Definitions

Acrylamide gels: A polymer gel used for electrophoresis of DNA or protein to measure their sizes (in daltons for proteins, or in base pairs for DNA). See "Gel Electrophoresis". Acrylamide gels are especially useful for high resolution separations of DNA in the range of tens to hundreds of nucleotides in length.

Agarose gels: A polysaccharide gel used to measure the size of nucleic acids (in bases or base pairs). See "Gel Electrophoresis". This is the gel of choice for DNA or RNA in the range of thousands of bases in length, or even up to 1 megabase if you are using pulsed field gel electrophoresis.

Antibiotic resistance: Plasmids generally contain genes which confer on the host bacterium the ability to survive a given antibiotic. If the plasmid pBR322 is present in a host, that host will not be killed by (moderate levels of) ampicillin or tetracycline. By using plasmids containing antibiotic resistance genes, the researcher can kill off all the bacteria which have not taken up his plasmid, thus ensuring that the plasmid will be propagated as the surviving cells divide.

AP-1 site: The binding site on DNA at which the transcription "factor" AP-1 binds, thereby altering the rate of transcription for the adjacent gene. AP-1 is actually a complex between c-fos protein and c-jun protein, or sometimes is just c-jun dimers. The AP-1 site consensus sequence is (C/G)TGACT(C/A)A. Also known as the TPA-response element (TRE). [TPA is a phorbol ester, tetradecanoyl phorbol acetate, which is a chemical tumor promoter]

ATG or AUG: The codon for methionine; the translation initiation codon. Usually, protein translation can only start at a methionine codon (although this codon may be found elsewhere within the protein sequence as well). In eukaryotic DNA, the sequence is ATG; in RNA it is AUG. Usually, the first AUG in the mRNA is the point at which translation starts, and an open reading frame follows - i.e. the nucleotides taken three at a time will code for the amino acids of the protein, and a stop codon will be found only when the protein coding region is complete.

BAC: Bacterial Artificial Chromosome — a cloning vector capable of carrying between 100 and 300 kilobases of target sequence. They are propagated as a mini-chromosome in a bacterial host. The size of the typical BAC is ideal for use as an intermediate in large-scale genome sequencing projects. Entire genomes can be cloned into BAC libraries, and entire BAC clones can be shotgun-sequenced fairly rapidly.

Bacteriophage lambda: A virus which infects E. coli , and which is often used in molecular genetics experiments as a vector, or cloning vehicle. Recombinant phages can be made in which certain non-essential l DNA is removed and replaced with the DNA of interest. The phage can accommodate a DNA "insert" of about 15-20 kb. Replication of that virus will thus replicate the investigator's DNA. One would use phage l rather than a plasmid if the desired piece of DNA is rather large.

Binding site: A place on cellular DNA to which a protein (such as a transcription factor) can bind. Typically, binding sites might be found in the vicinity of genes, and would be involved in activating transcription of that gene (promoter elements), in enhancing the transcription of that gene (enhancer elements), or in reducing the transcription of that gene (silencers). NOTE that whether the protein in fact performs these functions may depend on some condition, such as the presence of a hormone, or the tissue in which the gene is being examined. Binding sites could also be involved in the regulation of chromosome structure or of DNA replication.

Cap: All eukaryotes have at the 5' end of their messages a structure called a "cap", consisting of a 7-methylguanosine in 5'-5' triphosphate linkage with the first nucleotide of the mRNA. It is added post-transcriptionally, and is not encoded in the DNA.

Cap site: Two usages: In eukaryotes, the cap site is the position in the gene at which transcription starts, and really should be called the "transcription initiation site". The first nucleotide is transcribed from this site to start the nascent RNA chain. That nucleotide becomes the 5' end of the chain, and thus the nucleotide to which the cap structure is attached (see "Cap"). In bacteria, the CAP site (note the capital letters) is a site on the DNA to which a protein factor (the Catabolite Activated Protein) binds.

