Dictionary of DNA and Genome Technology

respiration (energy metab.) A specific energy-converting process in which a substrate is metabolized with the involvement of an exogenous (i.e. external) electron acceptor such as oxygen. In this process ATP is characteristically generated by oxidative phosphorylation.

In anaerobic respiration (a process carried out e.g. by some bacteria in the absence of oxygen) the electron acceptor may be e.g. a substance such as fumarate, nitrate, selenate, sulfur or sulfate.

(cf. FERMENTATION.)

response regulator See TWO-COMPONENT REGULATORY SYS- TEM.

restriction (DNA technol.) Cleavage of dsDNA by one or more

types of RESTRICTION ENDONUCLEASE.

In DNA technology, one normally uses particular type(s) of restriction enzyme so that cleavage occurs at specific sites in the target molecule(s).

restriction endonuclease (restriction enzyme; REase, RE) An endonuclease which binds to dsDNA, commonly at a specific sequence of nucleotides (termed the recognition sequence or recognition site), and which typically cuts each strand once if certain bases are unmethylated (for many REs) or methylated (for other REs); according to enzyme, the terminal regions at

a cleaved site are STICKY ENDS or BLUNT-ENDED DNA.

Restriction endonucleases are obtained from a wide range of prokaryotic microorganisms.

The heterogeneity of REs precludes a single definition that covers all cases. Thus, some REs have no precise recognition site while others have several sites; some bind to palindromic sequences, others to asymmetric sites; some need two copies of the recognition site, to be functional, while most need only one copy; some cut within the site and others cut outside it; some REs cut on both sides of the recognition site – excising part of the target molecule; some nicking enzymes (see later) resemble REs but cut only one strand.

A given sequence of nucleotides may be recognized by two or more different REs, derived from different species – see

ISOSCHIZOMER.

Under non-optimal conditions, the specificity of an RE, for a given recognition sequence, may become less stringent, i.e. the RE may be able to bind to certain other sequences. This phenomenon is called STAR ACTIVITY.

For some types of experiment it may be necessary to direct the activity of an RE to a particular recognition site, even when other copies of the given site are freely available elsewhere in the molecule: see e.g. ACHILLES’ HEEL TECHNIQUE

and PROGRAMMABLE ENDONUCLEASE.

The methylation of dsDNA (referred to above) is typically carried out in vivo by methyltransferases (= ‘methylases’): enzymes which add methyl groups to specific bases (the N6 of adenine, the C5 of cytosine) in prokaryotic DNA. In many cases, restriction (cutting) and methylation can be carried out by the same enzyme, i.e. some REs have a dual role.

The nomenclature currently recommended for the REs and methyltransferases [Nucleic Acids Res (2003) 31(7):1805–

1812] avoids the (former) use of italics. For example, EcoRI (instead of EcoRI) is used to refer to an RE from Escherichia coli strain R, the I indicating one particular RE from strain R. Sometimes the prefix ‘R’ is used – e.g. R.EcoRI, in which ‘R’ indicates a restriction – rather than a methylating – enzyme; enzymes which incorporate both restriction and methylating functions may have the prefix RM. Some other examples of REs are given in the table.

For online information on REs (and methyltransferases) see the entry REBASE.

REs are now classified into four main groups (types I–IV): Type I REs. This type of RE is a multisubunit enzyme with subunits for restriction, methylation and site-specificity (the latter specifying the recognition sequence); ATP-dependent cleavage occurs at a non-specific site outside the recognition sequence. An example of a type I RE is EcoKI.

[New type I REs from Escherichia coli: Nucleic Acids Res (2005) 33(13):e114.] (See also HSD GENES.)

[Mechanism of translocation of the type IC RE EcoR124I: EMBO J (2006) 25:2230–2239.]

Type II REs. These REs typically recognize a specific sequence of nucleotides; both of the strands are cleaved in an ATP-independent manner, at fixed locations in or near the recognition site – leaving a 5′-phosphate terminus and a 3′- hydroxyl terminus on both sides of the cut.

Precise cutting of DNA makes the type II RE very useful in recombinant DNA technology; over 3500 type II REs have been characterized.

Subtypes of the type II RE display various characteristics. Subtype IIP includes a large number of REs that recognize symmetric (palindromic) sequences and cleave at fixed sites within, or immediately adjacent to, their recognition sites. An example of a type IIP RE is EcoRI, whose recognition and cutting (↓) sites are:

5′-G↓AATTC-3′

and 3′-CTTAA↑G-5′ in the complementary strand. Note that the staggered cuts leave sticky ends: 5′-AATT overhangs on each strand.

