DNA is a directional molecule. 3’ refers to the end of a single-strand of DNA that has a hydroxyl group on the 3’ carbon of the sugar. DNA polymerases extend a DNA strand by adding nucleotides to the 3’ end (in the 5’ to 3’ direction).
The region at the 3’ end of a messenger RNA transcript, which is not translated.
A modified nucleotide group that is added to the 5’ end of a nascent transcript by the capping enzyme complex in order to protect it from degradation and target it for translation.
DNA is a directional molecule. 5’ refers to the end of a single-strand of DNA to which the phosphate group is attached.
The region of a messenger RNA transcript, which is not translated.
A type of electrophoresis gel made up of polyacrylamide used for resolving proteins (as in SDS-PAGE) and shorter DNA molecules in native electrophoresis.
A type of electrophoresis gel made up of agarose, a sugar derived from seaweed. It is used for resolving DNA molecules of a range of sizes. The agarose concentration can be varied to resolve different size ranges.
The genotype annotation for antibiotic resistance genes. Ampicillin, kanamycin, tetracycline, chloramphenicol, spectinomycin and streptomycin.
Ampicillin is a competitor for transpeptidase, which prevent bacteria from building their cell walls. This antibiotic is a bacteriostatic and therefore only prevents bacterial growth. For this reason, when bacteria have been transformed with an ampcillin resistance vector they can usually be plated onto the agar plates without a recovery period. This cannot be done for bactericidal antibiotics. It is typically used at a final concentration of 100µg/ml in LB agar plates and in LB media. It can be dissolved in water at a concentration of 10mg/ml and stored at -20 °C.
The ability to grow in the presence of a particular antibiotic. For instance ampicillin resistance.
An experiment to test for the presence and / or strength of some phenomenon.
The sequence, in single letter code, of the start codon, the first codon of a gene that becomes translated into the first amino acid of a polypeptide (methionine).
Bacterial Artificial Chromosome. A circular DNA molecule that is replicated and segregated by the same mechanisms as the chromosome. Due to its low copy number it can harbor much larger inserts than normal plasmids (inserts over 100 000 base pairs have been inserted)
A virus that infects bacteria.
Refers to an end of a double stranded-DNA molecule, which has neither a protruding 5’ nor 3’ strand end.
In a double-stranded DNA molecule the bases of matching nucleotides pair with one another by hydrogen bonding. The number of paired bases is a way of describing the length of a DNA molecule.
A DNA sequence which, when transcribed into mRNA, is recognized by the ribosome as a signal to initiate translation from the subsequent start codon 8 bases downstream.
DNA which has been reverse transcribed using RNA as a template. Normally made using reverse transcriptase.
A single molecule of cDNA cloned into a vector.
A collection of cDNA molecules extracted from a particular organism, tissue or developmental stage and cloned into a DNA vector population.
A Chromatin Immunoprecipitation assay uses an antibody directed against one of the histones to precipitate and enrich chromatin from cell extracts.
This antibiotic is a bacteriostatic agent that inhibits protein synthesis by binding to the 23S RNA of the 50S subunit of bacterial ribosomes. Resistance is acheived using the protein chloramphenicol acetyltransferase. The gene encoding this protein can also be used as a reporter gene in a CAT assay. In molecular biology it is typcally used in LB agar plates and LB media at a final concentration of approximately 25µg/ml. It can be dissolved and stored in Ethanol at a concentration of 25mg/ml.
A method of determining the DNA sequence of a chromosome by extending out from a known region, designing a new sequencing primer to read out from the newly sequenced region and continuing until the desired section of DNA has been sequenced.
Calf Intestinal alkaline Phosphatase, an enzyme for removing the phosphate group from the 5’ end of a strand of DNA.
To remove the phosphate group from the 5’ end of a strand of DNA using Calf Intestinal alkaline Phosphatase.
To purify a target DNA molecule from a reaction mixture (e.g. PCR or restriction digest) most often by using a diatomaceous earth based column.
A bacterium or a piece of DNA. A bacterial cell derived from another by cellular proliferation. A DNA molecule derived from another by DNA replication.
Making a clone of something. To insert a piece of DNA into a self-replicating plasmid so that it may be copied by the process of DNA replication in a host cell. Or, to make a copy of an organism.
