Glossary of terms used in molecular biology

5' or 3' end The nucleoside residues which form nucleic acids are joined by phosphodiester linkages between the 3' C atom of one ribose moiety and the 5' C atom of the next. Therefore each strand of DNA or RNA has a free 3' C at one end and a free 5' C at the other. The free 3' C normally carries a - OH group, and the 5' C a phosphate group.
Annealing Formation of double-stranded molecules from two single strands of nucleic acid by base pairing of complementary sequence. Usually achieved incubation at a favourable temperature.
Artificial chromosome A vector constructed from host cell chromosomal elements such as origin of replication, telomeres and centromere (in eukaryotes). So that it replicates and is segregated during cell division in the same way as a normal chromosome. The main uses are in preparation of genomic libraries since very large inserts (in the Mbp range) can be integrated. Has also been used in expression studies. Yeast artificial chromosomes (YACs) are the most commonly used form.
Auxotrophic mutant A bacterial strain which has a mutation in at least one of the enzymes in a biochemical pathway responsible for synthesising an essential substance, for example an amino acid. The mutant strain will be unable to grow on minimal salts medium with glucose as sole carbon source unless this is supplemented with the substance which is deficient in synthesis. The mutant phenotype can therefore be easily detected.
Back translation Use of the experimentally determined amino acid sequence of part or all of a polypeptide to determine the theoretical nucleic acid base sequence(s) which could code for it. This is normally done using a computer programme.
Bacteriophage Bacterial virus. Lambda bacteriophage is the basis of many E. coli vectors which are used for cDNA and genomic DNA libraries. Filamentous bacteriophages are used to produce single-stranded DNA for sequencing or in vitro mutagenesis (eg. M13), and as vectors for Phage display expression screening systems
Blotting The process of transferring nucleic acids or proteins from an unstable medium eg electrophoresis gel or agar plate onto nylon or nitrocellulose membrane. This allows the blotted material to be analysed by interaction with a specific, labelled probe to test for the presence of a specific molecular structure. For example a particular nucleic acid base sequence can be recognised by base pairing with a nucleic acid probe of complementary sequence.
Blunt end End of a DNA fragment produced by a restriction enzyme which cuts both strands of DNA at the same point, leaving no single-stranded sections. DNA with this type of end is more difficult to ligate
cDNA DNA which has been synthesised using an RNA template (cDNA = copy or complementary DNA). Most frequently used to describe double-stranded DNA copies of mRNA sequences eg. cDNA libraries. May be single or double stranded.
Clone A collection of cells or organisms which have identical genomes. Sometimes used to describe a preparation of identical vector molecules. Cloning is the term used to describe the production and isolation of large numbers of copies of a specific DNA sequence using a mixture of sequences as a starting point.
Cloning site A restriction enzyme site or group of sites (= multiple cloning site) located in a vector at the best position for insertion of DNA.
Colony lift Blot taken from colonies (normally bacterial) growing on an agar plate. Normally used for detection of a colony containing a plasmid with a specific inserted sequence (screening).
Cosmid An artificial hybrid vector into which large (~40kbp) DNA fragments can be inserted; this makes it useful in genomic library preparation. Cosmids replicate as plasmids in the host cell, but are inserted into the bacterium from lambda phage infectious particles, since transformation would be extremely inefficient with plasmids of this size. The cosmids carry cos sequences from lambda phage so that they can be packaged into phage heads (in vitro ), and circularise once in the cell.
Covalent bonds Links between atoms formed by sharing of two electrons, one from each atom. These bonds are strong, requiring significant energy for breakage. They are the bonds between the atoms forming the basic structures of biological molecules.