Other subtypes of the type II RE include:

IIA. These REs recognize asymmetric sequences.

IIB. These REs cleave on both sides of the recognition site. Examples include BaeI and BcgI. The recognition and cutting sites of BaeI can be written:

(10/15)AC(N4)GTAYC(12/7)

in which N4 refers to four (unspecified) nucleotides and Y is either C or T. 10/15 means that the strand shown is cut 10 nucleotides upstream and the complementary strand is cut 15 nucleotides upstream. 12/7 means that the strand shown is cut 12 nucleotides downstream and the complementary strand is cut 7 nucleotides downstream.

IIC. In these REs the cutting and methylation functions are

combined in one polypeptide; they include members of other subtypes.

IIE. These REs cut at the recognition site but need to interact with another copy of that site for ‘activation’. [DNA looping by two-site restriction enzymes: Nucleic Acids Res (2006) 34(10):2864–2877.] Studies on various ‘two-site’ REs have shown that these enzymes are sensitive to mechanical tension in the target DNA – enzymic activity being lost when the DNA is subjected to a few piconewtons [Proc Natl Acad Sci USA (2006) 103(31):11555–11560].

IIF. These REs interact with, and cut, two copies of the recognition site.

IIM. These REs cut at fixed locations where there is a specific pattern of methylation. An example is DpnI.

IIS. The type IIS REs cut one, or both, strands outside the recognition site.

Note. A given type II RE may belong to more than one subtype.

Type III REs. These REs interact with both copies of a nonpalindromic sequence that are orientated inversely; cleavage, which depends on ATP-dependent translocation, occurs at a specified distance from one of the sites. Examples of type III REs include EcoP1I and EcoP15I.

Type IV REs. These REs recognize a methylated site but, unlike the type IIM REs, their cutting sites apparently lack specificity. Example: EcoKMcrBC.

Putative REs. The existence of these REs (which have a prefix P) is inferred e.g. by comparing an open reading frame (ORF) in a sequenced genome with the sequence of a known RE.

Nicking enzymes. These are enzymes cut (‘nick’) only one of the strands of a DNA duplex; at least some of these enzymes resemble restriction endonucleases – e.g. BstNBI [Nucleic Acids Res (2001) 29:2492–2501].

The nicking enzymes have an ‘N’ prefix, e.g. N.BstNBI. Some (normal) REs can be made to act as nicking enzymes by using DNA with chemically modified bases: see e.g. SDA. At a low pH, the type IIS restriction enzyme BfiI becomes a nicking enzyme, selectively cutting a single strand [Proc Natl

Acad Sci USA (2003) 100(11):6410–6415].

restriction endonuclease analysis of plasmid DNA See REAP.

restriction enzyme Syn. RESTRICTION ENDONUCLEASE.

restriction enzyme analysis Syn. DNA fiNGERPRINTING.

used for scanning genomic DNA with the object of detecting e.g. DNA polymorphisms or altered patterns of DNA methylation.

In the original method, suitably prepared genomic DNA was initially digested with the RESTRICTION ENDONUCLEASE NotI, and the 5′ overhangs (at cleavage sites) filled in with labeled (32P) nucleotides. End-labeled (32P-labeled) DNA was then digested with another restriction enzyme, EcoRV, after which the first phase of gel electrophoresis was carried out.

DNA-containing gel was subsequently cut out and digested with the restriction enzyme MboI. Following this digestion with MboI the next phase of gel electrophoresis was carried out. The final bands of fragments were examined in the dried gel.

In studies on methylation patterns in genomic DNA the restriction enzyme NotI is particularly useful because its recognition site (5′-GC↓GGCCGC-3′) occurs primarily in CPG ISLANDS: >85% of NotI cutting sites within the human genome are found in CpG islands. The significance of this is that CpG islands are commonly associated with the promoter regions of genes, and aberrant patterns of methylation in such regions have been noted in cancer-related silencing of tumorsuppressor genes; hence, it is of particular interest to examine methylation patterns in fragments that include CpG islands. In this context, it is the enzyme NotI (rather than EcoRV) which determines the specific (CpG-associated) locations of the restriction fragments; hence, NotI is generally referred to as the landmark enzyme (or the restriction landmark) in this procedure.

