Dictionary of DNA and Genome Technology

in the preparation of peptide–oligonucleotide conjugates; this has been achieved by interacting diene-modified oligonucleotides and maleimide-derivatized peptides [Nucleic Acids Res (2006) 34(3):e24].

This procedure may be used e.g. for conjugating antisense oligonucleotides to appropriate peptides in order to facilitate their uptake by target cells.

diethylpyrocarbonate (DEPC) C2H5.O.CO.O.CO.C2H5 – an inactivator of RNases used e.g. for treating glassware etc. in order to avoid loss of sample RNA through exogenous RNase activity; autoclaving, after treatment, is used to remove any remaining DEPC.

DEPC can also modify the exposed N-7 position of an unpaired adenine in RNA or DNA.

(See also RNALATER.)

differential DNA cleavage See e.g. CPNPG SITES. differential display A method which is used e.g. for detecting

genes that are expressed in a given type of cell only under certain circumstances.

Essentially, this method compares mRNAs isolated from two or more populations of the given cell which are expected to reveal differential expression of genes. Selected mRNAs from each population being studied are initially converted to

cDNAs by reverse transcription; selection is achieved by the use of primers having a 3′ terminal base complementary to only a proportion of the mRNAs from a given population. The resulting cDNAs are amplified by PCR using the original (‘selective’) primer and a primer of arbitrary sequence; the products of PCR therefore represent the 3′ ends of selected mRNA molecules.

The amplicons from each population are subjected to gel electrophoresis in separate lanes, and the fingerprints of the different populations are compared. Any band of interest (e.g. one present in a given fingerprint but absent in others) can be extracted from the gel and amplified (using the same primers) prior to e.g. sequencing.

Differential display was used e.g. to study gene expression during the development of different zones of the neural tube in the chick embryo [BMC Dev Biol (2006) 6:9].

digoxigenin (DIG) A steroid (from the plant Digitalis) used e.g. for labeling PROBEs. DIG-labeled RNA probes are made e.g. by in vitro transcription on a DNA target sequence using labeled nucleotides.

DIG-dUTP can be incorporated into DNA probes during synthesis by various types of DNA polymerase – including Taq polymerase, phage T4 polymerase, Klenow fragment and

at least some types of reverse transcriptase.

Detection of DIG-labeled probes can be achieved with antidigoxigenin antibodies covalently linked to a fluorophore or linked to an enzyme such as alkaline phosphatase that gives a chromogenic reaction with appropriate substrates.

(See also EMEA.)

7,8-dihydro-8-oxoguanine See 8-OXOG.

dikaryon (1) A cell with two genetically dissimilar (haploid) nuclei, or a (fungal) mycelium comprising such cells.

(2) A pair of nuclei that may be genetically dissimilar.

dinB gene See DNA POLYMERASE IV. dioecism See HETEROTHALLISM. diphosphate See PYROPHOSPHATE.

diphosphopyridine nucleotide See NAD. diploid See PLOIDY.

direct amplification fingerprinting (DAF) See AP-PCR. direct repeat (DR) One of two (or more) identical or closely

similar sequences, in a given molecule of nucleic acid, which have the same polarity and orientation. Repeats may or may not be contiguous. An example:

5′.....GCCTA.....GCCTA.....3′

3′.....CGGAT.....CGGAT.....5′

The direct repeat sequences in the genome of Mycobacterium tuberculosis and related bacteria have been used in SPOLIGO-

TYPING.

(See also ITERON and TANDEM REPEAT.)

direct repeat (DR) locus (of Mycobacterium tuberculosis) A distinct region of the chromosome, found in members of the M. tuberculosis complex, consisting of a number of highly conserved 36-bp direct repeats (DRs) which are interspersed with spacers of 34–41 bp. The number of repeats, as well as the presence/length of the spacers, are strain-dependent characteristics; such variation may have arisen through intramolecular homologous recombination and/or integration of an insertion sequence.

Variability in the DR locus has been exploited in SPOLIGO-

TYPING.

direct selection Syn. POSITIVE SELECTION.

directed evolution Any technique whose object is, effectively, to enact a rapid evolutionary process in the laboratory with the object of selecting molecules with certain desired characteristics; this approach can be used e.g. with nucleic acids and proteins.

[Laboratory-directed protein evolution: Microbiol Mol Biol Rev (2005) 69(3):373–392.]