CCAAT box: (CAT box, CAAT box, other variants) A sequence found in the 5' flanking region of certain genes which is necessary for efficient expression. A transcription factor (CCAAT-binding protein, CBP) binds to this site.

cDNA clone: "complementary DNA"; a piece of DNA copied from an mRNA. The term "clone" indicates that this cDNA has been spliced into a plasmid or other vector in order to propagate it. A cDNA clone may contain DNA copies of such typical mRNA regions as coding sequence, 5'-untranslated region, 3' untranslated region or poly(A) tail. No introns will be present, nor any promoter sequences (or other 5' or 3' flanking regions). A "full-length" cDNA clone is one which contains all of the mRNA sequence from nucleotide #1 through to the poly(A) tail.

cDNA library : It usually consist of just a mixture of bacteria, where each bacteria carries a different plasmid. Inserted into the plasmids (one per plasmid) are thousands of different pieces of cDNA (each typ. 500-5000 bp) copied from some source of mRNA, for example, total liver mRNA. The basic idea is that if you have a large enough number of different liver-derived cDNAs carried in those bacteria, there is a 99% probability that a cDNA copy of any given liver mRNA exists somewhere in the tube. The real trick is to find the one you want out of that mess - a process called screening (see "Screening").

Clone (verb): To "clone" something is to produce copies of it. To clone a piece of DNA, one would insert it into some type of vector (say, a plasmid) and put the resultant construct into a host (usually a bacterium) so that the plasmid and insert replicate with the host. An individual bacterium is isolated and grown and the plasmid containing the "cloned" DNA is re-isolated from the bacteria, at which point there will be many millions of copies of the DNA - essentially an unlimited supply. Actually, an investigator wishing to clone some gene or cDNA rarely has that DNA in a purified form, so practically speaking, to "clone" something involves screening a cDNA or genomic library for the desired clone. See also "Probe" for a description of how one might start a cloning project, and "Screening" for how the probe in used.

One can also clone more complex organisms, with considerable difficulty. The much-publicized Scottish research that resulted in the sheep ‘Dolly’ exemplifies this approach.

Clone (noun): The term "clone" can refer either to a bacterium carrying a cloned DNA, or to the cloned DNA itself. If you receive a clone from a collaborator, you should first figure out if they send you DNA or bacteria. If it is DNA, your first job is to introduce it ("transform" it) into bacteria [see "Transformation (with respect to bacteria)"]. Occasionally, someone might send just the "insert", rather than the whole plasmid. "Your assignment, Jim, if you decide to accept it", is to splice that DNA into a convenient vector, and only then can you transform it into bacteria.

Coding sequence: The portion of a gene or an mRNA which actually codes for a protein. Introns are not coding sequences; nor are the 5' or 3' untranslated regions (or the flanking regions, for that matter - they are not even transcribed into mRNA). The coding sequence in a cDNA or mature mRNA includes everything from the AUG (or ATG) initiation codon through to the stop codon, inclusive.

Coding strand: an ambiguous term intended to refer to one specific strand in a double-stranded gene. See "Sense strand".

Codon: In an mRNA, a codon is a sequence of three nucleotides which codes for the incorporation of a specific amino acid into the growing protein. The sequence of codons in the mRNA unambiguously defines the primary structure of the final protein. Of course, the codons in the mRNA were also present in the genomic DNA, but the sequence may be interrupted by introns.

Consensus sequence: A ‘nominal’ sequence inferred from multiple, imperfect examples. Multiple lanes of shotgun sequence can be merged to show a consensus sequence. The optimal sequence of nucleotides recognized by some factor. A DNA binding site for a protein may vary substantially, but one can infer the consensus sequence for the binding site by comparing numerous examples. For example, the (fictitious) transcription factor ZQ1 usually binds to the sequences AAAGTT, AAGGTT or AAGATT. The consensus sequence for that factor is said to be AARRTT (where R is any purine, i.e. A or G). ZQ1 may also be able to weakly bind to ACAGTT (which differs by one base from the consensus).

Endonuclease: An enzyme which digests nucleic acids starting in the middle of the strand (as opposed to an exonuclease, which must start at an end). Examples include the restriction enzymes, DNase I and RNase A.

Endonuclease: An enzyme which digests nucleic acids starting in the middle of the strand (as opposed to an exonuclease, which must start at an end). Examples include the restriction enzymes, DNase I and RNase A.