Note that NotI is a methylation-sensitive enzyme, i.e. it will not cleave its recognition site if an internal cytosine residue is methylated. Consequently, an aberrantly methylated cutting site will not be cleaved and end-labeled – so that a particular band of RLGS fragments will be absent at the location in the gel which is normally occupied by a band.

While most RLGS studies have been carried out with the NotI landmark enzyme, it was reported that there are several advantages in complementing NotI with another restriction enzyme, AscI (cutting site: GG↓CGCGCC) [Genome Res (2002) 12(10):1591–1598].

A useful follow-up is to prepare a library by cloning the RLGS fragments, permitting further studies on particular loci of interest.

RLGS can be used not only to compare different genomes but also to compare DNA methylation patterns in different tissues within a given individual. One study found that many CpG islands are differentially methylated in a tissue-specific manner [Proc Natl Acad Sci USA (2005) 102(9):3336–3341].

restriction map In relation to a given DNA molecule or fragment: any diagram showing the positions of the recognition sequences of RESTRICTION ENDONUCLEASEs; other features, e.g. origin of replication, particular genes, direction of transcription etc. may also be shown on the map in order to give orientation.

(See also flASH CHEMILUMINESCENT GENE MAPPING KIT.)

restriction-minus cells (DNA technol.) Genetically engineered bacteria in which some, or all, of the restriction systems have been deleted.

An example of a restriction-minus strain is XL1-Blue MRF′ (Stratagene, La Jolla CA): ∆(mcrA)183, ∆(mcrCB-hsdSMR- mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac[F′ proAB, lacIqZ∆M15, Tn10(Tetr )].

Examples of the use of XL1-Blue MRF′ (e.g.): studies on MutH–MutL interaction in DNA mismatch repair [Nucleic

Acids Res (2006) 34(10):3169–3180] and on transcriptional responses to secretion stress in two species of fungi [BMC Genomics (2006) 7:32].

reverse transcription of an mRNA into DNA and subsequent insertion of the DNA into the genome (with an opportunity for expression); this type of process apparently occurs only rarely in evolution.

If the inserted sequence has no opportunity for expression, the resulting promoter-less, intron-less and function-deficient entity is referred to as a retropseudogene.

[Emergence of young human genes in primates: PLoS Biol (2005) 3(11):e357. Retrocopied genes in male fitness: PLoS Biol (2005) 3(11):e399. Functional displacement of a source gene by a retrocopy: Nucleic Acids Res (2005) 33(20):6654– 6661.]

(See also PSEUDOGENE.)

retrohoming See INTRON HOMING.

retron In certain bacteria – e.g. strains of Escherichia, Klebsiella, Proteus, Rhizobium, Salmonella: a chromosomal sequence encoding a REVERSE TRANSCRIPTASE similar to that encoded by retroviruses. These bacteria are able to synthesize a hybrid RNA/DNA molecule – called multicopy singlestranded DNA (msDNA) – in which the 5′ end of ssDNA is covalently bound (via a phosphodiester linkage) to the 2′- position of a non-terminal guanosine residue in the RNA. A single cell may contain as many as 500 copies of msDNA.

Retrons have been named according to the initials of the host species and the number of bases in the DNA moiety of the corresponding msDNA; thus, for example, in Myxococcus xanthus the msDNA contains 162 bases, and this molecule is designated Mx162.

retroposon Syn. RETROTRANSPOSON.

retropseudogene See RETROGENE.

retroregulation A type of post-transcriptional regulation which involves the influence of a cis-acting sequence downstream of the given gene. Ongoing transcription of the gene into the cis-acting sequence results in degradation of the transcript, thus blocking gene expression; if transcription stops before reaching the cis-acting sequence the transcript is available for translation.

retrotranscription Synthesis of a strand of DNA on an RNA template.

(See also REVERSE TRANSCRIPTASE.)

retrotransfer (bacteriol.) Transfer of DNA from the recipient cell, in CONJUGATION, to the donor cell.

retrotransposon (retroposon) A TRANSPOSABLE ELEMENT that

has a mode of transposition involving an RNA intermediate (and the activity of reverse transcriptase); the intermediate is reverse transcribed, and the DNA product is inserted into the target site. One example of a retrotransposon is the so-called

TY ELEMENT of the yeast Saccharomyces cerevisiae.

(See also ALU SEQUENCES.)

Retroviridae A family of ssRNA-containing viruses; members of this family are referred to, collectively, as RETROVIRUSES (q.v.).

retroviruses Enveloped, ssRNA viruses (family Retroviridae) which replicate via a dsDNA intermediate.