Degeneracy Backtranslation of amino acid sequences usually leads to a collection of possible base sequences which can code for the amino acid sequence, due to the degeneracy of the genetic code. In practice, when synthesising an oligonucleotide primer or probe based on this information a mixture of all the possible sequences is made, and referred to as a degenerate oligonucleotide.
Denaturation Separation of two complementary strands of nucleic acid by breakage of the hydrogen bonds involved in base pairing. This is necessary prior to probe hybridisation and most methods involving enzymic DNA synthesis on a DNA template e.g. sequencing, PCR, some labelling methods. Denaturation may be achieved by heating or treatment with NaOH.
Differential display A form of RT-PCR in which primers are used to select a subset of the total mRNA population. This allows comparison of mRNAs from different cells. leading to identification of those mRNAs which are expressed only in certain situations e.g. after stimulation.
dNTP deoxyribonucleoside triphosphate
Dot Blot The simplest form of blot. Prepared by pipetting known volumes of DNA or RNA mixtures onto a blotting membrane. The blot is probed in the normal way, allowing detection and quantitation of molecules carrying specific sequence in the original mixtures.
End-labelling Transfer of 32P to the 5' end(s) of a DNA or RNA molecule, using polynucleotide kinase.
Exons The coding sections of eukarotic genes, separated by introns.
Expression vector A vector which is designed to allow expression (transcription and translation) of the inserted section of DNA. The vector carries a promoter (normally inducible) on one side of the cloning site, and a transcription terminator on the other side. These must be recognisable by the intended host cell. The vector may also carry a ribosome binding sequence (for bacterial expression) and a start codon, depending on the nature of the inserted DNA. Some expression vectors produce fusion proteins.
Filling-in In vitro  synthesis of DNA (by DNA polymerase) to convert an overhanging end of a double-stranded DNA fragment to a blunt end.
Fusion protein A recombinant protein which is composed of amino acid sequences derived from more than one gene. Produced by insertion of coding DNA sequence "in frame" within a gene, followed by expression from the promoter of the original gene..
Genomic DNA The DNA contained in the chromosomes of a cell.

Homopolymer tailing Extension of the 3' ends of a piece of double-stranded DNA using the enzyme terminal transferase and a single deoxynucleoside triphosphate as substrate, resulting in a 3' overhang composed of a single base repeat. If DNA which is to be annealed to this is tailed with the complementary base, mutually cohesive ends are produced. The tailed molecules are unable to circularise because their ends are not cohesive. Sometimes used for insertion of DNA into vectors.
Hybridisation Formation of a double-stranded nucleic acid molecule by complementary base pairing of two single strands from different sources. DNA-RNA hybrids are possible. Describes the process in which a nucleic acid probe is allowed to bind to and detect a complementary sequence on a blot.
Hydrogen bond A weak bond between molecules, or different parts of the same molecule, formed by a restricted form of sharing of electrons between atoms. Readily formed between -OH (hydroxyl) and NH2 (amino) groups and oxygen or nitrogen atoms. They are important in proteins and are the mechanism for base-pairing in nucleic acids. They are easily disrupted by heat. They are responsible for base-pairing in nucleic acids and for the maintenance of 3D structure in proteins.
Introns Sequences of non-coding bases found in eukaryotic genes. They may make up a large proportion of the total gene length. Introns are spliced out of the RNA transcript during its processing to mRNA.
kbp kilobase pairs. A measure of size of double-stranded DNA.
Kinasing = phosphorylation. Addition of a phosphate group to the 5' ends of DNA or RNA molecules, using polynucleotide kinase.

Library A collection of clones or DNA fragments which contains all of the sequences present in the source. Thus a genomic library contains all of the sequences present in the genome and a cDNA library contains copies of all the mRNA molecules present in the extracted cells.
Ligation In vitro reaction used to make the covalent phosphodiester linkage between two double stranded DNA ends. The reaction requires the presence of a phosphate group on the free 5' end and a -OH group on the free 3' end of the strands to be joined. The reaction is catalysed by the enzyme DNA ligase.
Lysogen Bacterial cell or strain which carries a copy of a bacteriophage genome in its chromosome. Genes within the prophage will be expressed providing their promoters are active.
Marker A gene which, on expression, allows easy identification of cells which carry it. Normally used to describe genes carried by a vector which are used to detect vector presence or state in a host cell.
Mitochondrial DNA Mitochondria, and chloroplasts in plants, carry their own small chromosomes, usually in multiple copies per organelle. These carry a limited number of genes which code for rRNA, tRNA and a few organelle proteins. This DNA is maternally inherited.
N- or C-terminal The amino acids which form polypeptides are joined by peptide bonds between the amino group of one amino acid and the carboxy group of the next. Therefore each polypeptide has a free amino group at one end (N-terminal) and a free carboxy group at the other (C-terminal).
Northern Blot RNA blot taken from an electrophoresis gel.
Nucleic acid A polymeric molecule composed of nucleotide monomers joined by phosphodiester bonds. These link the 5' ribose carbon atom of one nucleotide residue with the 3' ribose carbon atom of the neighbouring nucleotide residue, through a phosphate group. Each phosphate group ionises by losing a hydrogen ion thus becoming negatively charged, and giving the molecule its acidic character. The negative charge of nucleic acids is made use of in electrophoretic separations.
Nucleoside In molecular biology; a molecule composed of a sugar (2' deoxyribose in DNA; ribose in RNA) which is linked to a purine (adenine or guanine) or a pyrimidine (thymine (DNA), cytidine or uridine (RNA)). The link is through the 1' carbone atom in ribose or deoxyribose.
Nucleotide In molecular biology; the phosphate ester of a nucleoside, it may carry one, two or three phosphates linked to each other e.g. adenosine mono-, di- and triphosphates (AMP, ADP and ATP). The phosphates are carried on the 5' carbon atom of the ribose or deoxyribose part of the molecule. See also nucleic acid and oligonucleotide.

