MMBio 240 4

Northern blotting

An adaptation of the Southern blot procedure used to detect specific sequences of RNA by hybridization with complementary DNA or RNA probes.

Chargaff's rule

Chargaff's rules states that DNA from any cell of all organisms should have a 1:1 ratio (base Pair Rule) of pyrimidine and purine bases and, more specifically, that the amount of guanine is equal to cytosine and the amount of adenine is equal to thymine.

base pair

Noun - a pair of complementary bases in a double-stranded nucleic acid molecule, consisting of a purine in one strand linked by hydrogen bonds to a pyrimidine in the other. Cytosine always pairs with guanine, and adenine with thymine (in DNA) or uracil (in RNA). Verb - the act of hydrogen bond formation between a complementary purine and pyrimidine base

guanine

Guanine (G) is one of the four main nitrogenous bases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine (uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is called guanosine.

deamination of cytosine

The amine group of cytosine can be spontaneously hydrolysed with uracil and ammonia as products.

Southern blotting

A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British biologist Edwin Southern. Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern's name. Hybridization of the probe to a specific DNA fragment on the filter membrane indicates that this fragment contains DNA sequence that is complementary to the probe. The transfer step of the DNA from the electrophoresis gel to a membrane permits easy binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the fixation of the target-probe hybrids, required for analysis by autoradiography or other detection methods. Southern blots performed with restriction enzyme-digested genomic DNA may be used to determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes only to a single DNA segment that has not been cut by the restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., those that may be the result of sequence duplication). Modification of the hybridization conditions (for example, increasing the hybridization temperature or decreasing salt concentration) may be used to increase specificity and decrease hybridization of the probe to sequences that are less than 100% similar.

deoxyribonuclease (Dnase)

A deoxyribonuclease (DNase, for short) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. Some DNases cut, or "cleave", only residues at the ends of DNA molecules (exodeoxyribonucleases, a type of exonuclease). Others cleave anywhere along the chain (endodeoxyribonucleases, a subset of endonucleases). Some are fairly indiscriminate about the DNA sequence at which they cut, while others, including restriction enzymes, are very sequence-specific. Some cleave only double-stranded DNA; others are specific for single-stranded molecules; and still others are active toward both.

deoxyribonucleotide

A deoxyribonucleotide is the monomer, or single unit, of DNA, or deoxyribonucleic acid. Each deoxyribonucleotide comprises three parts: a nitrogenous base, a deoxyribose sugar, and one phosphate group. The nitrogenous base is always bonded to the 1' carbon of the deoxyribose, which is distinguished from ribose by the presence of a proton on the 2' carbon rather than an -OH group. The phosphate groups bind to the 5' carbon of the sugar.

glycosidic bond

A glycosidic bond or glycosidic linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group. In DNA, a glycosidic bond exits at the nitrogen-carbon linkage between the 9' nitrogen of purine bases or 1' nitrogen of pyrimidine bases and the 1' carbon of the sugar group.

heteroduplex

A heteroduplex is a double-stranded (duplex) molecule of nucleic acid originated through the basepairing of single complementary strands derived from different sources, such as from different homologous chromosomes or even from different organisms.

polynucleotide

A linear polymer composed of many nucleotide units

molecular weight markers

A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix. Therefore, when used in gel electrophoresis, markers effectively provide a logarithmic scale by which to estimate the size of the other fragments (providing the fragment sizes of the marker are known).

nucleoside

A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a 5-carbon sugar (either ribose or deoxyribose), whereas a nucleotide is composed of a nucleobase, a five-carbon sugar, and one or more phosphate groups. In a nucleoside, the base is bound to either ribose or deoxyribose via a beta-glycosidic linkage. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.

nucleotide

A nucleotide is made of a nucleobase (also termed a nitrogenous base), a five-carbon sugar (either ribose or 2-deoxyribose, depending on if it is RNA or DNA), and one or, depending on the definition, more than one phosphate groups. Authoritative chemistry sources such as the ACS Style Guide and IUPAC Gold Book clearly state that the term nucleotide refers only to a molecule containing one phosphate. However, common usage in molecular biology often extends this definition to include molecules with two or three phosphate groups.Thus, the term "nucleotide" generally refers to a nucleoside monophosphate, but a nucleoside diphosphate or nucleoside triphosphate could be considered a nucleotide as well.