Retroviruses are found in a wide range of species, including e.g. mammals, birds and reptiles. (See also COPIA ELEMENT.) Infection of a given host by a retrovirus may be symptomless or may be associated with any of a number of diseases which range from pneumonia and tumors to leukemia and AIDS.

Transmission of retroviruses may occur horizontally and/or vertically.

The following is a generalized account of the retroviruses.

Classification of retroviruses

The retroviruses have been placed in subfamilies on the basis of e.g. the type(s) of pathogenic effect they cause. Viruses of the LENTIVIRINAE (including HIV-1) replicate in – and kill – host cells. The subfamily ONCOVIRINAE includes oncogenic viruses, while viruses of the subfamily SPUMAVIRINAE are characteristically non-pathogenic.

The virion

The heat-sensitive and detergent-sensitive virion, 80–120 nm in diameter, consists of a nucleoprotein core (which includes the viral REVERSE TRANSCRIPTASE) and an envelope of hostderived lipid from which project virus-encoded glycoproteins that determine the types of cell which can be infected by the virus.

All the genetic information is encoded in a single-stranded, positive-sense RNA of about 3–10 kb, according to virus; the virion contains two identical (or similar) copies of this RNA linked non-covalently at the 5′ ends.

The viral RNA has regions of TERMINAL REDUNDANCY flanked, externally, by a 5′ CAP and a 3′ poly(A) tail.

U5 and U3 are sequences unique to the 5′ and 3′ regions of the RNA, respectively; each is adjacent to its corresponding region of terminal redundancy.

Coding regions of the RNA molecule include the gag, pol and env sequences. The gag region encodes proteins of the viral core. The pol region encodes e.g. reverse transcriptase and the viral integrase (the latter involved in integration of the dsDNA provirus into the chromosome of the host cell). The env region encodes e.g. major glycoprotein components of the viral envelope (gp120 and gp41). There is also e.g. a ψ (psi) sequence (which promotes packaging of viral RNA into virions).

Infection of cells

Infection of cells involves interaction between viral envelope proteins and specific type(s) of cell-surface receptor. During infection by HIV-1, the envelope glycoprotein gp120 binds to cell-surface antigen CD4; target cells for this virus therefore include e.g. the (CD4+) ‘helper’ subset of T lymphocytes (i.e. helper T cells). (In addition to the CD4 receptor, infection by HIV-1 is reported to depend on the presence of certain other cell-surface receptor molecules such as receptors for CXCtype and CC-type chemokines – see e.g. HIV-1.)

A PCR-based study reported that, during infection of blood leukocytes by HIV-1 ex vivo, blood dendritic cells were preferentially infected [J Virol (2007) 81(5):2297–2306].

(See also DC-SIGN.)

Reverse transcription and integration of provirus

Within the cell, viral RNA is converted to DNA by the viral reverse transcriptase. Priming involves a host tRNA molecule which binds at the 3′ boundary of the U5 sequence; in many cases tRNApro is used, but tRNAlys is used e.g. by HIV-1.

Following extension of the (first) strand of DNA to the 5′ terminus of the RNA template, the RNA corresponding to the 5′ terminal redundancy region is degraded by RNase H. The resulting (single-stranded) sequence of DNA then hybridizes with the 3′ terminal redundancy region at the other end of the RNA template; extension of the DNA strand then continues on this end of the template.

Because synthesis of the second strand of DNA begins at a staggered site, each end of the final dsDNA product contains a so-called long terminal repeat (LTR) in which U3 and U5 are separated by a sequence corresponding to one copy of the terminal redundancy.

One or more copies of the dsDNA provirus may integrate into the host cell’s DNA. Integration of the provirus DNA is mediated by retroviral integrase, a tetramer of which forms a stable complex with the two ends of the viral DNA; the two ends of the prophage are inserted, sequentially, into the host cell’s chromosome [EMBO J (2006) 25(6):1295–1304].

Expression of the viral genome (the provirus)

Transcription of proviral DNA, processing of transcripts and the subsequent formation of virions are complex processes involving e.g. synthesis of polycistronic pre-mRNAs and the essential contribution of certain virus-encoded proteins. For example, the small early intronless transcripts can translocate readily from the nucleus to the cytoplasm, but larger introncontaining transcripts, formed later, require the REV PROTEIN (in HIV-1) for translocation to the cytoplasm via the CRM1 nuclear export pathway.

Other virus-encoded proteins (in HIV-1) include e.g. the transcription regulatory factor Tat, the Vif (‘viral infectivity factor’) protein and the Vpu protein (which promotes efficient budding of the virion during productive infection of the cell).