Enhancer: An enhancer is a nucleotide sequence to which transcription factor(s) bind, and which increases the transcription of a gene. It is NOT part of a promoter; the basic difference being that an enhancer can be moved around anywhere in the general vicinity of the gene (within several thousand nucleotides on either side or even within an intron), and it will still function. It can even be clipped out and spliced back in backwards, and will still operate. A promoter, on the other hand, is position- and orientation-dependent. Some enhancers are "conditional" - in other words, they enhance transcription only under certain conditions, for example in the presence of a hormone.

Exon: Those portions of a genomic DNA sequence which WILL be represented in the final, mature mRNA. The term "exon" can also be used for the equivalent segments in the final RNA. Exons may include coding sequences, the 5' untranslated region or the 3' untranslated region.

Exonuclease: An enzyme which digests nucleic acids starting at one end. An example is Exonuclease III, which digests only double-stranded DNA starting from the 3' end.

Expression: To "express" a gene is to cause it to function. A gene which encodes a protein will, when expressed, be transcribed and translated to produce that protein. A gene which encodes an RNA rather than a protein (for example, a rRNA gene) will produce that RNA when expressed.

Gel electrophoresis: A method to analyze the size of DNA (or RNA) fragments. In the presence of an electric field, larger fragments of DNA move through a gel slower than smaller ones. If a sample contains fragments at four different discrete sizes, those four size classes will, when subjected to electrophoresis, all migrate in groups, producing four migrating "bands". Usually, these are visualized by soaking the gel in a dye (ethidium bromide) which makes the DNA fluoresce under UV light. 

Gene: A unit of DNA which performs one function. Usually, this is equated with the production of one RNA or one protein. A gene contains coding regions, introns, untranslated regions and control regions.

Genome: The total DNA contained in each cell of an organism. Mammalian genomic DNA (including that of humans) contains 6x109 base pairs of DNA per diploid cell. There are somewhere in the order of a hundred thousand genes, including coding regions, 5' and 3' untranslated regions, introns, 5' and 3' flanking DNA. Also present in the genome are structural segments such as telomeric and centromeric DNAs and replication origins, and intergenic DNA.

Genomic library: It is similar in concept to a cDNA library, but differs in three major ways - 1) the library carries pieces of genomic DNA (and so contains introns and flanking regions, as well as coding and untranslated); 2) you need bacteriophage l or cosmids, rather than plasmids, because... 3) the inserts are usually 5-15 kb long (in a l library) or 20-40 kb (in a cosmid library). Therefore, a genomic library is most commonly a tube containing a mixture of l phages. Enough different phages must be present in the library so that any given piece of DNA from the source genome has a 99% probability of being present.

hnRNA: Heterogeneous nuclear RNA; refers collectively to the variety of RNAs found in the nucleus, including primary transcripts, partially processed RNAs and snRNA. The term hnRNA is often used just for the unprocessed primary transcripts, however.

Host strain (bacterial): The bacterium used to harbor a plasmid. Typical host strains include HB101 (general purpose E. coli strain), DH5a (ditto), JM101 and JM109 (suitable for growing M13 phages), XL1-Blue (general-purpose, good for blue/white lacZ screening). Note that the host strain is available in a form with no plasmids (hence you can put one of your own into it), or it may have plasmids present (especially if you put them there). Hundreds, perhaps thousands, of host strains are available.

Intron: Introns are portions of genomic DNA which ARE transcribed (and thus present in the primary transcript) but which are later spliced out. They thus are not present in the mature mRNA. Note that although the 3' flanking region is often transcribed, it is removed by endonucleolytic cleavage and not by splicing. It is not an intron. 

Klenow fragment: Refers to large protein fragment of DNA polymerase - I formed due to selective proteolysis, it retains polymerization and 3'→ 5' exonuclease activity, but has lost 5'→ 3' exonuclease activity. Klenow protein fragment retains the polymerization fidelity of the holoenzyme without degrading 5' termini. 

Kozak sequence - It is a conserved nucleotide sequence (5′- GCC(A/G)CCAUGG -3′) in eukaryotic mRNA that surrounds the start codon (AUG) and helps the ribosome identify the correct site for translation initiation. 

