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Order in which computer file records are sorted. The sequence is based on a field in the record, such as ZIP code or customer number. The ZIP code is used most often to sequence records because it facilitates lookup from the name and address information on customer correspondence and the printing of mailing labels in zip code sequence.

noun

1. Something brought about by a cause: aftermath, consequence, corollary, effect, end product, event, fruit, harvest, issue, outcome, precipitate, ramification, result, resultant, sequel, sequent, upshot. See cause/effect.
2. A way in which things follow each other in space or time: consecution, order, procession, succession. See order/disorder, precede/follow.
3. A way or condition of being arranged: arrangement, categorization, classification, deployment, disposal, disposition, distribution, formation, grouping, layout, lineup, order, organization, placement. See order/disorder.
4. A number of things placed or occurring one after the other: chain, consecution, course, order, procession, progression, round, run, series, string, succession, suite, train. Informal streak. See order/disorder.

Sequencing refers to the techniques used to determine the order of the constituent bases (i.e., adenine, thymine, guanine, and cytosine) of deoxyribonucleic acid (DNA) or protein. Protein sequencing determines the order of the constituent amino acids. Sequencing is increasingly important in forensic science and in the rapid and positive identification of potential pathogens that can be exploited by bioterrorists.

DNA is typically sequenced for several reasons: to determine the sequence of the protein encoded by the DNA, the location of sites at which restriction enzymes can cut the DNA, the location of DNA sequence elements that regulate the production of messenger RNA, or alterations in the DNA.

The sequencing of DNA is accomplished by stopping the lengthening of a DNA chain at a known base and at a known location in the DNA. Practically, this can be done in two ways. In the first method, called the Sanger-Coulson procedure, a small amount of a specific so-called dideoxynucleoside base is incorporated in along with a mixture of the other four normal bases. This base is slightly different from the normal base and is radioactively labeled. The radioactive base becomes incorporated into the growing DNA chain instead of the normal base, growth of the DNA stops. This stoppage is done four times, each time using one of the four different dideoxynucleosides. This generates four collections of DNA molecule. Also, because replication of the DNA always begins at the same point, and because the amount of altered base added is low, for each reaction many DNA pieces of different length will be generated. When the sample is used for gel electrophoresis, the different sized pieces can be resolved as radioactive bands in the gel. Then, with the location of the bases known, the sequence of the DNA can be deduced. The second DNA sequencing technique is known as the Maxam-Gilbert technique, after its co-discoverers. In this technique, both strands of double-stranded DNA are radioactively labeled using radioactive phosphorus. Upon heating, the DNA strands separate and can be physically distinguished from each other, as one strand is heavier than the other. Both strands are then cut up using specific enzymes, and the different sized fragments of DNA are separated by gel electrophoresis. Based on the pattern of fragments the DNA sequence is determined.

The Sanger-Coulsom is the more popular method. Various modifications have been developed and it has been automated for very large-scale sequencing. During the sequencing of the human genome, a sequencing method called shotgun sequencing was very successfully employed. Shotgun sequencing refers to a method that uses enzymes to cut DNA into hundreds or thousands of random bits. So many fragments are necessary since automated sequencing machines can only decipher relatively short fragments of DNA about 500 bases long. The many sequences are then pieced back together using computers to generate the entire DNA genome sequence.

Protein sequencing involves determining the arrangement of the amino acid building blocks of the protein. It is common to sequence a protein by the DNA sequence encoding the protein. This, however, is only possible if a cloned gene is available. It still is often the case that chemical protein sequencing, as described subsequently, must be performed in order to manufacture an oligonucleotide probe that can then be used to locate the target gene. The most popular direct protein chemical sequencing technique in use today is the Edman degradation procedure. This is a series of chemical reactions, that remove one amino acid at a time from a certain end of the protein (the amino terminus). Each amino acid that is released has been chemically modified in the release reaction, allowing the released product to be detected using a technique called reverse phase chromatography. The identity of the released amino acids is sequentially determined, producing the amino acid sequence of the protein.