(See also DNA SHUFflING and HOMING ENDONUCLEASE.)

directional cloning Syn. FORCED CLONING.

directional TOPO® pENTR™ vectors Vectors (Invitrogen, Carlsbad CA) used for cloning an insert, in a specific orientation, using the TOPOISOMERASE I CLONING approach.

Directional insertion of a fragment is achieved by using an asymmetric arrangement in which one end of the (linearized) vector is blunt-ended and the other end has a four-nucleotide

overhang; as in other forms of topo cloning the enzyme topoisomerase I is bound to phosphate at each end of the vector – on opposite strands – and carries out the same function.

These vectors include att sequences, permitting recombination with any of a range of Gateway® destination vectors –

see the table in GATEWAY SITE-SPECIfiC RECOMBINATION

SYSTEM; they also include sites suitable for sequencing. disjunction Separation of chromosomes during the process of

nuclear division.

displacement loop (D loop) See D LOOP. dissymmetry ratio Syn. BASE RATIO. distal box See RNASE III.

ditag See SAGE.

dithiothreitol (DDT, Cleland’s reagent) A reagent used e.g. to reduce disulfides to thiols and to maintain thiol groups in the reduced state. High concentrations of dithiothreitol facilitate denaturation of proteins by chaotropes and detergents.

In DNA technology, the reagent has also been used e.g. as a mucolytic agent for the treatment of mucoid specimens of sputum prior to examination by nucleic-acid-based tests for

Mycobacterium tuberculosis.

divergent transcription Transcription from two closely adjacent promoters, in opposite directions.

DNA Deoxyribonucleic acid: typically, a double-stranded (i.e. ‘duplex’) molecule or a single-stranded molecule – referred to as dsDNA and ssDNA, respectively – that consists of linearly polymerized deoxyribonucleotides, with adjacent nucleotides linked by a phosphodiester bond. The major bases found in the nucleotides of DNA are adenine, guanine, cytosine and thymine; this differs from RNA (ribonucleic acid) – in which the major bases are adenine, guanine, cytosine and uracil.

(See also TRIPLEX DNA and QUADRUPLEX DNA.)

DNA encodes the genetic information in cells and in some types of virus. In prokaryotes, DNA is found in the nucleoid (see also PLASMID), while in eukaryotes it occurs primarily in the nucleus, in mitochondria and (in photosynthetic species) in chloroplasts.

In linear molecules of DNA each strand has polarity: a 5′ end and a 3′ end. At one end of a strand, 5′ refers to the 5′ carbon atom in a ribose residue which is not linked to another nucleotide, while 3′ at the other end of the strand refers to the 3′ carbon atom in the ribose residue which, similarly, is not linked to another nucleotide.

In linear dsDNA molecules the two strands are arranged in an antiparallel mode: a 5′-to-3′ strand is bound to a 3′-to-5′ strand. The hybridization (binding) between the two strands involves hybrogen bonding between the bases in opposite strands. Hydrogen bonding occurs between specific pairs of bases: adenine base-pairs with thymine, while cytosine basepairs with guanine; adenine and thymine are complementary bases, as are cytosine and guanine. One strand of the duplex is said to be complementary to the other (when all the bases are correctly paired).

In so-called ‘circular’ molecules of dsDNA the polymer is continuous, i.e. there are no free 5′ and 3′ ends. Such mole-

cules may be in either a relaxed or a supercoiled state. (See also TOPOISOMERASE.) ssDNA can also occur as a circular molecule. (See also DNA NANOCIRCLE.)

(See also PNA.)

DNA amplification (in vitro) Any of various procedures in which, typically, a defined sequence of nucleotides in DNA is replicated – often with the production of a large number of copies.

(See also NUCLEIC ACID AMPLIfiCATION and cf. WHOLE- GENOME AMPLIfiCATION.)

Isothermal amplification (amplification at a fixed temperature) can be carried out by methods such as strand displacement amplification (see SDA). (cf. NASBA.)

Other methods, such as the LIGASE CHAIN REACTION and the polymerase chain reaction (see PCR), involve repetitive temperature cycling (e.g. for ~25–40 cycles). Temperatures used in such processes include an initial high temperature (e.g. 94°C) that is designed to separate the strands of dsDNA in order to expose target sequence(s). These methods require the use of thermostable enzymes (e.g. a thermostable ligase in LCR and a thermostable polymerase in PCR).