Library: A library might be either a genomic library, or a cDNA library. In either case, the library is just a tube carrying a mixture of thousands of different clones - bacteria or l phages. Each clone carries an "insert" - the cloned DNA.

Ligase: An enzyme, T4 DNA ligase, which can link pieces of DNA together. The pieces must have compatible ends (both of them blunt, or else mutually compatible sticky ends), and the ligation reaction requires ATP. 

Molecular weight size marker: a piece of DNA of known size, or a mixture of pieces with known size, used on electrophoresis gels to determine the size of unknown DNA’s by comparison.

Genetic marker: A known site on the chromosome. It might for example be the site of a locus with some recognizable phenotype, or it may be the site of a polymorphism that can be experimentally discerned. See 'Microsatellite', 'SNP', 'Genotyping'.

mRNA: "messenger RNA" or sometimes just "message"; an RNA which contains sequences coding for a protein. The term mRNA is used only for a mature transcript with polyA tail and with all introns removed, rather than the primary transcript in the nucleus. As such, an mRNA will have a 5' untranslated region, a coding region, a 3' untranslated region and (almost always) a poly(A) tail. Typically about 2% of the total cellular RNA is mRNA. 

Nick translation: A method for incorporating radioactive isotopes (typically 32P) into a piece of DNA. The DNA is randomly nicked by DNase I, and then starting from those nicks DNA polymerase I digests and then replaces a stretch of DNA. Radiolabeled precursor nucleotide triphosphates can thus be incorporated. 

Nuclease: An enzyme which degrades nucleic acids. A nuclease can be DNA-specific (a DNase), RNA-specific (RNase) or non-specific. It may act only on single stranded nucleic acids, or only on double-stranded nucleic acids, or it may be non-specific with respect to strandedness. A nuclease may degrade only from an end (an exonuclease), or may be able to start in the middle of a strand (an endonuclease). To further complicate matters, many enzymes have multiple functions; for example, Bal31 has a 3'-exonuclease activity on double-stranded DNA, and an endonuclease activity specific for single-stranded DNA or RNA. 

Open reading frame: Any region of DNA or RNA where a protein could be encoded. In other words, there must be a string of nucleotides (possibly starting with a Met codon) in which one of the three reading frames has no stop codons. See "Reading frame" for a simple example.

Origin of replication: Nucleotide sequences present in a plasmid which are necessary for that plasmid to replicate in the bacterial host. (Abbr. "ori")

PolyA tail: After an mRNA is transcribed from a gene, the cell adds a stretch of A residues (typically 50-200) to its 3' end. It is thought that the presence of this "polyA tail" increases the stability of the mRNA (possibly by protecting it from nucleases). Note that not all mRNAs have a polyA tail; the histone mRNAs in particular do not.

Polymerase: An enzyme which links individual nucleotides together into a long strand, using another strand as a template. There are two general types of polymerase — DNA polymerases (which synthesize DNA) and RNA polymerase (which makes RNA). Within these two classes, there are numerous sub-types of polymerase, depending on what type of nucleic acid can function as template and what type of nucleic acid is formed. A DNA-dependant DNA polymerase will copy one DNA strand starting from a primer, and the product will be the complementary DNA strand. A DNA-dependant RNA polymerase will use DNA as a template to synthesize an RNA strand.

Polymerase chain reaction: A technique for replicating a specific piece of DNA in-vitro , even in the presence of excess non-specific DNA. Primers are added (which initiate the copying of each strand) along with nucleotides and Taq polymerase. By cycling the temperature, the target DNA is repetitively denatured and copied. A single copy of the target DNA, even if mixed in with other undesirable DNA, can be amplified to obtain billions of replicates. PCR can be used to amplify RNA sequences if they are first converted to DNA via reverse transcriptase. This two-phase procedure is known as ‘RT-PCR’.

Polymerase Chain Reaction (PCR) is the basis for a number of extremely important methods in molecular biology. It can be used to detect and measure vanishingly small amounts of DNA and to create customized pieces of DNA. It has been applied to clinical diagnosis and therapy, to forensics and to vast numbers of research applications. It would be difficult to overstate the importance of PCR to science.