Sequence which may be translated into a sequence of amino acids.
A gene is comprised of two DNA strands: the template strand and a coding strand. An mRNA molecule is produced by base pairing RNA nucleotides with the template strand. Therefore the coding strand base sequence matches that of the mRNA molecule. However, uracil is used in mRNA in place of thymidine in DNA.
The genetic code is a triplet code and a codon is a three-base stretch, which may be translated into a single amino acid. Start codons (ATG and GUG) and stop codons (TAA, TAG and TGA) instruct the ribosome to begin or end translation respectively.
Redundancy of the genetic code is due to the fact that there are 64 possible combinations of the four bases in a triplet codon, yet there are only 22 amino acids. So, each of the amino acids is encoded by two or three different codons. For some amino acids, one of the codons may be used more frequently than the others and the tRNA levels in the cell for that codon will be higher. This varies between species meaning that while the genetic code is universal a gene expressing a gene from one species (say human) in another (say E. coli) may be suboptimal. Codon optimization means altering the codons used (but not amino acid sequence) in a target gene to match those preferred by the recipient organism to enhance the gene’s expression.
For some amino acids, one of the codons may be used more frequently than the others and the tRNA levels in the cell for that codon will be higher. This can vary between species (see codon optimization).
When the end of a double stranded DNA molecule has a protruding 5’ or 3’ strand it is called cohesive or sticky. If two cohesive ends have protruding strands that can base-pair with one another it means they are compatible. Two compatible cohesive ends may be covalently joined by DNA ligase (see ligation).
Two or more similar (homologous) DNA or amino acid sequences from different species may be aligned using an alignment tool. The consensus sequence reports the most frequent residue at each position in such an alignment.
Contiguous sequence. When two or more overlapping sequence reads are joined to form a continuous sequence strand.
A cosmid is a DNA plasmid that contains a cohesive end site (Cos) sequences that allow the packing of the plasmid into bacteriophage lambda (λ) capsids. They can be used to grow large plasmids (30-40Kb) and select out small plasmids. This is because the phage packages concatemers of DNA efficiently only if the Cos sites are spaced approximately 30-50 kilbobases apart. In DNA plasmids below this size the Cos sites are too close together. A cosmid normally also has a bacterial origin of replication and a standard bacterial selectable marker.
In molecular biology, dephosphorylation is the removal of a phosphate group from the 5’ end of a DNA strand. This is useful because it prevents the two ends of a linearized vector from joining together, thereby reducing the frequency of non-recombinant products in a ligation reaction.
A stretch of DNA specifically recognized by a DNA binding protein. For instance, in E. coli, the protein DnaA recognizes DnaA boxes in the OriC region of the chromosome.
A short linear stretch of double stranded DNA.
A stretch of dsDNA intended for cloning (or inserting) into a vector.
An enzyme which degrades DNA, usually by hydrolysis.
Are used to describe the position of DNA elements relative to a gene. Downstream being towards the 3’ end of the coding strand and upstream being towards the 5’ end.
Escherichia coli, a species of bacteria and the workhorse of molecular cloning.
The most common method of separating macromolecules (biological polymers) (DNAs, RNAs and proteins) based on their length and molecular mass.
An enhancer element is a linear stretch of DNA that can enhance the level of transcription from a promoter. Normally, this is achieved through the binding of transcription factors to specific sites within the enhancer DNA element. They normally act in cis (on the same peice of DNA) on a promoter close by, rather than in trans. Common enhancer elements include the CMV and SV40 enhancers but much of the early work was discovered whilst investigating B cell antibody transcription.
When a gene is expressed, the new RNA transcript must be processed into a mature messenger RNA molecule before translation can begin. In eukaryotes, part of this processing is the removal of stretches of intervening RNA sequence (introns) and the splicing together of the remaining regions (exons), which encode the amino acid sequence.
A DNA degrading enzyme (nuclease) that acts at the ends of linear DNA molecules.
A type of assay used to detect a protein binding to a DNA molecule. When the protein is bound, the DNA migrates more slowly during gel electrophoresis shifting the band up the gel.
The basic unit of heredity. A gene usually means a portion of an organism’s genome, which may be transcribed and translated to give a functional protein.