All of these methods may yield false-positive and/or falsenegative results under inappropriate conditions. In particular, contamination by extraneous nucleic acids must be rigorously

excluded (see e.g. AMPLICON CONTAINMENT and AMPLICON INACTIVATION).

The methods mentioned above are well established and have been available for some years. More recent methods are

HELICASE-DEPENDENT AMPLIfiCATION and the MOLECULAR ZIPPER.

Recombinase polymerase amplification is a method which combines recombinase-mediated binding of primers with the strand-displacement synthesis of DNA: see RPA.

(See also in situ solid-phase DNA amplification in SOLID-

PHASE PCR.)

DNA-binding proteins (in assays) See CHROMATIN IMMUNO-

PRECIPITATION, EMSA, FOOTPRINTING and SCINTILLATION PROXIMITY ASSAY.

DNA caging See CAGED DNA.

DNA chip See CHIP; see also MICROARRAY.

DNA clean-up resin See e.g. STRATACLEAN RESIN. DNA cloning See CLONING.

DNA combing Any procedure used for preparing DNA in a condition in which individual molecules are made available for analysis (e.g. fluoroscopic analysis) in a stretched state. In some methods, the molecules of DNA are anchored at one end to a solid (e.g. glass) surface, and stretching is carried out e.g. by the forces present in a receding meniscus.

[Simple method for stretching linear DNA molecules (of up to 18 kb) for fluorescent imaging: Nucleic Acids Res (2006) 34(17):e113.]

DNA database A phrase that frequently refers to a DATABASE containing a record of the DNA sequences from a number of individuals; such databases are often maintained e.g. by lawenforcement agencies.

(See also CODIS and FORENSIC APPLICATIONS.)

DNA delay mutant (of phage T4) A strain with mutation(s) in topoisomerase-encoding gene(s) which exhibits delayed synthesis of DNA and a smaller burst size. For replication, a T4 DNA delay mutant depends on a functional gyrase in the host cell.

DNA demethylation See DEMETHYLATION. DNA dendrimer See DENDRICHIP.

DNA-dependent DNA polymerase (DNA polymerase) Any enzyme which polymerizes dNTPs (deoxyribonucleoside triphosphates) in the 5′-to-3′ direction on a DNA template, with elimination of pyrophosphate.

Escherichia coli encodes two main DNA polymerases that are designated I and III. (See also DNA POLYMERASE II, DNA

POLYMERASE IV and DNA POLYMERASE V.)

Eukaryotes typically have a number of different DNA polymerases which include separate enzymes for replicating mitochondrial and nuclear DNA. Nuclear polymerases include DNA polymerases α and δ (see also OKAZAKI FRAGMENT).

(See also Y FAMILY.)

There is an extensive range of commercial DNA polymerases, many of which are thermostable enzymes intended for

use in PCR (see e.g. ACCUPRIME GC-RICH DNA POLYMERASE, PFUTURBO, PLATINUM TAQ DNA POLYMERASE and RTTH DNA POLYMERASE).

Various forms of the well-known TAQ DNA POLYMERASE (including recombinant forms) are available.

An error-prone DNA polymerase (Mutazyme™) is used for generating random mutations during PCR (in the GENE-

MORPH PCR MUTAGENESIS KIT).

DNA-dependent RNA polymerase See RNA POLYMERASE. DNA DIRECT™ See DYNABEADS.

DNA enzyme See DEOXYRIBOZYME.

DNA extraction See DNA ISOLATION.

DNA fingerprinting (restriction enzyme analysis) A method used e.g. for TYPING bacteria. In the original method, the genomic DNA from a test strain is cleaved by certain restriction endonucleases and the resulting fragments separated by gel electrophoresis. The bands of fragments are stained in situ or, alternatively, denatured in the gel and blotted onto a membrane before staining. The pattern of bands in the gel, or on the membrane, is the fingerprint; strains are compared and classified on the basis of similarities and differences in their fingerprints.

One disadvantage of this method is that it may generate too many fragments and thus make a complex fingerprint which is difficult to interpret. Fewer, larger fragments are generated if a so-called ‘rare-cutting’ restriction enzyme is used for the initial restriction. These large fragments can be separated by PFGE, although this procedure requires 2–3 days and may be susceptible to endogenous nucleases.