ORF Open reading frame. A continuous sequence of nucleotide triplets (codons) in which each triplet codes for an amino acid.
Oligonucleotide A short nucleic acid molecule; normally refers to molecules between 5 and 200 nucleotide residues(bases) long.
Origin of replication (ori ) Section of DNA sequence which is recognised by a cell's DNA replication proteins, allowing initiation of new DNA synthesis. DNA molecules which do not carry an ori  recognised by the host cell will be eventually lost from a growing population unless they are incorporated into the cell's genome.
Overhang Single-stranded section of DNA at the end of a double-stranded fragment, for example in a sticky end. Overhangs may be 3' or 5' i.e. the end of the single-stranded section may carry a free 3' or 5' end. This will depend on how the overhang was created.

PCR Polymerase chain reaction. An in vitro  technique to produce many copies of a specific section of DNA sequence. PCR is normally used to amplify sections up to ~2kbp in length, although routine PCR of sections up to 20kbp is becoming possible. PCR amplification is possible from complex sequence background e.g. a short sequence from an entire chromosome, and from impure DNA. The technique is widely used in many applications.
Phage display Phage display vectors express the inserted DNA as a protein at a prominent position on their capsid. This allows capture and isolation of recombinant phage clones by immobilised interacting proteins (eg. antibodies). This screening method is called biopanning.
Phagemid A plasmid-based vector which carries a filamentous phage ori , leading to production of single-stranded copies of the phagemid when the cell is infected with the relevant helper phage. Under normal circumstances the phagemid behaves in exactly the same way as a plasmid
Phosphatasing = dephosphorylating. Removal of 5' phosphate from an RNA or DNA molecule, using alkaline phosphatase.
Plaque lift Blot taken from plaques (usually bacteriophage on a bacterial lawn) growing on an agar plate. Normally used for detection of a plaque containing a phage with a specific inserted sequence (screening).
Plasmid Double stranded, circular DNA molecule found in bacteria and some fungi. Size range from ~5 - 200kbp, contains genes which may be useful to host, but are not essential. Plasmids can be eliminated from bacterial populations by growth under selective conditions ("curing"). Carry an origin of replication (ori ) which allows replication in a specific host species. Plasmids used in recombinant DNA work are generally smaller than 10kbp in size and have a copy number of up to 500/cell depending on plasmid type.
Polishing In vitro  digestion of the single-stranded section of an overhanging end (by exonuclease) to produce a blunt end.

Probe A labelled molecule which will recognise and bind to a specific target molecule, thus allowing detection of the target. DNA probes are used to locate and quantitate DNA (or RNA) of complementary sequence. Antibody probes may also be used in aspects of recombinant DNA work, e.g. screening an expression library. The label may be radioactive (32P), biotin or digoxygenin (DIG).
Promoter A short base sequence which is positioned close to the 5" end of a gene and acts as a recognition and binding site for the RNA polymerase complex prior to transcription of the gene.
RAPD Random amplification of polymorphic DNA. A method for identifying differences between genomes of different individuals by PCR with a single short (usually 10-base) primer, which will anneal with complementary sequence at undetermined positions in the genome. The products form a type of "genetic fingerprint".
Recombinant A DNA or protein molecule produced as a result of assembling and joining DNA sequences from different sources. Sometimes used to refer to an organism carrying such a gene.
Recombination A natural cellular process through which DNA molecules of similar or identical sequence can be exchanged. This process is the molecular basis of crossing over during meiosis and some DNA repair mechanisms. Another form of recombination, site-specific recombination, is found where transfer, rearrangement or insertion of specific sections of DNA occurs e.g. insertion of viral DNA into chromosomes, gene switching etc.
Repeat (repetitive) sequence DNA  Sections of DNA in which short base sequences are repeated from several to many times.

