Nucleic acid sequence

The sequence, or primary structure of a nucleic acid is the exact specification of its atomic composition and the chemical bonds connecting those atoms. As nucleic acids, e.g. DNA and RNA, are unbranched polymers, this is equivalent to specifying exact sequence of nucleotides that comprise the whole molecule. This sequence is written as a succession of letters representing a real or hypothetical DNA molecule or strand. By convention, the primary structure of a DNA or RNA molecule is reported from the 5' end to the 3' end.

The sequence has capacity to carry information. When used in reference to biological DNA, which carries the information which directs the functions of living beings, the term genetic sequence is often used. Sequences can be read from the biological raw material through DNA sequencing methods.

Primary structure is sometimes mistakenly termed primary sequence, but there is no such term, as well as no parallel concept of secondary or tertiary sequence.

Nucleotides

Chemical structure of RNA

Nucleic acids consist of a chain of linked units called nucleotides. Each nucleotide consists of three subunits: a phosphate group and a sugar (ribose in the case of RNA, deoxyribose in DNA) make up the backbone of the nucleic acid strand, and attached to the sugar is one of a set of nucleobases. The nucleobases are important in base pairing of strands to form higher-level secondary and tertiary structure such as the famed double helix.

The possible letters are A, C, G, and T, representing the four nucleotide bases of a DNA strand — adenine, cytosine, guanine, thymine — covalently linked to a phosphodiester backbone. In the typical case, the sequences are printed abutting one another without gaps, as in the sequence AAAGTCTGAC, read left to right in the 5' to 3' direction. With regards to transcription, a sequence is on the coding strand if it has the same order as the transcribed RNA.

One sequence can be complementary to another sequence, meaning that they have the base on each position is the complementary (i.e. A to T, C to G) and in the reverse order. For example, the complementary sequence to TTAC is GTAA. If one strand of the double-stranded DNA is considered the sense strand, then the other strand, considered the antisense strand, will have the complementary sequence to the sense strand.

Notation

While A, T, C, and G represent a particular nucleotide at a position, there are also letters that represent ambiguity. Of all the molecules sampled, there is more than one kind of nucleotide at that position. The rules of the International Union of Pure and Applied Chemistry (IUPAC) are as follows:^[1]

• A = adenine
• C = cytosine
• G = guanine
• T = thymine
• R = G A (purine)
• Y = T C (pyrimidine)
• K = G T (keto)
• M = A C (amino)
• S = G C (strong bonds)
• W = A T (weak bonds)
• B = G T C (all but A)
• D = G A T (all but C)
• H = A C T (all but G)
• V = G C A (all but T)
• N = A G C T (any)

These symbols are also valid for RNA, except with U (uracil) replacing T (thymine).^[1]

Apart from adenosine (A), cytidine (C), guanosine (G), thymidine (T) and uridine (U), DNA and RNA also contain bases that have been modified after the nucleic acid chain has been formed. In DNA, the most common modified base is 5-methylcytidine (m5C). In RNA, there are many modified bases, including pseudouridine (Ψ), dihydrouridine (D), inosine (I), ribothymidine (rT) and 7-methylguanosine (m7G).^[2][3] Hypoxanthine and xanthine are two of the many bases created through mutagen presence, both of them through deamination (replacement of the amine-group with a carbonyl-group). Hypoxanthine is produced from adenine, xanthine from guanine.^[4] Similarly, deamination of cytosine results in uracil.

Biological significance

A depiction of the genetic code, by which the information contained in nucleic acids are translated into amino acid sequences in proteins.

In biological systems, nucleic acids contain information which is used by a living cell to construct specific proteins. The sequence of nucleobases on a nucleic acid strand is translated by cell machinery into a sequence of amino acids making up a protein strand. Each group of three bases, called a codon, corresponds to a single amino acid, and there is a specific genetic code by which each possible combination of three bases corresponds to a specific amino acid.