The problem of too many fragments can also be addressed by using a labeled probe which binds only to those fragments that contain the probe’s target sequence; in this case, only fragments which bind the probe are visible in the gel – so that

the fingerprint consists of only a limited number of bands. This principle is exploited in RIBOTYPING.

DNA-free cell A cell, modified in vitro, in which the chromosomal DNA has undergone cleavage and extensive degradation. This can be achieved in at least two ways. First, treatment of certain types of cell with ULTRAVIOLET RADIATION (see e.g. MAXICELL). Second, treatment of dam mutants of Escherichia coli with 2-aminopurine, resulting in mismatch repair-related degradation of the chromosome [see J Bacteriol (2006) 188(1):339–342].

DNA glycosylase Any enzyme which cleaves an aberrant base from a nucleotide in DNA; DNA glycosylases are active in

the BASE EXCISION REPAIR pathway.

(See also 8-OXOG.)

DNA glycosylase I (in Escherichia coli) Syn. Tag protein (see

DNA REPAIR).

DNA glycosylase II (in Escherichia coli) Syn. AlkA (see DNA

REPAIR).

DNA helicase See HELICASE.

DNA identification tag See IDENTIfiER DNA.

DNA isolation (DNA extraction) Any procedure for removing and concentrating DNA from cells/tissues. The methods are influenced by factors such as the type of DNA sought (genomic, plasmid DNA etc.), the required quality of DNA, amount of sample available, and nature of sample (e.g. fungal, bacterial, mammalian cells). [Isolation of ancient DNA (optimization of procedures): BioTechniques (2007) 42(3):343–352.]

(See also entry NUCLEIC ACID ISOLATION.)

Protocols include a wide range of ad hoc in-house methods and commercial systems.

For ‘difficult’ tissues (such as skin) the method may include an initial stage of mechanical disaggregation of the tissue followed by digestion with a proteinase; DNA may then be recovered by phenol–chloroform extraction and precipitated by ethanol (or e.g. isopropanol).

DNA can be extracted from some tissues (e.g. brain, liver) without organic solvents. The RecoverEase™ DNA isolation procedure (Stratagene, La Jolla CA) employs an initial stage of physical disaggregation and coarse filtration. The nuclei are separated by centrifugation. Incubation with proteinase K is continued overnight within a dialysis cup, yielding purified high-molecular-weight DNA.

DNA from whole blood can be obtained rapidly e.g. with the QIAamp® blood kit (QIAGEN, Hilden, Germany). After buffer-mediated lysis, the lysate is loaded into a spin column. On centrifugation, lysate passes through a specialized membrane to which DNA is adsorbed. Following spin washes, the DNA can be eluted from the membrane. This system can be used with blood containing common anticoagulants such as citrate and heparin. (Other types of QIAamp kit are available for different sources of DNA.)

Plasmid DNA may be recovered from bacteria e.g. with the QIAGEN® plasmid mini kit. The starting point is a pellet of cells of (e.g.) Escherichia coli. The pellet is resuspended in a buffer which disrupts the bacterial outer membrane. The use of controlled alkaline lysis with NaOH and the detergent

sodium dodecyl sulfate (SDS) then releases soluble proteins, RNA and plasmids from the cells. The RNA is degraded by RNase A. Conditions are optimized for maximum release of plasmid DNA without release of the chromosomal DNA; the protocol is also designed to avoid irreversible denaturation of plasmid DNA. Neutralization by a high-salt buffer (pH 5.5) precipitates the SDS; proteins are entrapped in the salt–SDS complexes – but plasmids renature and remain in solution. Centrifugation removes the proteins etc., leaving plasmids in the supernatant. The supernatant is then passed through a QIAGEN anion-exchange resin; given the pH and electrolyte concentration of the supernatant, plasmid DNA is retained by the resin. After washing, the plasmid DNA is eluted in buffer containing 1.25 M NaCl (pH 8.5), desalted, and concentrated by precipitation with isopropanol.

Some of the problems (and possible causes) experienced in DNA isolation are listed in the table.

DNA ladder See GEL ELECTROPHORESIS.

DNA ligase Any LIGASE which can catalyze a phosphodiester bond between the 3′-OH terminal of a strand of DNA and the correctly juxtaposed 5′-phosphate terminal of the same strand or of a different strand.