The maturation of retroviral RNA and post-transcriptional events leading to production of virions have been reviewed [Retrovirology (2006) 3:18].

Retroviruses in DNA technology

Recombinant (engineered) retroviral vectors have been used e.g. in clinical trials in GENE THERAPY – and also for various studies involving genetic modification of cultured cells.

Factors favorable to the use of retroviruses:

•retroviruses can effect gene transfer to (sensitive) cells with high frequency;

•once integrated in the cell’s genome, the retroviral provirus can usually be expressed on a long-term basis;

•proviral DNA is transmitted to daughter cells;

•high-titer preparations of infectious, replication-deficient

viral particles can be produced;

•retroviruses can be pseudotyped to control host range. Factors unfavorable to the use of retroviruses:

•most retroviruses infect only actively dividing cells; however, lentiviruses can infect both dividing and non-dividing cells (and are used as vectors);

•the retroviral provirus integrates randomly into the DNA of the host cell – with potential lethality; thus, in a gene therapy setting, a knock-out insertion in a tumor-suppressor gene may lead e.g. to tumorigenesis;

•in a laboratory setting retroviral particles are rather labile. Replication-competent retroviral vectors are currently being

studied [J Virol (2007) 81(13):6973–6983].

Rett syndrome An X-linked neurodevelopmental disorder (in humans) commonly resulting from mutation in MECP2, a gene (Xq28) encoding methyl-CpG-binding protein 2, but it can also result from mutation in gene CDKL5 (Xp22), which encodes a cyclin-dependent kinase.

(See also GENETIC DISEASE (table).)

Rev protein A regulatory protein encoded by human immunodeficiency virus type 1 (HIV-1). Activity of the Rev protein is essential for ongoing productive infection by HIV-1; thus, Rev is required e.g. for transporting various intron-containing mRNAs (encoding e.g. envelope and certain other proteins) from the nucleus to the cytoplasm. Rev binds to a complex secondary structure (called the Rev response element, RRE) in appropriate transcripts and mediates their transport from the nucleus.

The Rev protein itself incorporates a nuclear localization signal (NLS) and a nuclear export signal (NES).

Rev activity was found to be inhibited by a dsRNA-binding protein (nuclear factor 90) [Retrovirology (2006) 3:83].

The Rev protein has been studied in a live-cell assay for the simultaneous monitoring of expression and interaction of proteins [BioTechniques (2006) 41(6):688–692].

(See also transdominant negative proteins in the entry GENE

THERAPY.)

reverse genetics Essentially: working/studying in the direction genotype → phenotype, as opposed to the usual (or ‘classic’) approach: phenotype → genotype. Experimental procedures used in reverse genetics include (for example) KNOCK-OUT

MUTATIONS and RNAI.

reverse gyrase A hyperthermophile-specific TOPOISOMERASE, found in the archaean Sulfolobus and in some bacteria, which can introduce positive supercoiling into a ccc dsDNA molecule. It appears to have a helicase-like domain and homology with a bacterial topo I enzyme; reverse gyrase may mediate positive supercoiling in the plasmids of hyperthermophilic organisms.

This enzyme may have a supercoiling-independent, heatprotective function [Nucleic Acids Res (2004) 32(12):3537– 3545]. Other studies indicate that it is not essential for growth at 90°C [J Bacteriol (2004) 186(14):4829– 4833].

Selective degradation of reverse gyrase (and fragmentation of DNA) by the alkylating agent methylmethane sulfonate

(MMS) was reported in the archaean Sulfolobus solfataricus [Nucleic Acids Res (2006) 34(7):2098–2108].

reverse hybridization The procedure (used e.g. in some diagnostic tests) in which target sequences of nucleic acid bind to a set of immobilized probes, i.e. the reverse of the procedure in which the target is immobilized. (cf. REVERSE SOUTHERN

HYBRIDIZATION.)

(See also LINE PROBE ASSAY.)

reverse mutation Syn. BACK MUTATION. reverse primer See FORWARD PRIMER.

reverse Southern hybridization A method used to detect a specific sequence of DNA, in solution, by hybridization to a labeled probe. The probe is initially modified with a psoralen derivative such that, when bound to its target and exposed to ultraviolet radiation, the probe and target sequence become covalently cross-linked. Bound and unbound probes can then be separated e.g. by gel electrophoresis.

reverse transcriptase (RNA-dependent DNA polymerase) An enzyme which can synthesize a strand of DNA on an RNA template. Reverse transcriptases are encoded e.g. by retroviruses as an essential requirement for their intracellular development and chromosomal integration, and are also encoded by some retrovirus-like elements, such as the TY ELEMENT in the yeast Saccharomyces cerevisiae. Genes encoding these enzymes are found in certain bacteria, including Escherichia coli. (See also RETRON.) The DNA polymerase I of E. coli is reported to have (poorly processive) reverse transcriptase activity.

