of an inch apart.

As the DNA pieces move through the gel, the sequencing machine reads the order of DNA bases and stores this information in its computer memory. But just like the slab-gel machines, capillary machines read the base sequence as DNA moves through the gel. Capillary sequencers can sequence each piece of DNA about twice as fast as slab-gel machines.

Most large-scale sequencing projects use a combination of slab-gel and capillary machines. Sequencing machines can't "see" DNA directly, so scientists must use a complex set of procedures to prepare DNA for sequencing.

When DNA is finally in a form that the machines can read, it has been chopped up, copied, chemically modified, and tagged with fluorescent dyes corresponding to the four different DNA bases, or genetic letters.

Before it is sequenced, a piece of DNA is copied many times, then divided into four batches in preparation for another round of copying. When one of these modified bases is incorporated into a DNA molecule, the chain of bases stops growing.

The result of all this is that one batch of DNA will contain only pieces that end in T, another only pieces that end in A, a third only pieces that end in G, and the fourth batch only pieces that end in C. In the second round of copying, a different fluorescent dye is also added to each batch of DNA.

Unlike sequencing methods currently in use, nanopore DNA sequencing means researchers can study the same molecule over and over again. Researchers now are able to compare large stretches of DNA - 1 million bases or more - from different individuals quickly and cheaply. Such comparisons can yield an enormous amount of information about the role of inheritance in susceptibility to disease and in response to environmental influences. In addition, the ability to sequence the genome more rapidly and cost-effectively creates vast potential for diagnostics and therapies.

Although routine DNA sequencing in the doctor's office is still many years away, some large medical centers have begun to use sequencing to detect and treat some diseases. In cancer, for example, physicians are increasingly able to use sequence data to identify the particular type of cancer a patient has. This enables the physician to make better choices for treatments.

Other researchers are studying its use in screening newborns for disease and disease risk. Another National Institutes of Health program examines how gene activity is controlled in different tissues and the role of gene regulation in disease. Ongoing and planned large-scale projects use DNA sequencing to examine the development of common and complex diseases, such as heart disease and diabetes, and in inherited diseases that cause physical malformations, developmental delay and metabolic diseases.

Comparing the genome sequences of different types of animals and organisms, such as chimpanzees and yeast, can also provide insights into the biology of development and evolution. Draft and Complete Genomes. Figure 3: Examples of cyclic array sequencing and sequencing by hybridization.

Left: repeated cycles of polymerase extension with a single nucleotide at each step. The means of detecting incorporation events at individual array features varies from method to method.

Right: an example of raw data from Pyrosequencing, a cyclic-array method. The identity of nucleotides used at each extension step are listed along the x-axis. The y-axis depicts the measured signal at each cycle for one sequence; both single and multiple such as homopolymeric incorporations can be distinguished from non-incorporation events. The decoded sequence is listed along the top. To resequence a given base, four features are present on the microarray, each identical except for a different nucleotide at the query position the central base of bp oligonucleotides.

Genotyping data at each base are obtained through the differential hybridization of genomic DNA to each set of four features. Cutler, D. High-throughput variation detection and genotyping using microarrays.

Genome Research 11 , — Figure Detail. References and Recommended Reading Carlson, R. Analytical Biochemistry , — Metzker, M. Genome Research 15 , — Reinders, J. Article History Close. Share Cancel. Revoke Cancel. Keywords Keywords for this Article. Save Cancel. Flag Inappropriate The Content is: Objectionable. Flag Content Cancel. Email your Friend. Submit Cancel. This content is currently under construction. Explore This Subject.

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