Refers to either transcription of DNA into RNA or transcription followed by translation into polypeptide.
An organism’s total hereditary DNA, the bulk of which will be in the form of a chromosome(s).
In molecular biology a library refers to a collection of DNA molecules cloned into a population of vectors. A genomic library is collection of a particular organism’s genomic DNA.
The genetic make-up of an organism, as opposed to phenotype, the characteristics associated with a particular genotype.
When two or more genes are derived from the same ancestral gene, they are homologous.
An organism used in the lab for holding or propagating a clone or construct.
A region of DNA between genes.
When two or more restriction endonucleases recognise the same sequence, they are called isoschizomers.
This antibiotic is bacteriocidal and binds to the 30S subunit of the prokaryotic ribosomes. Cells that have been transformed with a plasmid that have kanamycin resistance must be allowed to recover before being plated onto Kanamycin containing plates. It is typically used in molecular biology at a final concentration of 50µg/mL in LB agar plates or LB media. It can be dissolved and stored in water at concentration of 50mg/mL and stored at -20 °C.
One thousand bases.
Eukaryotic ribosome binding sequence found around the start codon on an mRNA.
An E. coli bacteriophage virus.
An enzyme used to join two ends of DNA together.
A reaction joining two ends of DNA together using ligase.
A short stretch of DNA used to join two others together.
A bacteriophage that can be used to produce single stranded DNA from plasmid carrying the M13 origin of replication.
A mixture of linear DNA molecules of known length, which have the appearance of rungs on a ladder when run on an agarose gel. Can be used to estimate the length of a DNA molecule of interest by comparing it with the ladder.
Megabase. One million bases.
A DNA gene is transcribed into RNA, which is further processed (for instance by splicing) into mRNA. The mRNA base sequence is recognised and translated by a ribosome along with tRNAs into an amino acid sequence in a polypeptide.
A region of mRNA that is not translated into amino acid sequence.
A method for detecting a specific RNA(s). A sample is run on an agarose gel and then blotted onto a membrane by capillary action. The membrane is then soaked in a solution containing a labeled probe for a sequence of interest. The probe binds specifically to the RNA of interest and may then be visualised.
The monomeric unit of DNA or RNA, a nucleotide consists of a phosphate group, a sugar (ribose or deoxyribose) and a base (A, T, C, G or U).
RNA that is in the nucleus.
An enzyme that digests DNA or RNA.
A short (~ <150) polymer of nucleotides.
A stretch of DNA or RNA with a start codon and a stop codon, which could be translated to give a polypeptide.
The place(s) on a chromosome or plasmid from which DNA replication is initiated.
A low copy E. coli plasmid origin of replication.
A reaction used to generate many copies of a DNA molecule of interest. PCR is based on the ability of a DNA polymerase to extend an oligonucleotide primer bound to a template strand. The use of opposing primers means that the product of one cycle of synthesis becomes a template for the next, resulting in an exponential chain reaction.
A phagemid is a DNA plasmid that contains the origin of replication from a bacteriophage, normally M13 or F1, and allows the creation of single stranded DNA in bacteria that have been infected with the bactriophage.
A self-replicating circular DNA molecule.
An RNA molecule that has a string of adenines at the end. This can be protein coding mRNA or a non-coding RNA (as in microRNA primary transripts). In eukaryotic systems this tail stabilises the RNA in the cytosol and is present on all transcripts produced by RNA polymerase II. RNA polymerase 1 and 3 transcripts are not polyadenylated. In bacterial systems it can be signal for degradation rather than stabilisation.
A poly A site is made up of a GT rich region that is just downstream of the poly A signal. The consensus sequence of the Poly A signal AATAAA but the downstream GT rich region is less well conserved, but ist typically begins 22-23 nucleotides after the poly A signal, although this is very approximate and does vary.
A series of restriction sites placed in close proximity to aid cloning into a vector.
An enzyme that extends a DNA or RNA strand. DNA polymerases usually require a template strand and extend an existing strand in the 5’ to 3’ direction.
A modification to an RNA molecule after it has been transcribed. For instance an RNA transcript may be polyadenylated.
A modification to a polypeptide after is has been translated. For instance an amino acid may be phosphorylated.