Reporter gene Gene coding for an easily assayed protein which is used to detect expression of the gene under different conditions; usually to test the activity of a promoter. The ß- galactosidase, luciferase and Green Fluorescent Protein genes are examples of reporter genes.
Restriction enzyme More correctly called a Type II Restriction Endonuclease. A bacterial enzyme which forms part of a system to protect the cell against infection by bacteriophage, and unregulated influx of foreign DNA. Binds to DNA at a short specific base sequence, and cuts both strands between specific bases in this sequence.
Restriction map A diagram of all or part of a dsDNA molecule which indicates the positions (in base pairs) of restriction enzyme recognition/cutting sites. Used extensively in identification of DNA fragments and in developing procedures for manipulating cloned DNA.
RFLP Restriction fragment length polymorphism. A difference in restriction fragment length between individuals due to loss or gain of a restriction enzyme site due to point mutation, or insertion or deletion between consecutive sites. Normally detected by Southern blotting and probing. Used in detection of genetic disease alleles etc.
RT-PCR PCR amplification from an RNA template. The first step involves synthesis of a single strand of cDNA on the RNA template using reverse transcriptase. The cDNA is then used as the template for PCR to produce a DNA product.

Satellite DNA A genomic DNA fraction which has a different density to the main body of genomic DNA and therefore forms a separate band (satellite) during density gradient ultracentrifugation. The difference in density is due to a bias in base content caused by the presence of repeat sequence DNA.
Selectable marker A gene which is usually constitutively expressed and allows the selection of cells which carry it through growth on a selective medium. The most common example is the use of the ß-lactamase gene in plasmid vectors to confer ampicillin resistance on the host cell.
Sequence database A computer-based collection of nucleic acid or amino acid sequences containing all published and some unpublished sequences. These can be searched to check for similarities with newly determined sequences. Examples are Genbank and Swissprot.
Southern Blot DNA blot taken from an electrophoresis gel. The original blot type named after its originator Ed Southern,
Sticky end Short section of single-stranded DNA produced at the end of a double-stranded DNA fragment produced by DNA digestion with certain restriction enzymes. The single- stranded section will base-pair and "stick" loosely to another sticky end produced by the same enzyme, facilitating ligation..
Synthetic oligonucleotide A single strand of DNA made by machine and used for priming DNA synthesis in sequencing or PCR, or as a probe. The maximum achievable length is around 50 bases, but longer molecules can be constructed using oligonucleotides as building blocks.
Transcription terminator A short base sequence found at the 3' end of a gene which causes the RNA polymerase to stop transcription.
Transfection A general term to describe the introduction of recombinant or vector DNA into host cells.
Transformation Usually refers to passive uptake of DNA by cells, although in nature some bacterial species have a specific active uptake system. Transformation is normally used to introduce plasmids into bacterial cells, but the process is increasingly inefficient with increasing size of plasmid. E. coli cells must be made competent before transformation can be carried out.
Transgenic Refers to an organism which carries a stable copy of a gene originating from another species introduced by DNA technology.
Transposable element Section of DNA which can copy itself and insert the copy into another position in the chromosome or another chromosome. Free transposons are not found; they spread via transposition into mobile DNA molecules such as plasmids. Bacterial transposonsmay carry, in addition to genes encoding enzymes needed for transposition, genes for antibiotic resistance or other functions.
Vector Generally a carrier nucleic acid molecule which allows transfer of inserted (recombinant) DNA into a host cell. Normally refers to molecules such as plasmids and bacteriophages which replicate so that they are maintained in the host cell population. Vector replication which is independent of the host cell DNA replication may allow amplification of the vector to produce large numbers of copies (as in cloning).
VNTR Variable number tandem repeat. A type of DNA sequence found in eukaryotic genomes, in which a short sequence is repeated. The number of repeats varies between individuals and is used as a basis for genetic identification.
Western Blot Protein blot taken from an electrophoresis gel. Normally probed with a specific antibody.

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