palindrome

A palindromic sequence is a nucleic acid sequence on double-stranded DNA or RNA wherein reading 5' (five-prime) to 3' (three prime) forward on one strand matches the sequence reading 5' to 3' on the complementary strand with which it forms a double helix. This definition of palindrome thus depends on complementary strands being palindromic of each other. The meaning of palindrome in the context of molecular biology is slightly different from the definition used for words and sentences. Since a double helix is formed by two paired strands of nucleotides that run in opposite directions in the 5'-to-3' sense, and the nucleotides always pair in the same way (Adenine (A) with Thymine (T) for DNA, with Uracil (U) for RNA; Cytosine (C) with Guanine (G)), a (single-stranded) nucleotide sequence is said to be a palindrome if it is equal to its reverse complement. For example, the DNA sequence ACCTAGGT is palindromic because its nucleotide-by-nucleotide complement is TGGATCCA, and reversing the order of the nucleotides in the complement gives the original sequence. A palindromic nucleotide sequence can form a hairpin.

phosphodiester bond

A phosphodiester bond occurs when exactly two of the hydroxyl groups in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. Phosphodiester bonds are central to all life on Earth as they make up the backbone of the strands of nucleic acid. In DNA and RNA, the phosphodiester bond is the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another, deoxyribose in DNA and ribose in RNA.

purine

A purine is a heterocyclic aromatic organic compound that consists of a pyrimidine ring fused to an imidazole ring.

ribonucleotide

A ribonucleotide or ribotide is a nucleotide containing ribose as its pentose component.

inverted repeat

A sequence of nucleotides followed downstream by its reverse complement. The intervening sequence of nucleotides between the initial sequence and the reverse complement can be any length including zero. When the intervening length is zero, the composite sequence is a palindromic sequence.

adenine

Adenine is one of the two purines (the other being guanine) used in forming the nucleotides of nucleic acids. In DNA, adenine binds (basepairs) with thymine via two hydrogen bonds to assist in stabilizing the nucleic acid structures. In RNA, adenine basepairs to uracil.

colony blot hybridization

Colony blot hybridization is applied to DNA or RNA released from blotted microbial colonies. The microbial colonies are transferred (blotted) to a membrane. The cells are lysed in place to release the nucleic acids. The RNA or DNA (after denaturation) is fixed to the filter and hybridized with a labelled probe. Blocking reagent may be added prior to the probe to prevent unspecific binding. Excess probe is washed away and the membrane is visualized by UV or autoradiography. Colony blot hybridization can be used for screening clones or bacterial isolates.

cruciform

Cruciform structures are important regulators of biological processes. Both stem-loops and cruciforms are capable of forming from inverted repeats. Cruciform structures consist of a branch point, a stem and a loop, where the size of the loop is dependent on the length of the gap between inverted repeats

cytosine

Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine. It is a pyrimidine derivative with a heterocyclic aromatic ring. The nucleoside of cytosine is cytidine.

hypochromic and hyperchromic effect

DNA absorbs very strongly at wavelengths close to UV light (~260 nm). A single stranded DNA will absorb more UV light than that of double-stranded DNA - this is called the hypochromic effect.. DNA UV absorption decreases when it forms a double strand, this characteristic is an indication of DNA stability. With the increase in light energy, its structure and therefore its function will still remain intact since there is low disturbance to its structure. When denatured, the DNA solution of now single strand molecules will absorb more UV light - the hyperchromic effect.

methylation (DNA)

DNA methylation is a process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription.

hybridization

Hybridization (or hybridisation) occurs when single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. Though a double-stranded DNA sequence is generally stable under physiological conditions, changing these conditions in the laboratory (generally by raising the surrounding temperature) will cause the molecules to separate into single strands. These strands are complementary to each other but may also be complementary to other sequences present in their surroundings. Lowering the surrounding temperature allows the single-stranded molecules to anneal or "hybridize" to each other.

major and minor groove of DNA

If the glycosidic bonds (which attach the nucleic base to the sugar in the backbone) stuck straight out at 90 degree angles on both sides, then the grooves in double-stranded DNA would be symmetrical. Because the glycosidic bonds are at an angle (relative to the interface between the AT or GC pairs), one of the "faces" of the base pair is larger than the other. The major groove is on the side opposite the glycosidic bonds.

