How to determine the sequence of bases in a DNA Sequenceing molecule and Key principles

DNA sequencing is the process of determining the exact order of the bases A, T, C and G in a piece of DNA. In essence, the DNA is used as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA.

The most commonly used method of sequencing DNA - the dideoxy or chain termination method - was developed by Fred Sanger in 1977 . The key to the method is the use of modified bases called dideoxy bases; when a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, sequencing DNA stops the replication reaction. The wells are cleaned by circulating buffer into the wells with a syringe and, immediately prior to the loading of each sample, the urea in each well is suctioned out with a mouth pipette.

A DNA molecule carries information in the form of four chemical groups or bases, represented by the letters A, C, G and T. The order of bases on a DNA strand is the DNA sequence. Most DNA sequencing is carried out using the chain termination method. This involves the synthesis of new DNA strands on a single stranded template and the random incorporation of chain-terminating nucleotide analogues sequencing DNA. Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid real-time sequence determination. This technique employs four enzymatic reactions in a single tube to monitor DNA synthesis.

The most obvious application of DNA sequencing technology is the accurate sequencing of genes and genomes. Only about 500-800 bases can be sequenced in one experiment so larger DNA molecules, including whole genomes, must be broken into smaller fragments before sequencing and then reassembled by searching for overlaps. Accuracy is achieved by sequencing each template several times of sequencing DNA.

details on how DNA Sequencing works- Methods for Sequencing DNA

DNA sequencing is one of the most important platforms for the study of biological systems today.DNA Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids.

One of the major problems in DNA cycle sequencing is that when fluorescent primers are used the reaction conditions are such that the nested fragment set distribution is highly dependent upon the template concentration in the reaction mix. DNA Sequence Prior to taping, these glass plates are cleaned with Alconox detergent and hot water, are rinsed with double distilled water, and dried with a Kimwipe. Typically, the notched glass plate is treated with a silanizing reagent and then rinsed with double distilled water. After pouring, the gel immediately is laid horizontally and a well forming comb is inserted into the gel and held in place by metal clamps.

We have recently observed that the nested fragment set distribution for the DNA cycle sequencing reactions using the fluorescent labeled terminators (8) is much less sensitive to DNA concentration than that obtained with the fluorescent labeled primer reactions as described above. In addition, the fluorescent terminator reactions require only one reaction tube per template while the fluorescent labeled primer reactions require one reaction tube for each of the four terminators.

DNA sequencing reactions are just like the PCR reactions for replicating DNA (refer to the previous page DNA Denaturation, Annealing and Replication). The reaction mix includes the template DNA, free nucleotides, an enzyme and a 'primer' - a small piece of single-stranded DNA about 20-30 nt long that can hybridize to one strand of the template DNA.