Post-transcriptional regulation: Any process occurring after transcription which affects the amount of protein a gene produces. Includes RNA processing efficiency, RNA stability, translation efficiency, protein stability. For example, the rapid degradation of an mRNA will reduce the amount of protein arising from it. Increasing the rate at which an mRNA is translated will increase the amount of protein product.

Post-translational processing: The reactions which alter a protein's covalent structure, such as phosphorylation, glycosylation or proteolytic cleavage.

Post-translational regulation: Any process which affects the amount of protein produced from a gene, and which occurs AFTER translation in the grand scheme of genetic expression. Actually, this is often just a buzz-word for regulation of the stability of the protein. The more stable a protein is, the more it will accumulate.

Primary transcript: When a gene is transcribed in the nucleus, the initial product is the primary transcript, an RNA containing copies of all exons and introns. This primary transcript is then processed by the cell to remove the introns, to cleave off unwanted 3' sequence, and to polyadenylate the 3' end. The mature message thus formed is then exported to the cytoplasm for translation.

Primer: A small oligonucleotide (anywhere from 6 to 50 nt long) used to prime DNA synthesis. The DNA polymerases are only able to extend a pre-existing strand along a template; they are not able to take a naked single strand and produce a complementary copy of it de-novo. A primer which sticks to the template is therefore used to initiate the replication. Primers are necessary for DNA sequencing and PCR.

Promoter: The first few hundred nucleotides of DNA "upstream" (on the 5' side) of a gene, which control the transcription of that gene. The promoter is part of the 5' flanking DNA, i.e. it is not transcribed into RNA, but without the promoter, the gene is not functional. Note that the definition is a bit hazy as far as the size of the region encompassed, but the "promoter" of a gene starts with the nucleotide immediately upstream from the cap site, and includes binding sites for one or more transcription factors which can not work if moved farther away from the gene. 

Pulsed field gel electrophoresis: (PFGE) A gel technique which allows size-separation of very large fragments of DNA, in the range of hundreds of kb to thousands of kb. As in other gel electrophoresis techniques, populations of molecules migrate through the gel at a speed related to their size, producing discrete bands. In normal electrophoresis, DNA fragments greater than a certain size limit all migrate at the same rate through the gel. In PFGE, the electrophoretic voltage is applied alternately along two perpendicular axes, which forces even the larger DNA fragments to separate by size. 

Repetitive DNA: A surprising portion of any genome consists not of genes or structural elements, but of frequently repeated simple sequences. These may be short repeats just a few nt long, like CACACA etc. They can also range up to a few hundred nt long. Examples of the latter include Alu repeats, LINEs, SINEs. The function of these elements is often unknown. In shorter repeats like di- and tri-nucleotide repeats, the number of repeating units can occasionally change during evolution and descent. They are thus useful markers for familial relationships and have been used in paternity testing, forensic science and in the identification of human remains 

Reverse transcriptase: An enzyme which will make a DNA copy of an RNA template - a DNA-dependant RNA polymerase. RT is used to make cDNA; one begins by isolating polyadenylated mRNA, providing oligo-dT as a primer, and adding nucleotide triphosphates and RT to copy the RNA into cDNA. 

RNAi: 'RNA interference' (a.k.a. 'RNA silencing') is the mechanism by which small double-stranded RNAs can interfere with expression of any mRNA having a similar sequence. Those small RNAs are known as 'siRNA', for short interfering RNAs. The mode of action for siRNA appears to be via dissociation of its strands, hybridization to the target RNA, extension of those fragments by an RNA-dependent RNA polymerase, then fragmentation of the target. Importantly, the remnants of the target molecule appears to then act as an siRNA itself; thus the effect of a small amount of starting siRNA is effectively amplified and can have long-lasting effects on the recipient cell. The RNAi effect has been exploited in numerous research programs to deplete the call of specific messages, thus examining the role of those messages by their absence.

rRNA: "ribosomal RNA"; any of several RNAs which become part of the ribosome, and thus are involved in translating mRNA and synthesizing proteins. They are the most abundant RNA in the cell (on a mass basis). 

Sense strand: A gene has two strands: the sense strand and the anti-sense strand. The Sense strand is, by definition, the same 'sense' as the mRNA; that is, it can be translated exactly as the mRNA sequence can. 