A thermostable ligase, Ampligase®, from a thermophilic prokaryote, has uses which include e.g. the LIGASE CHAIN

REACTION and REPEAT-EXPANSION DETECTION; however, it

is not used for blunt-ended ligation.

The phage T4 DNA ligase (EC 6.5.1.1) and the (NAD+- dependent) Escherichia coli DNA ligase (EC 6.5.1.2) are used e.g for closing nicks.

DNA looping See LOOPING. DNA melting See MELTING.

DNA methylation See METHYLATION.

DNA methyltransferase (MTase) Any enzyme that catalyzes the METHYLATION of (specific) bases in DNA.

One example of a prokaryotic methyltransferase is M.SssI, an enzyme that carries out SYMMETRIC METHYLATION at the 5-position in the cytosine residues in the sequence 5′-CG-3′.

Many prokaryotic RESTRICTION ENDONUCLEASEs carry out specific methylation as well as restriction.

The primary role of methylation in prokaryotes is generally taken to be the protection of genomic DNA from the cell’s own restriction enzymes (enzymes which degrade DNA, e.g. ‘foreign’ DNA lacking the particular pattern of methylation characteristic of the cell’s own DNA).

[Use of prokaryotic DNA methyltransferases as analytical and experimental tools in biology: Anal Biochem (2005) 338 (1):1–11.]

Mammals have a range of DNA methyltransferases, and it appears that the activities of these enzymes are essential for normal function or survival. The mammalian DNA methyltransferases are apparently involved in maintaining an overall pattern of methylation that determines the normal functioning of cells. The dysregulation of methylation – either aberrant demethylation or aberrant hyper-methylation – can cause disorders or death; thus, for example, mutation in DNMT3B, the

gene encoding DNA methyltransferase 3B (Dnmt3B), results in the disorder ICF SYNDROME.

Methyltransferases Dnmt3A and Dnmt3B appear to be required for de novo methylation; Dnmt1 is generally viewed as a ‘maintenance’ enzyme.

[Methyltransferases of mammals: Hum Mol Genet (2000) 9(16):2395–2402.]

(See also DEMETHYLATION and TAGM.)

DNA nanocircle A small, synthetic circular ssDNA molecule (up to ~120 nt) which can be transcribed in vitro or in bacteria (after insertion by heat shock); RNA synthesis occurs by the ROLLING CIRCLE mechanism. RNA polymerase may be able to initiate transcription with GTP at any cytosine residue in the circle, although the conformation of the template can strongly inhibit or promote transcription.

A nanocircle containing an optimized promoter sequence, and encoding a self-cleaving monomeric hammerhead RIBO- ZYME, was able to express the active ribozyme in cells of Escherichia coli [Proc Natl Acad Sci USA (2002) 99(1):54– 59].

DNA notation See e.g. DNA SKYLINE.

DNA-only transposon Any TRANSPOSABLE ELEMENT that can

transpose without the involvement of an RNA intermediate.

(cf. RETROTRANSPOSON.)

DNA photolyase See PHOTOLYASE.

DNA polymerase See DNA-DEPENDENT DNA POLYMERASE for

a definition and for some commercial examples.

DNA polymerase I (Kornberg enzyme) In Escherichia coli, the

DNA-DEPENDENT DNA POLYMERASE which is involved e.g.

in EXCISION REPAIR and in the degradation of RNA primers during DNA replication; it has proof-reading (3′-to-5′ exonuclease) and 5′-to-3′ exonuclease activities. It can also act as a reverse transcriptase, but with low efficiency.

Proteolytic cleavage of the polymerase (e.g. with subtilisin) gives rise to the 75-kDa Klenow fragment, an enzyme with polymerase and proof-reading activities but lacking the 5′ exonuclease activity. It is used, for example, in the Sanger (chain-termination) method of DNA sequencing and for 3′- end-labeling of dsDNA, second-strand synthesis of cDNA, and filling in 5′ overhangs; its strand-displacement activity can be employed in SDA.

(See also EXO KLENOW FRAGMENT.)

DNA polymerase II In Escherichia coli, a DNA-DEPENDENT DNA POLYMERASE that is induced following damage to DNA or inhibition of replication (in the so-called SOS response).

(See also DNA POLYMERASE IV.)

DNA polymerase III In Escherichia coli, the major DNA- DEPENDENT DNA POLYMERASE which is used e.g. in chromosomal replication; it has both 3′ and 5′ exonuclease activities.