Another protein sequencing technique is called fast atom bombardment mass spectrometry, or FAB-MS. This is a powerful technique in which the sample is bombarded with a stream of fast atoms, such as argon. The protein becomes charged and fragmented in a sequence-specific manner. The fragments can be detected and their identify determined. The expense and relative scarcity of the necessary equipment can be a limitation to the technique.

Still another protein sequencing strategy is the digestion of the protein with specialized protein-degrading enzymes called proteases. The shorter fragments that are generated, called peptides, can then be sequenced. The problem then is to order the peptides. This is done by the use of two proteases that cut the protein at different points, generating overlapping peptides. The peptides are separated and sequenced, and the patterns of overlap and the resulting protein sequence can be deduced.

Further Reading

Books

Cirincione, Joseph, Jon B. Wolfsthal, Miriam Rajkuman, Jessica T. Mathews. Deadly Arsenals: Tracking Weapons of Mass Destruction. Washington, DC: Carnegie Endowment for International Peace, 2002.

Periodicals

Balding D. J. "The DNA Database Search Controversy." Biometrics 2002 Mar; 58 (1): 241–4.

Henderson J. P. "The Use of DNA Statistics in Criminal

Trials." Forensic Sci Int. 2002 Aug 28; 128 (3): 183–6.

Mullis, K. B. and F. A. Faloona."Specific Synthesis of DNA in vitro via a Polymerase catalysed Chain Reaction."Methods in Enzymology no. 155 (1987): 335–50.

1. Repetition of the same basic melodic theme at a different pitch. 2. A type of Gregorian chant with non-biblical texts, lines grouped in rhymed pairs, and one note per syllable.

The order in which monomers occur in polymeric molecules; the order of amino acids in a polypeptide chain or of nucleotides in nucleic acid.

• autonomously replicating s. — usually plasmids that replicate independently of chromosomal DNA.
• coding s's — sections of DNA which code for the amino acids of a protein.
• consensus s. — a sequence of nucleotides that is always present in a large set of independently determined sequences. See also box.
• enhancer s. — in DNA transcription, an upstream cis-acting DNA sequence that enhances expression of a particular gene and forms part of a complex array of upstream sequences that control gene expression.
• expressed s's — in eukaryotic pre-messenger RNA the noncoding sequences, also called intervening sequences or introns, are removed in the nucleus; the mRNA is transported to the cytoplasm where the exons are translated to a protein.
• intervening s. — see intron.
• palindromic s. — see palindrome.
• signal s. — a collection of hydrophobic amino acid residues at the amino terminus of secretory or integrated membrane proteins that direct the protein to cell membranes, particularly endoplasmic reticulum where the proteins are modified, e.g. glycosylated, and the signal sequence is removed prior to secretion or integration of the protein into the lumen of the endoplasmic reticulum.
• temporal s. — in protein synthesis, is from the amino to the carboxyl end.

Order of occurrence or performance.

• Cinematography Tools and Techniques - sequence: series of scenes or shots tied together by single idea, concept, or event
• Order, Hierarchy, and Systems - sequence: continuous, connected series

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For the sense of "sequencing" used in electronic music, see the music sequencer article. For sequence learning in cognitive science, see sequence learning.

In genetics and biochemistry, sequencing means to determine the primary structure (sometimes falsely called primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.

Contents 1 DNA sequencing 1.1 Sanger sequencing 1.2 Pyrosequencing 2 RNA sequencing 3 Protein sequencing 4 Polysaccharide sequencing 5 See also 6 References

DNA sequencing

Main article: DNA sequencing

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. Thus far, most DNA sequencing has been performed using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, new sequencing technologies such as Pyrosequencing are gaining an increasing share of the sequencing market. More genome data is now being produced by pyrosequencing than Sanger DNA sequencing. Pyrosequencing has enabled rapid genome sequencing. Bacterial genomes can be sequenced in a single run with several X coverage with this technique. This technique was also used to sequence the genome of James Watson recently.