A reverse transcriptase synthesizes the DNA strand 5′-to-3′ from a short RNA primer. Divalent cations are required for enzymic activity. The rTth enzyme (Perkin Elmer/Applied Biosystems) can function as a reverse transcriptase in the presence of Mn2+; on chelation of Mn2+ (with EGTA), the enzyme can behave as a DNA polymerase in the presence of Mg2+. Alternatively, this enzyme can be used in a specialized single-buffer system.

The lack of 3′-to-5′ exonuclease (proof-reading) activity in a reverse transcriptase results in error-prone DNA synthesis.

Some reverse transcriptases have RNase H-like activity – being able to degrade the RNA strand in a DNA/RNA hybrid.

A reverse transcriptase may be inhibited by agents such as

AZT (see NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBIT- ORS) as well as NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS. (See also SURAMIN.)

reverse transcriptase PCR (rtPCR, rt-PCR, RT-PCR; formerly RNA PCR) A PCR-based procedure for making copies of a sequence from an RNA template; rt-PCR has various uses – including studies on gene expression (e.g. detecting mRNAs that are in low concentration in cell lysates), and studies on RNA viruses.

In an early ‘two-tube’ protocol, a strand of DNA is initially synthesized on the RNA target by REVERSE TRANSCRIPTASE activity at ~50°C. The RNA strand is then degraded (e.g. by RNase H) and a complementary strand of DNA is synthesized on the first strand, forming a dsDNA template which is

then amplified by routine PCR methodology.

The above protocol, carried out at ~50°C, can be problematic if the target RNA contains secondary structures which are stable at these temperatures and are able to inhibit DNA synthesis by physically blocking the polymerase. In a more recent protocol, RT-PCR is conducted at higher temperatures using a thermostable enzyme (e.g. RTTH POLYMERASE) that is able to act as both reverse transcriptase and DNA polymerase under appropriate conditions.

A different method for amplifying an RNA target sequence (NASBA) was found to be less inhibited by factors that were inhibitory in an RT-PCR assay of noroviruses in environmental waters [Appl Environ Microbiol (2006) 72(8):5349– 5358].

RF (1) Recombination frequency.

(2) The replicative form of the genome in certain types of bacteriophage: see e.g. PHAGE M13.

RFI The supercoiled replicative form of the genome in certain types of bacteriophage: see e.g. PHAGE M13.

RFLP analysis Restriction fragment length polymorphism analysis: a method used for comparing related sequences of nucleotides (e.g. wild-type and mutant forms of a particular sequence of DNA) by comparing the subfragments produced when these molecules are cleaved by the same RESTRICTION ENDONUCLEASE; different numbers and/or sizes of fragments are produced from a given molecule if that molecule has lost or gained a restriction site (e.g. through a point mutation) or if it has undergone any insertion or deletion of nucleotides. The sets of subfragments from each sample are compared by gel electrophoresis. Differences between samples, in terms of the number and/or position of restriction sites or the existence of insertions or deletions, are manifested by the development of bands at different positions in the gel.

Various types of sample can be examined by this method – for example: viral genomes, plasmids, PCR-generated copies of particular sequences of nucleotides in bacterial and other genomes.

One factor in the sensitivity of RFLP analysis is the choice of restriction endonuclease; this, in turn, is influenced by the composition of the target sequence. Any variation among the samples is more likely to be detected if the chosen enzyme is one which has a greater number of restriction sites within the target sequence. (See also GC%.)

RFLP analysis has various uses which include the typing of bacteria (comparison of corresponding sequences of nucleotides from various strains) and the detection of mutations or polymorphisms (or methylation of specific bases) by detecting their effects on the recognition site of a given restriction enzyme.

The use of RFLP analysis for TYPING bacteria presupposes an appropriate level of stability in the target sequence. This condition may not be satisfied in some cases. Thus, in some strains of Mycobacterium tuberculosis the validity of results from RFLP analysis was questioned because it appeared that the target sequence was evolving too rapidly for the reliable