DNA polymerase IV In Escherichia coli, a DNA polymerase (product of the dinB gene) involved in mutagenesis when induced in the SOS response.

(See also DNA POLYMERASE II and DNA POLYMERASE V.)

In recA730 strains of Escherichia coli, constitutive expression of the produces a mutator phenotype in

which DNA polymerase IV is reported to contribute significantly [J Bacteriol (2006) 188(22):7977–7980].

(See also Y FAMILY.)

DNA polymerase V In Escherichia coli, a DNA polymerase consisting of two UmuD′ proteins and one UmuC protein (products of the umuD and umuC genes respectively), which is involved in error-prone repair of DNA (TRANSLESION SYNTHESIS) when induced in the SOS response.

(See also DNA POLYMERASE IV, Y FAMILY, TOL PLASMID.)

DNA Polymorphism Discovery Resource A collection of 450 samples of DNA from US residents, with ancestry from all major regions of the world, intended for studies to facilitate discovery of human genetic variation [Genome Res (1998) 8(12):1229–1231; erratum: Genome Res (1999) 9(2):210].

[Example of use of resource (discovery of rare nucleotide polymorphisms): Nucleic Acids Res (2006) 34(13):e99.]

DNA profile Data specific to the DNA of a given individual. For example, a profile may consist of details of the number of short tandem repeats at given genomic loci (e.g. CODIS).

DNA quadruplex See QUADRUPLEX DNA.

DNA repair Any of various (normal) physiological processes which recognize and repair damaged/abnormal DNA.

Some repair processes remove the damaged (or abnormal) part of a strand and replace it by synthesis (see e.g. EXCISION

REPAIR).

By contrast, direct reversal of damage caused by a methylating agent in Escherichia coli can be carried out by the Ada protein; this protein is a bifunctional methyltransferase which can transfer a methyl group from e.g. O6-methylguanine to a cysteine residue on the Ada protein itself. Methylation of a phosphodiester bond can also be reversed by Ada: the methyl group is transferred to a different cysteine residue on the Ada protein.

Some repair systems are constitutive (i.e. usually present in the cell). By contrast, the Ada protein is part of an inducible repair system (the so-called ‘adaptive response’) produced in E. coli in response to the presence of low levels of certain alkylating agents (e.g. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU)). Another enzyme produced in the adaptive response is 3-methyladenine DNA glycosylase II (the AlkA protein) which can cleave the methylated bases from N3-methyladenine, N3-methylguanine and N7-methylguanine as well as from O2-methylcytosine.

The Tag (TAG) protein (tag gene product) is a constitutive glycosylase that specifically cleaves N3-methyladenine. [Activity of Tag: EMBO J (2007) 26:2411–2420.]

(See also BASE EXCISION REPAIR.)

The DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) is used as a partner in certain fusion proteins (see GENE FUSION (uses)).

DNA sampling See, for example, BUCCAL CELL SAMPLING and

FORENSIC APPLICATIONS.

DNA sequencing Determining the identity and position of each of the constituent nucleotides in a molecule or fragment of DNA. Sequencing provides definitive information on a given

molecule or fragment; such information is required e.g. for identification, characterization and taxonomic studies.

Once determined, the sequence of nucleotides in a given sample of DNA may or may not give a direct indication of the composition of any protein(s) encoded by the DNA; this is because the actual sequence of amino acid residues in a protein can be influenced by factors such as INTRONS and by events such as FRAMESHIFTING during translation – that is, a given sequence of nucleotides in genomic DNA does not necessarily indicate a series of codons (see CODON).

As the strands in duplex DNA are normally complementary (so that the sequence of one strand can be deduced from the sequence of the other), it might be supposed that sequencing of only one strand would be adequate; however, sequencing of both strands increases the level of accuracy.

Single-stranded templates (of the sample to be sequenced) may be prepared e.g. by asymmetric PCR or by cloning in a filamentous phage (such as phage M13).

Methods used for sequencing DNA

A commonly used procedure is the DIDEOXY METHOD (q.v.) which can be used for sequencing templates of up to ~500 nt in length. Longer templates may be sequenced by using a solid-phase approach (see PRIMER WALKING). The dideoxy method can be automated (see AUTOMATED SEQUENCING).

The MAXAM–GILBERT METHOD (= the chemical cleavage method) is a mechanistically distinct approach involving the base-specific cleavage of end-labeled fragments and analysis of the resulting products.