The sequence of DNA encodes the necessary information for living things to survive and reproduce. Determining the sequence is therefore useful in fundamental research into why and how organisms live, as well as in applied subjects. Because of the key nature of DNA to living things, knowledge of DNA sequence may come in useful in practically any biological research. For example, in medicine it can be used to identify, diagnose and potentially develop treatments for genetic diseases. Similarly, research into pathogens may lead to treatments for contagious diseases. Biotechnology is a burgeoning discipline, with the potential for many useful products and services.

Sanger sequencing

Part of a radioactively labelled sequencing gel

In chain terminator sequencing (Sanger sequencing), extension is initiated at a specific site on the template DNA by using a short oligonucleotide 'primer' complementary to the template at that region. The oligonucleotide primer is extended using a DNA polymerase, an enzyme that replicates DNA. Included with the primer and DNA polymerase are the four deoxynucleotide bases (DNA building blocks), along with a low concentration of a chain terminating nucleotide (most commonly a di-deoxynucleotide). Limited incorporation of the chain terminating nucleotide by the DNA polymerase results in a series of related DNA fragments that are terminated only at positions where that particular nucleotide is used. The fragments are then size-separated by electrophoresis in a slab polyacrylamide gel, or more commonly now, in a narrow glass tube (capillary) filled with a viscous polymer.

View of the start of an example dye-terminator read (click to expand)

An alternative to the labelling of the primer is to label the terminators instead, commonly called 'dye terminator sequencing'. The major advantage of this approach is the complete sequencing set can be performed in a single reaction, rather than the four needed with the labeled-primer approach. This is accomplished by labelling each of the dideoxynucleotide chain-terminators with a separate fluorescent dye, which fluoresces at a different wavelength. This method is easier and quicker than the dye primer approach, but may produce more uneven data peaks (different heights), due to a template dependent difference in the incorporation of the large dye chain-terminators. This problem has been significantly reduced with the introduction of new enzymes and dyes that minimize incorporation variability. This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. The major reason for this is that the primers do not have to be separately labelled (which can be a significant expense for a single-use custom primer), although this is less of a concern with frequently used 'universal' primers. [This is changing rapidly due to the increasing cost-effectiveness of 2nd and 3rd generation systems from Illumina, 454, ABI, Helicos & Dover.]

Pyrosequencing

Pyrosequencing, which was developed by Pål Nyhren and Mostafa Ronaghi, has been commercialized by Biotage (for low throughput sequencing) and 454 Life Sciences (for high-throughput sequencing). The latter platform sequences roughly 100 megabases [now up to 400 megabases] in a 7-hour run with a single machine. In the array-based method (commercialized by 454 Life Sciences), single-stranded DNA is annealed to beads and amplified via EmPCR. These DNA-bound beads are then placed into wells on a fiber-optic chip along with enzymes which produce light in the presence of ATP. When free nucleotides are washed over this chip, light is produced as ATP is generated when nucleotides join with their complementary base pairs. Addition of one (or more) nucleotide(s) results in a reaction that generates a light signal that is recorded by the CCD camera in the instrument. The signal strength is proportional to the number of nucleotides, for example, homopolymer stretches, incorporated in a single nucleotide flow. [1]

RNA sequencing

RNA is less stable in the cell, and also more prone to nuclease attack experimentally. As RNA is generated by transcription from DNA, the information is already present in the cell's DNA. However, it is sometimes desirable to sequence RNA molecules. In particular, in Eukaryotes RNA molecules are not necessarily co-linear with their DNA template, as introns are excised. To sequence RNA, the usual method is first to reverse transcribe the sample to generate cDNA fragments. This can then be sequenced as described above.

