Electron Microscopy-based DNA Sequencing

DNA Sequencing sprung to life in 1972, when Frederick Sanger at the University of Cambridge, in England began work on the genome sequence using a variation of the recombinant DNA method. The full DNA sequence of a viral genome was completed by Sanger in 1977. However, Sanger's technique of DNA sequencing was inefficient and no serious work beyond this attempt was even considered, due to the vast resources needed to compute a single genome. At the same time, Maxam and Gilbert publish their own "DNA sequencing by chemical degradation" which became an important method of sequencing for many years thereafter.

During the bulk of the 80's little work was done on furthering the science of sequential analysis, but by 1992, most of the computer technology and lab equipment was in place to allow large companies to sequence up to 100,000 base pair DNA strands - but the cost was very high for any sequencing. While progress was not at a standstill it was clear that no massive work could be done without tremendous effort.

A genome map describes the order of genes or other markers and the spacing between them on each chromosome. Human genome maps are constructed on several different scales or levels of resolution. At the coarsest resolution are genetic linkage maps, which depict the relative chromosomal locations of DNA markers (genes and other identifiable DNA sequences) by their patterns of inheritance. Physical maps describe the chemical characteristics of the DNA molecule itself.

The Human Genome Project began in the late 90's as an attempt to sequence what was considered the ultimate achievement, the human genome. Engineers and scientists worldwide gathered to create new methods in the field. Their goals were twofold: to reduce the overall pricetag associated with performing sequencing and to improve the speed and reliability of these techniques.

Each DNA molecule contains many genes -- the basic physical and functional units of heredity. A gene is a specific sequence of nucleotide bases, whose sequences carry the information required for constructing proteins, which provide the structural components of cells and tissues as well as enzymes for essential biochemical reactions. The human genome is estimated to comprise more than 30,000 genes.

The Need for DNA Sequencing

The process of DNA sequencing translates the DNA of a specific organism into a format that is decipherable by researchers and scientists. DNA sequencing has given a massive boost to numerous fields such as forensic biology, biotechnology and more. By mapping the basic sequence of nucleotides, DNA sequencing has allowed scientists to better understand genes and their role in the creation of the human body.

Dye-terminator sequencing utilizes labelling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labeled-primer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labeled with fluorescent dyes, each of which with different wavelengths of fluorescence and emission. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing.

Its limitations include dye effects due to differences in the incorporation of the dye-labeled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace chromatogram after capillary electrophoresis (see figure to the right). This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs". The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects.

orensic biology uses DNA sequences to identify the organism which it is unique to. Although identifying an individual is less accurate currently, but as the processes evolves further, direct comparisons of large DNA segments, and maybe even genomes, will be more practical and viable and will allow precise identification of an individual. Scientists will be able to isolate the genes responsible for genetic diseases like Cystic Fibrosis, Alzheimer’s disease, myotonic dystrophy, etc., which are caused by the inability of genes to work properly.

orensic biology uses DNA sequences to identify the organism which it is unique to. Although identifying an individual is less accurate currently, but as the processes evolves further, direct comparisons of large DNA segments, and maybe even genomes, will be more practical and viable and will allow precise identification of an individual. Scientists will be able to isolate the genes responsible for genetic diseases like Cystic Fibrosis, Alzheimer’s disease, myotonic dystrophy, etc., which are caused by the inability of genes to work properly.