Visualization of sequencing products in gels (which originally involved the use of radioactive labels) can be achieved with e.g. fluorescent labels; alternatively, use can be made of

CHEMILUMINESCENCE systems.

Pyrosequencing™ (see PYROSEQUENCING) can be used for relatively short templates (e.g. ~40 nt).

HYBRIDIZATION-BASED SEQUENCING is a further option.

DNA sequencing has been reported with a MALDI-TOF- based approach [Nucleic Acids Res (2007) 35(8):e62].

An approach to sequencing that may be possible in the future depends on translocation of DNA molecules through nanopores under conditions which allow the identification of individual nucleotides in transit [see e.g. Biophys J (2004) 87 (3):2086–2097].

DNA sex determination assay Any DNA-based asssay used to determine the gender of the individual from which a given sample has been obtained.

One widely used assay (e.g. in archeological and forensic work) involves amplification of a specific sequence in each of the two genes encoding AMELOGENIN (q.v.). In each of the genes, a sequence in the region of the first intron is targeted by a pair of PCR primers; the targets are chosen so that easily distinguishable products of different sizes and sequences are obtained from the X and Y chromosomes. With this arrangement, the formation of products of one type only (from the X chromosome) generally indicates a female source – products of two types (from the X and Y chromosomes) indicating a

male source.

Various commercial assay kits are available, and different sets of primers are used, but the same principle applies in all of the assays.

Failures in accuracy of the amelogenin assay can occur as a result of mutation. For example, mutation in the Y allele, blocking amplification of the specific sequence from the Y chromosome, could cause misinterpretation of the result (i.e. products from only the X chromosome) as an indication of a female source.

Failures in the amelogenin assay may be affected by ethnic group/geographical location. Thus, in one study on an Indian population, relevant deletions were observed in the Y copy of the amelogenin gene in 10 of 4257 males [BMC Med Genet (2006) 7:37].

(See also FORENSIC APPLICATIONS.)

DNA shuffling (gene shuffling; sometimes referred to as ‘sexual PCR’) A technique in which the properties of a given protein

can be modified by extensive in vitro recombination between the encoding gene and a number of allelic or related forms of that gene.

The isolated genes are initially cleaved by DNase I to form fragments of ~50 bp. The fragments from all of these genes are then used in a primer-less form of PCR in which the fragments prime each other; fragment–fragment binding occurs owing to high-level homology among the genes. During this process there is frequent switching of templates (successive priming of different fragments), giving rise to a wide range of chimeric genes. These chimeric genes are then amplified by conventional PCR, and the resulting library of recombinant genes can be screened for those products with the desired characteristics.

One or more further rounds of shuffling can be carried out if required.

(See also PSEUDORECOMBINATION.)

DNA Skyline A form of notation in which a sequence of

nucleotides is shown as a series of symbols – rather than by a series of the letters A, C, G and T; in this system, the bases adenine, cytosine, guanine and thymine are represented by four symbols of different height, and these symbols are used instead of the usual letters (see figure). Skyline was designed to facilitate visual comparison of strand complementarity and conserved motifs etc. [BioTechniques (2006) 40:740].

DNA staining A procedure in which a dye, often a fluorophore, is bound to DNA. Such binding may be non-covalent – for

example, by INTERCALATING AGENTS such as ETHIDIUM

BROMIDE, or by minor-groove-binding agents such as DAPI and Hoechst 33342 (see also BOXTO) – or covalent (e.g. following amine modification: see PROBE LABELING).

DNA may be stained e.g. to detect bands of amplification products in gel electrophoresis strips, to study preparations of chromosomes or in situ genomes, to demonstrate chromosome banding, to facilitate flow cytometry and to distinguish between living and dead cells; the latter role depends on the ability of some dyes to penetrate the cell membrane in living cells (and of other dyes to be excluded): see e.g. LIVE/DEAD

BACLIGHT BACTERIAL VIABILITY KIT.

The (many) DNA-staining dyes include e.g. ACRIDINES,

BENA435, BOXTO, DAPI, ETHIDIUM BROMIDE and ETHIDIUM MONOAZIDE, HEXIDIUM IODIDE, PROPIDIUM IODIDE, QUIN- ACRINE, SYBR GREEN I and various SYTO DYES.

ssDNA may be quantitated e.g. with OLIGREEN.