The central dogma of molecular biology outlines the mechanism by which proteins are constructed using information contained in nucleic acids. DNA is transcribed into mRNA molecules, which travels to the ribosome where the mRNA is used as a template for the construction of the protein strand. Since nucleic acids can bind to molecules with complementary sequences, there is a distinction between "sense" sequences which code for proteins, and the complementary "antisense" sequence which is by itself nonfunctional, but can bind to the sense strand.

Sequence determination

Electropherogram printout from automated sequencer for determining part of a DNA sequence

DNA sequencing is the process of determining the nucleotide sequence of a given DNA fragment. 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.

RNA is not sequenced directly. Instead, it is copied to a DNA by reverse transcriptase, and this DNA is then sequenced.

Current sequencing methods rely on the discriminatoy ability of DNA polymerases, and can therefore only distinguish four bases. An inosine (created from adenosine during RNA editing) will be read as a G, and 5-methyl-cytosine (created from cytosine by DNA methylation) will be read as a C. It is also currently difficult to sequence small amounts of DNA, as the signal will be to weak to measure. This is overcome by PCR amplification.

Sequence analysis

Once a nucleic acid sequence has been obtained from an organism, it may be analyzed using the tools of bioinformatics to attempt to determine its function.

Genetic testing

The DNA in an organism's genome can be analyzed to diagnose vulnerabilities to inherited diseases, and can also be used to determine a child's paternity (genetic father) or a person's ancestry. Normally, every person carries two copies of every gene, one inherited from their mother, one inherited from their father. The human genome is believed to contain around 20,000 - 25,000 genes. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders.

Genetic testing identifies changes in chromosomes, genes, or proteins.^[5] Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance of developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed.^[6][7]

Sequence alignment

In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences.^[8] If two sequences in an alignment share a common ancestor, mismatches can be interpreted as point mutations and gaps as indels (that is, insertion or deletion mutations) introduced in one or both lineages in the time since they diverged from one another. In sequence alignments of proteins, the degree of similarity between amino acids occupying a particular position in the sequence can be interpreted as a rough measure of how conserved a particular region or sequence motif is among lineages. The absence of substitutions, or the presence of only very conservative substitutions (that is, the substitution of amino acids whose side chains have similar biochemical properties) in a particular region of the sequence, suggest ^[9] that this region has structural or functional importance. Although DNA and RNA nucleotide bases are more similar to each other than are amino acids, the conservation of base pairs can indicate a similar functional or structural role.

Computational phylogenetics makes extensive use of sequence alignments in the construction and interpretation of phylogenetic trees, which are used to classify the evolutionary relationships between homologous genes represented in the genomes of divergent species. The degree to which sequences in a query set differ is qualitatively related to the sequences' evolutionary distance from one another. Roughly speaking, high sequence identity suggests that the sequences in question have a comparatively young most recent common ancestor, while low identity suggests that the divergence is more ancient. This approximation, which reflects the "molecular clock" hypothesis that a roughly constant rate of evolutionary change can be used to extrapolate the elapsed time since two genes first diverged (that is, the coalescence time), assumes that the effects of mutation and selection are constant across sequence lineages. Therefore it does not account for possible difference among organisms or species in the rates of DNA repair or the possible functional conservation of specific regions in a sequence. (In the case of nucleotide sequences, the molecular clock hypothesis in its most basic form also discounts the difference in acceptance rates between silent mutations that do not alter the meaning of a given codon and other mutations that result in a different amino acid being incorporated into the protein.) More statistically accurate methods allow the evolutionary rate on each branch of the phylogenetic tree to vary, thus producing better estimates of coalescence times for genes.

Sequence motifs

Frequently the primary structure encodes motifs that are of functional importance. Some examples of sequence motifs are: the C/D^[10] and H/ACA boxes^[11] of snoRNAs, Sm binding site found in spliceosomal RNAs such as U1, U2, U4, U5, U6, U12 and U3, the Shine-Dalgarno sequence,^[12] the Kozak consensus sequence^[13] and the RNA polymerase III terminator^[14].

See also

• Single-nucleotide polymorphism (SNP)

References

External links

• A bibliography on features, patterns, correlations in DNA and protein texts