antiparallel

In biochemistry, two biopolymers are antiparallel if they run parallel to each other but with opposite alignments. An example is the two complementary strands of a DNA double helix, which run in opposite directions (determined by 5' and 3' ends) alongside each other.

probe

In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length (usually 100-1000 bases long) which can be radioactively labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe-target base pairing due to complementarity between the probe and target. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ. To detect hybridization of the probe to its target sequence, the probe is tagged (or "labeled") with a molecular marker of either radioactive or (more recently) fluorescent molecules; 32P (a radioactive isotope of phosphorus incorporated into the phosphodiester bond in the probe DNA) is a commonly used marker. DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Detection of sequences with moderate or high similarity depends on how stringent the hybridization conditions were applied — high stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar. Hybridization probes used in DNA microarrays refer to DNA covalently attached to an inert surface, such as coated glass slides or gene chips, to which a mobile cDNA target is hybridized.

denaturation (nucleic acids)

Nucleic acid denaturation occurs when hydrogen bonding between nucleotides is disrupted, and results in the separation of previously annealed strands. For example, denaturation of DNA due to high temperatures results in the disruption of Watson and Crick base pairs and the separation of the double stranded helix into two single strands. Nucleic acid strands are capable of re-annealling when "normal" conditions are restored, but if restoration occurs too quickly, the nucleic acid strands may re-anneal imperfectly resulting in the improper pairing of bases.

oligonucleotide

Oligonucleotides are short DNA or RNA molecules, oligomers. Commonly made by in vitro nucleic acid synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, library construction and as molecular probes. Oligonucleotides are characterized by the sequence of nucleotide residues that make up the entire molecule. The length of the oligonucleotide is usually denoted by "-mer" (from Greek meros, "part"). For example, an oligonucleotide of six nucleotides (nt) is a hexamer, while one of 25 nt would usually be called a "25-mer". Oligonucleotides readily bind, in a sequence-specific manner, to their respective complementary oligonucleotides, DNA, or RNA to form duplexes or, less often, hybrids of a higher order. This basic property serves as a foundation for the use of oligonucleotides as probes for detecting DNA or RNA.

ribonuclease (Rnase)

Ribonuclease (commonly abbreviated RNase) is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases

hairpin structure

Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop. The resulting structure is a key building block of many RNA secondary structures.

B-DNA (B-form of DNA)

The DNA double helix polymer of nucleic acid, held together by nucleotides which base pair together. In B-DNA, the most common double helical structure found in nature, the double helix is right-handed with about 10-10.5 base pairs per turn. This translates into about 20-21 nucleotides per turn. The double helix structure of DNA contains a major groove and minor groove. In B-DNA the major groove is wider than the minor groove. Given the difference in widths of the major groove and minor groove, many proteins which bind to B-DNA do so through the wider major groove.

DNA methyltransferase

The DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA.

annealing

The creation of a region of double-stranded nucleic acid when single-stranded DNA or RNA molecules basepair with DNA or RNA molecules via hydrogen bonds. The term is often used to describe the binding of a DNA probe, or the binding of a primer to a DNA strand during polymerase chain reaction.

melting point (Tm)

The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the single-stranded (ssDNA) state. Tm depends on the length of the DNA molecule and its specific nucleotide sequence.

in vitro synthesis of nucleic acids

The process of synthesizing polynucleic acids in vitro without the need for DNA or RNA template.

hydrophobic stacking

The two strands of double-stranded DNA are held together by a number of weak interactions such as hydrogen bonds, stacking interactions, and hydrophobic effects. Of these, the stacking interactions between nitrogenous bases are the most significant. The strength of base stacking interactions depends on the bases. It is strongest for stacks of G-C base pairs and weakest for stacks of A-T base pairs. The hydrophobic effect stacks the bases on top of one another. The stacked base pairs attract one another through Van der Waals forces.. In addition, base stacking in DNA is favored by the conformations of the somewhat rigid five membered rings of the backbone phosphate-sugars. The base-stacking interactions, which are largely nonspecific with respect to the identity of the stacked base, make the major contribution to the stability of the double helix.

thymine

Thymine (T) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G-C-A-T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. In RNA, thymine is replaced by the nucleobase uracil.

triplex DNA

Triple-stranded DNA is a DNA structure in which three oligonucleotides wind around each other and form a triple helix. In this structure, one strand binds to a B-form DNA double helix through Hoogsteen or reversed Hoogsteen hydrogen bonds.