A primary transcript is the RNA that has been produced by transcription before it has undergone any further modification, such as splicing.
DNA polymerases cannot initiate DNA synthesis de novo; they can only extend the existing 3’ end of a DNA strand. This means that an oligonucleotide primer is needed for a DNA polymerase to work.
An oligonucleotide designed to hybridise to a specific DNA or RNA sequence of interest. Probes are often labeled with an additional group (32P for instance) to allow visualization or detection.
After an RNA transcript is produced it is processed to yield a mature mRNA. This may involve splicing, polyadenylation and 5’ capping.
A region of DNA upstream of a gene for directing the basic transcription machinery and other transcription factors.
An E. coli plasmid replication origin.
The genetic code is a triplet code, meaning that nucleotide sequences are translated into amino acid sequences in three-base pair steps (see codon). There are three possible reading frames in one direction of sequence, for instance:
g cgg ggc gcg
gc ggg gcg cg
gcg ggg cgc g
The reading frame that is used is determined by the position of the start codon.
A response element is a DNA sequence that is bound by a transcription factor under certain environmental or physiological conditions. For example, during hypoxia certain transcription factors (HIFs) will bind to hypoxia inducible elements in promoters that are upstream of genes that can help the cell handle low oxygen. The sequences bound by the HIFs are the response elements.
A type of enzyme that recognizes a specific DNA sequence and cleaves the DNA strands at a precise position relative to the recognition sequence. Some restriction endonucleases generate blunt ends (see blunt ends) and some generate cohesive ends (see compatible cohesive ends).
An enzyme complex made up of various polypeptide and RNA subunits that, in conjunction with aminoacyl tRNAs, initiates and extends a growing polypeptide chain in an mRNA directed manner. (See translation).
A procedure for artificially reducing the expression of a gene of interest by causing the degradation of its mRNA using the cellular machinery.
An enzyme that degrades RNA.
An enzyme usually derived from a retrovirus that produces a new DNA strand from an RNA template. Often useful when studying an organism’s mRNA.
The process of using reverse transcriptase to copy a sample of RNA into DNA, which is then termed complementary DNA or cDNA.
RT-PCR is reverse transcription (see above) followed by PCR using the cDNA as template. QPCR is quantitative PCR, a method that measures the concentration of a DNA of interest in real time to quantify the amount of starting template material.
A type of nuclease that degrades single stranded DNA or RNA. It is more active against DNA than RNA.
Satellite colonies are normally only found when using bacteriostatic antibiotics such as Ampicillin. They appear because the beta-lactamase enzyme (in the case of AmpR) produced by the bacteria to confer resistance will slowly deplete the local concentration of the antibiotic. As the antibiotic concentration gets lower in region close to the colony, bacteria that do not contain a resistance plasmid are then able to grow. This leads to satellite colonies forming. This can be reduced by using higher concentrations of antibiotic if required (see antibiotic concentrations).
Refers to the process of testing colonies for the presence of recombinant versus non-recombinant plasmids.
A component added to media to inhibit growth of non-transformants while permitting proliferation of transformant cells.
Refers to the order of bases in a polynucleotide or the order of amino acid residues in a polypeptide.
Is the ribosome binding site and translation initiation signal in bacteria.
Sequencing the DNA library created by shotgun cloning.
A Short Hairpin RNA can refer to the precursor of an RNA that will be processed to produce a microRNA, or small inhibitory RNA (siRNA). These molecules reduce protein expression from an mRNA by complementary binding. A shRNA is normally an exogenous molecule that is designed in a shRNA expression vector for the purpose of knocking down the expression of a gene of interest. They have strong self-complementarity in their RNA structure, hence the term hairpin.
A vector that has the necessary elements to replicate in two or more different hosts.
A silencer element is a linear stretch of DNA that can reduce the level of transcription from a promoter. Normally, this is achieved through the binding of transcription factors to specific sites within the silencer DNA element. They normally act in cis (on the same peice of DNA) on a promoter close by, rather than in trans.
Small inhibitory RNA molecules that bind to complementary sequences in mRNA molecules and reduce the amount of protein translated.
Small nucleolar RNA.
A polymorphism is a naturally occurring variation between organisms in a population.
