sequencing DNA - Wikipedia, the free encyclopedia
12/8/07 3:17 PM
reactions are still required, but DNA fragments with dye labels can be read using an optical system, facilitating faster and more economical analysis and automation. This approach is known as 'dye-primer sequencing'. The later development by L Hood and coworkers[6][7] of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing. The different chain-termination methods have greatly simplified the amount of work and planning needed for DNA sequencing. For example, the chain-termination-based DNA fragments can be "Sequenase" kit from USB Biochemicals labeled by using a radioactive contains most of the reagents needed for or fluorescent tag on the primer (1), in the new DNA sequencing, prealiquoted and ready to use. strand with a labeled dNTP, Some sequencing problems can occur with or with a labeled ddNTP. the Sanger Method, such as non-specific (click to expand) binding of the primer to the DNA, affecting accurate read out of the DNA sequence. In addition, secondary structures within the DNA template, or contaminating RNA randomly priming at the DNA template can also affect the fidelity of the obtained sequence. Other contaminants affecting the reaction may consist of extraneous DNA or inhibitors of the DNA polymerase.
Dye-terminator sequencing
Sequence ladder by radioactive sequencing compared to fluorescent peaks (click to expand)
An alternative to primer labelling is labelling of the chain terminators, a method commonly called 'dye-terminator sequencing'. The major advantage of this method is that the sequencing can be performed in a single reaction, rather than four reactions as in the labelledprimer method. In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with a different fluorescent dye, each fluorescing at a different wavelength. This method is attractive Capillary electrophoresis because of its greater expediency and speed and is now the mainstay in (click to expand) automated sequencing with computer-controlled sequence analyzers (see below). Its potential limitations include dye effects due to differences in the incorporation of the dye-labelled 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 largely been overcome with the introduction of new DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for eliminating "dye blobs", caused by certain chemical characteristics of the dyes that can result in artifacts in DNA sequence traces. The dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is now being used for the vast majority of sequencing projects, as it is both easier to perform and lower in cost than most previous sequencing methods.
Automation and sample preparation
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DNA sequencing - Wikipedia, the free encyclopedia
12/8/07 3:17 PM
Modern automated DNA sequencing instruments (DNA sequencers) can sequence up to 384 fluorescently labelled samples in a single batch (run) and perform as many as 24 runs a day. However, DNA automated sequencers carry out only DNA size separation by capillary electrophoresis, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms. Sequencing reactions by thermocycling, cleanup and resuspension in a buffer solution before loading onto the sequencer are performed separately.
View of the start of an example dye-terminator read (click to expand)
Large-scale sequencing strategies
Current methods can directly sequence only relatively short (300-1000 nucleotides long) DNA fragments in a single reaction. [3] (http://www.appliedbiosystems.com/catalog/myab/StoreCatalog/products/CategoryDetails.jsp? hierarchyID=102&category1st=a50&category2nd=a51&category3rd=111907) . The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide. Limitations on ddNTP incorporation were largely solved by Tabor at Harvard Medical, Carl Fuller at USB biochemicals, and their coworkers[8] . Large-scale sequencing aims at sequencing very long DNA fragments. Even relatively small bacterial genomes contain millions of nucleotides, and the human chromosome 1 alone contains about 246 million bases. Therefore, some approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA is cloned into a DNA vector, usually a bacterial plasmid, and amplified in Escherichia coli. The amplified DNA can then be purified from the bacterial cells (a disadvantage of bacterial clones for sequencing is that some DNA sequences may be inherently un-clonable in some or all available bacterial strains, due to deleterious effect of the cloned sequence on the host bacterium or other effects). These short DNA fragments purified from individual bacterial colonies are then individually and completely sequenced and assembled electronically into one long, contiguous sequence by identifying 100%-identical overlapping sequences between them Genomic DNA is fragmented (shotgun sequencing). This method does not require any pre-existing into random pieces and cloned information about the sequence of the DNA and is often referred to as de as a bacterial library. DNA novo sequencing. Gaps in the assembled sequence may be filled by Primer from individual bacterial walking, often with sub-cloning steps (or transposon-based sequencing clones is sequenced and the sequence is assembled by depending on the size of the remaining region to be sequenced). These using overlapping strategies all involve taking many small reads of the DNA by one of the regions.(click to expand) above methods and subsequently assembling them into a contiguous sequence. The different strategies have different tradeoffs in speed and accuracy; the shotgun method is the most practical for sequencing large genomes, but its assembly process is complex and potentially error-prone - particularly in the presence of sequence repeats. Because of this, the assembly of the human genome is not literally complete -- the repetitive sequences of the centromeres, telomeres, and some other parts of chromosomes result in gaps in the genome assembly. Despite having only
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