Shine -Dalgarno sequence: It is a short, purine-rich sequence of nucleotides (usually AGGAGG) found in the 5′ untranslated region (UTR) of prokaryotic mRNA. It lies a few bases upstream of the start codon (AUG) and pairs with a complementary sequence on the 16S rRNA of the small ribosomal subunit. This base pairing helps position the ribosome correctly at the start codon, ensuring accurate initiation of translation in prokaryotes. 

Stoffel fragment: Highly active 544 amino acid length Taq DNA polymerase without 5' to 3' exonuclease activity. Stoffel Fragment works optimally at a broader range of MgCl2 concentrations (2-10 mM) as compared to original Taq polymerase. It is easier and faster to optimize and useful for multiplex reactions. 

Taq polymerase: A DNA polymerase isolated from the bacterium Thermophilis aquaticus and which is very stable to high temperatures. It is used in PCR procedures and high temperature sequencing.

TATA box: A sequence found in the promoter (part of the 5' flanking region) of many genes. Deletion of this site (the binding site of transcription factor TFIID) causes a marked reduction in transcription, and gives rise to heterogeneous transcription initiation sites.

Tautomer - It is one of two (or more) structural isomeric forms of the same molecule that can interconvert by the movement of a proton (H⁺) and a shift of a double bond.

Transcription factor: A protein which is involved in the transcription of genes. These usually bind to DNA as part of their function (but not necessarily). A transcription factor may be general (i.e. acting on many or all genes in all tissues), or tissue-specific (i.e. present only in a particular cell type, and activating the genes restricted to that cell type). Its activity may be constitutive, or may depend on the presence of some stimulus; for example, the glucocorticoid receptor is a transcription factor which is active only when glucocorticoids are present.

Transcription: The process of copying DNA to produce an RNA transcript. This is the first step in the expression of any gene. The resulting RNA, if it codes for a protein, will be spliced, polyadenylated, transported to the cytoplasm, and by the process of translation will produce the desired protein molecule.

Transfection: A method by which experimental DNA may be put into a cultured mammalian cell. Such experiments are usually performed using cloned DNA containing coding sequences and control regions (promoters, etc) in order to test whether the DNA will be expressed. Since the cloned DNA may have been extensively modified (for example, protein binding sites on the promoter may have been altered or removed), this procedure is often used to test whether a particular modification affects the function of a gene.

Transformation (with respect to bacteria): The process by which a bacteria acquires a plasmid and becomes antibiotic resistant. This term most commonly refers to a bench procedure performed by the investigator which introduces experimental plasmids into bacteria.

Translation: The process of decoding a strand of mRNA, thereby producing a protein based on the code. This process requires ribosomes (which are composed of rRNA along with various proteins) to perform the synthesis, and tRNA to bring in the amino acids. Sometimes, however, people speak of "translating" the DNA or RNA when they are merely reading the nucleotide sequence and predicting from it the sequence of the encoded protein. This might be more accurately termed "conceptual translation" 

tRNA: "transfer RNA"; one of a class of rather small RNAs used by the cell to carry amino acids to the enzyme complex (the ribosome) which builds proteins, using an mRNA as a guide. Fairly abundant.

Upstream activator sequence: A binding site for transcription factors, generally part of a promoter region. A UAS may be found upstream of the TATA sequence (if there is one), and its function is (like an enhancer) to increase transcription. Unlike an enhancer, it can not be positioned just anywhere or in any orientation.

Upstream/Downstream: In an RNA, anything towards the 5' end of a reference point is "upstream" of that point. This orientation reflects the direction of both the synthesis of mRNA, and its translation - from the 5' end to the 3' end. In DNA, the situation is a bit more complicated. In the vicinity of a gene (or in a cDNA), the DNA has two strands, but one strand is virtually a duplicate of the RNA, so it's 5' and 3' ends determine upstream and downstream, respectively. NOTE that in genomic DNA, two adjacent genes may be on different strands and thus oriented in opposite directions. Upstream or downstream is only used on conjunction with a given gene.

References and Courtesy

Lyons R.H. "A Molecular Biology Glossary." A quick and dirty reference to terms used in Molecular Biology.   Online accessed on 06-05-2021.