Small nuclear ribonuclear proteins.
A method for detecting a specific DNA(s). A sample is run on an agarose gel and then blotted onto a membrane by capillary action. The membrane is then soaked in a solution containing a labeled DNA probe for a sequence of interest. The probe binds specifically to the target DNA and may then be visualised. (See northern blotting).
This antibiotic interrupts protein synthesis in bacterial cells by binding to the 30S subunit of the bacterial ribosomes. At low concentrations it is a bacteriostatic agent but it is bactericidal at high concentrations. Cells transformed with a plasmid encoding a spectinomycin resistance gene should probably be allowed to recover before plating, although we have not tested whether this is necessary or not. It is typically used at a final concentration of 50µg/mL. It can be dissolved in water at a stock concentration of 50mg/mL and stored at -20 ºC.
The genetic code is a triplet code and a codon is a three-base stretch, which may be translated into a single amino acid. Start codons (ATG and GUG) and instruct the ribosome to initiate translation. (See also codon).
When the end of a double stranded DNA molecule has a protruding 5’ or 3’ strand it is called cohesive or sticky. Many restriction endonucleases produce sticky ends. If two cohesive ends have protruding strands that can base-pair with one another it means they are compatible. Two compatible cohesive ends may be covalently joined by DNA ligase (see ligation).
The genetic code is a triplet code and a codon is a three-base stretch, which may be translated into a single amino acid. Stop codons (TAA, TAG and TGA) instruct the ribosome to end translation. (See also codon).
This antibiotic interrupts protein synthesis in bacterial cells by binding to the 30S subunit of the bacterial ribosomes. At low concentrations it is a bacteriostatic agent but it is bactericidal at high concentrations. It is closely related to spectinomycin. Cells transformed with a plasmid encoding a streptomycin resistance gene should probably be allowed to recover before plating, although we have not tested whether this is necessary or not. It is typically used at a final concentration of 50µg/m. It can be dissolved in water at a stock concentration of 50mg/mL and stored at -20 ºC.
The process of transferring a stretch of DNA from one vector to another.
A DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus. Widely used in polymerase chain reactions.
The name for the consensus sequence in many eukaryotic promoters and archaea promoters. It is also sometimes called the Goldberg-Hogness box. Its is bound by the TATA bind protein (TBP) and most promoters containing a TATA box are transcripbed by RNA polymerase II, although not all. For example, some RNA polymerase III promoters (such as U6) contain TATA sequences.
A gene is comprised of two DNA strands: the template strand and a coding strand. An mRNA molecule is produced by base pairing RNA nucleotides with the template strand.
This antibiotic is bactericidal and is a broad sprectrum protein synthesis inhibitor. It binds to the 30S subunit of bacterial ribosomes. Its potency sometimes means that bacteria with this resistance cassette can grow slower than those with resistance to bacteriostatic antibiotics. It also means that recovery time following DNA transformation is essential in order to allow the resistance gene to express. Typically, it is used at final concentration of 10µg/mL in LB agar plates and LB media. It can be dissolved and stored in methanol at -20 °C at a concentration of 10mg/mL.
Used to indicate when gene expression is limited to a specific tissue.
The temperature at which an oligonucleotide dissociates from a complementary strand.
Not connected to. A diffusible factor that may act on a different DNA molecule. As opposed to cis.
The process of using a DNA strand as a template for synthesizing a complementary RNA strand by an RNA polymerase enzyme.
A protein (other than RNA polymerase) involved in transcription. A general transcription factor contributes to the transcription of all of an organism’s genes, whereas specific transcription factors stimulate (or inhibit) transcription of a subset of genes.
Artificial introduction of a DNA construct(s) into mammalian cells in culture.
In molecular biology transformation means to insert DNA into a cell.
The process of synthesizing a polypeptide chain using the information contained in the mRNA sequence. Aminoacyl tRNA molecules base pair with codons in the mRNA and a ribosome covalently links amino acids to the growing chain.
Transfer RNA, small RNA molecules which are covalently linked to each amino acid to give an amino acyl tRNA. They match each amino acid up with the correct codon.
Something used to introduce DNA into a cell or organism. For instance a plasmid can be used to introduce cloned DNA into E. coli.
Yeast artificial chromosome.