(See also ULTRAVIOLET ABSORBANCE.)

DNA stretching Syn. DNA COMBING.

DNA thermal cycler Syn. THERMOCYCLER.

DNA toroid A highly compacted form of DNA found e.g. in the nucleoid of the bacterium Deinococcus radiodurans and in bacterial endospores. The toroidal structure may contribute to the resistance of D. radiodurans (and of endospores) to DNA-damaging agents such as certain types of radiation; it may facilitate the repair of double-stranded breaks in DNA in a template-independent, RecA-independent way [J Bacteriol (2004) 186:5973–5977].

The DNA in human sperm cells occurs in nucleoprotamine toroids [DNA structure in human sperm cells: J Cell Sci (2005) 118(19):4541–4550].

DNA unwinding protein See HELICASE.

DNA uptake site See TRANSFORMATION.

DNA vaccine Any parenterally administered vaccine containing DNA encoding specific antigen(s) that are synthesized in the vaccinated individual; the object of such vaccination is to induce humoral (that is, antibody) and cell-mediated immune responses to given antigen(s). (See also pVAX™200-DEST in the table of destination vectors in GATEWAY SITE-SPECIfiC

RECOMBINATION SYSTEM.)

A DNA vaccine may be administered intradermally by a biolistic approach (see also GENE GUN). Administration may also be achieved by intramuscular injection, by aerosol, or by intravenous delivery.

The efficacy/immunogenicity of a vaccine can be improved e.g. by optimization of codon usage [Infect Immun (2005)

73(9):5666–5674] and by the use of a DNA vector encoding certain proteins in addition to the target antigen(s) – e.g. CALRETICULIN [J Virol (2004) 78(16):8468–8476] or VP22 protein of bovine herpesvirus 1 [J Virol (2005) 79(3):1948– 1953].

The effective induction of specific antibodies in laboratory animals has been achieved by the intravenous delivery of an antibody-specifying plasmid [BioTechniques (2006) 40(2): 199–208]. [See also J Virol (2007) 81(13):6879–6889.]

Bacteriophages have been used as vehicles for the delivery of DNA vaccines in a mammalian setting [e.g. Infect Immun (2006) 74(1):167–174].

(See also GENE THERAPY.)

dnaA gene (Escherichia coli) A gene whose product (DnaA) is required for DNA replication. During initiation of replication, copies of DnaA bind to so-called ‘DnaA boxes’ located in the origin or replication (oriC); this is a prelude to strand separation (‘melting’), an essential feature of the replication process. The Orc1–6 proteins provide a similar function in eukaryotic cells, while in archaeans the function involves the Orc1 and CdC6 proteins.

Some early reports indicated that DnaA may be needed for the replication of certain plasmids (including pSC101), but it now appears that this is not the case.

DNase (deoxyribonuclease) Any enzyme that cleaves phosphodiester bonds in DNA. An exodeoxyribonuclease is a DNase that cleaves terminal bonds, while an endodeoxyribonuclease is a DNase that cleaves internal bonds. A few examples of

DNases: DNASE I, DNASE II, ENDONUCLEASE S1, EXONUC- LEASE III, EXONUCLEASE IV.

DNases are produced by various microorganisms and e.g. by the human pancreas.

Microbial DNases include e.g. the staphylococcal ‘thermonuclease’ – a Ca+-dependent thermostable enzyme with both exonuclease and endonuclease activity.

(See also STREPTODORNASE.)

To detect DNase production by bacteria, the test strain is plated on an agar medium that contains DNA and a calcium salt. Following development of colonies, the plate is flooded with hydrochloric acid (1 N HCl) to precipitate non-degraded DNA; any DNase-producing colonies are surrounded by a clear halo in which the DNA has been degraded.

DNase I A DNASE whose products have 5′-phosphate terminal groups (compare DNASE II). This enzyme is EC 3.1.21.1.

(See also STREPTODORNASE.)

DNase II A DNASE whose products have 3′-phosphate terminal groups (compare DNASE I). This enzyme is EC 3.1.22.1.

DNAzyme Syn. DEOXYRIBOZYME.

Dnmt1 (DNMT1) DNA METHYLTRANSFERASE 1. (See also DEMETHYLATION.)

DNMT3B The gene encoding (mammalian) DNA methyltransferase 3B.