For more information on the capabilities of next-generation sequencing applied to whole transcriptomes see: RNA-Seq and MicroRNA Sequencing.

Protein sequencing

Main article: protein sequencing

Methods for performing protein sequencing include:

• Edman degradation
• Peptide mass fingerprinting
• Mass spectrometry
• Protease digests

If the gene encoding the protein can be identified it is currently much easier to sequence the DNA and infer the protein sequence. Determining part of a protein's amino-acid sequence (often one end) by one of the above methods may be sufficient to enable the identification of a clone carrying the gene.

Polysaccharide sequencing

Though polysaccharides are also biopolymers, it is not so common to talk of 'sequencing' a polysaccharide, for several reasons. Although many polysaccharides are linear, many have branches. Many different units (individual monosaccharides) can be used, and bonded in different ways. However, the main theoretical reason is that whereas the other polymers listed here are primarily generated in a 'template-dependent' manner by one processive enzyme, each individual join in a polysaccharide may be formed by a different enzyme. In many cases the assembly is not uniquely specified; depending on which enzyme acts, one of several different units may be incorporated. This can lead to a family of similar molecules being formed. This is particularly true for plant polysaccharides. Methods for the structure determination of oligosaccharides and polysaccharides include NMR spectroscopy and methylation analysis.^[1]

See also

• Genetic code
• Full Genome Sequencing
• Pathogenomics
• RNA-Seq
• MicroRNA Sequencing
• Sequence motif

References

1. ^ A practical guide to structural analysis of carbohydrates

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Dansk (Danish)
n. - rækkefølge, følge, række, kontinuitet, sekvens, orden
v. tr. - anbringe i rækkefølge, bestemme rækkefølgen af

Nederlands (Dutch)
opeenvolging, reeks, achter elkaar zetten

Français (French)
n. - succession, série, séquence, ordre (des événements), numéro de danse, (Mus) séquence, (Comput, Math) séquence, séquence (jeux)
v. tr. - organiser/disposer dans l'ordre d'une séquence

Deutsch (German)
n. - Reihenfolge, Sequenz
v. - ordnen, die Sequenz feststellen

Ελληνική (Greek)
n. - ακολουθία, αλληλουχία, (κινημ.) σεκάνς
v. - βάζω σε σειρά

Italiano (Italian)
sequela, catena, sequenza, serie, successione

Português (Portuguese)
n. - seqüência (f), sucessão (f), resultado (m)
v. - sequenciar

Русский (Russian)
последовательность, последствия, несколько сцен, составляющих эпизод, серия

Español (Spanish)
n. - sucesión, serie, secuencia
v. tr. - seguir una cosa a la otra

Svenska (Swedish)
n. - ordningsföljd, ordning, följd, räcka, rad, serie
v. - följa efter, komma i ordningsföljd, ordna i följd, placera i ordningsföljd

中文（简体）(Chinese (Simplified))
序列, 顺序, 继起的事, 按顺序排好

中文（繁體）(Chinese (Traditional))
n. - 序列, 順序, 繼起的事
v. tr. - 按順序排好

한국어 (Korean)
n. - 잇달아 일어남, 연재물, 순서
v. tr. - (컴퓨터) 배열하다, 차례로 나열하다

日本語 (Japanese)
n. - 連続, 法則に従った順序, 順序, 筋道, 連続物, 続き札, 数列, 帰結, 結果
v. - 順番に並べる

العربيه (Arabic)
‏(الاسم) سلسله متعاقبه, ألمتتاليه (فعل) يرتب بالتعاقب‏

עברית (Hebrew)
n. - ‮רצף, המשך, סדרה, עוקב, סדר, חזרה על אותו רצף-צלילים בדרגת-גובה נמוכה או גבוהה יותר, שם של המנון כנסייתי, סדרת שירים על נושא אחד‬
v. tr. - ‮סידר בסדר מסוים‬

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