Misplaced Pages

#216783

100-459: In molecular biology it is used as a restriction enzyme . Eco RI creates 4 nucleotide sticky ends with 5' end overhangs of AATT. The nucleic acid recognition sequence where the enzyme cuts is G↓AATTC, which has a palindromic complementary sequence of CTTAA↓G. Other restriction enzymes, depending on their cut sites, can also leave 3' overhangs or blunt ends with no overhangs. EcoRI is an example of type II restriction enzymes which now has more

200-446: A 2D gel electrophoresis . The Bradford assay is a molecular biology technique which enables the fast, accurate quantitation of protein molecules utilizing the unique properties of a dye called Coomassie Brilliant Blue G-250. Coomassie Blue undergoes a visible color shift from reddish-brown to bright blue upon binding to protein. In its unstable, cationic state, Coomassie Blue has a background wavelength of 465 nm and gives off

300-413: 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. The double-helix model of DNA structure was first published in the journal Nature by James Watson and Francis Crick in 1953, (X,Y,Z coordinates in 1954 ) based on

400-739: A plasmid ( expression vector ). The plasmid vector usually has at least 3 distinctive features: an origin of replication, a multiple cloning site (MCS), and a selective marker (usually antibiotic resistance ). Additionally, upstream of the MCS are the promoter regions and the transcription start site, which regulate the expression of cloned gene. This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means

500-501: A triple-stranded conformation . The realization that the structure of DNA is that of a double-helix elucidated the mechanism of base pairing by which genetic information is stored and copied in living organisms and is widely considered one of the most important scientific discoveries of the 20th century. Crick, Wilkins, and Watson each received one-third of the 1962 Nobel Prize in Physiology or Medicine for their contributions to

600-543: A 10.4 x 30 = 312 base pair molecule will circularize hundreds of times faster than 10.4 x 30.5 ≈ 317 base pair molecule. The bending of short circularized DNA segments is non-uniform. Rather, for circularized DNA segments less than the persistence length, DNA bending is localised to 1-2 kinks that form preferentially in AT-rich segments. If a nick is present, bending will be localised to the nick site. Longer stretches of DNA are entropically elastic under tension. When DNA

700-553: A cell. Twisting-torsional stiffness is important for the circularisation of DNA and the orientation of DNA bound proteins relative to each other and bending-axial stiffness is important for DNA wrapping and circularisation and protein interactions. Compression-extension is relatively unimportant in the absence of high tension. DNA in solution does not take a rigid structure but is continually changing conformation due to thermal vibration and collisions with water molecules, which makes classical measures of rigidity impossible to apply. Hence,

800-451: A density gradient, which separated the DNA molecules based on their density. The results showed that after one generation of replication in the N medium, the DNA formed a band of intermediate density between that of pure N DNA and pure N DNA. This supported the semiconservative DNA replication proposed by Watson and Crick, where each strand of the parental DNA molecule serves as a template for

900-467: A force, straightening it out. Using optical tweezers , the entropic stretching behavior of DNA has been studied and analyzed from a polymer physics perspective, and it has been found that DNA behaves largely like the Kratky-Porod worm-like chain model under physiologically accessible energy scales. Under sufficient tension and positive torque, DNA is thought to undergo a phase transition with

1000-526: A host's immune system cannot recognize the bacteria and it kills the host. The other, avirulent, rough strain lacks this polysaccharide capsule and has a dull, rough appearance. Presence or absence of capsule in the strain, is known to be genetically determined. Smooth and rough strains occur in several different type such as S-I, S-II, S-III, etc. and R-I, R-II, R-III, etc. respectively. All this subtypes of S and R bacteria differ with each other in antigen type they produce. The Avery–MacLeod–McCarty experiment

1100-456: A labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression

SECTION 10

#1732776360217

1200-518: A mixture of proteins. Western blots can be used to determine the size of isolated proteins, as well as to quantify their expression. In western blotting , proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE . The proteins in the gel are then transferred to a polyvinylidene fluoride (PVDF), nitrocellulose, nylon, or other support membrane. This membrane can then be probed with solutions of antibodies . Antibodies that specifically bind to

1300-433: A nucleic acid molecule relative to its predecessor along the axis of the helix. Together, they characterize the helical structure of the molecule. In regions of DNA or RNA where the normal structure is disrupted, the change in these values can be used to describe such disruption. For each base pair, considered relative to its predecessor, there are the following base pair geometries to consider: Rise and twist determine

1400-412: A preferred direction to bend, i.e., anisotropic bending. This is, again, due to the properties of the bases which make up the DNA sequence - a random sequence will have no preferred bend direction, i.e., isotropic bending. Preferred DNA bend direction is determined by the stability of stacking each base on top of the next. If unstable base stacking steps are always found on one side of the DNA helix then

1500-436: A reddish-brown color. When Coomassie Blue binds to protein in an acidic solution, the background wavelength shifts to 595 nm and the dye gives off a bright blue color. Proteins in the assay bind Coomassie blue in about 2 minutes, and the protein-dye complex is stable for about an hour, although it is recommended that absorbance readings are taken within 5 to 20 minutes of reaction initiation. The concentration of protein in

1600-443: A section of DNA is somewhat dependent on its sequence, and this can cause significant variation. The variation is largely due to base stacking energies and the residues which extend into the minor and major grooves . At length-scales larger than the persistence length , the entropic flexibility of DNA is remarkably consistent with standard polymer physics models, such as the Kratky-Porod worm-like chain model. Consistent with

1700-408: A single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence . A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified ( expression profiling ). In this technique the RNA in a tissue is isolated and converted to labeled complementary DNA (cDNA). This cDNA is then hybridized to the fragments on

1800-463: A strand (such as heating). The task of un-knotting topologically linked strands of DNA falls to enzymes termed topoisomerases . These enzymes are dedicated to un-knotting circular DNA by cleaving one or both strands so that another double or single stranded segment can pass through. This un-knotting is required for the replication of circular DNA and various types of recombination in linear DNA which have similar topological constraints. For many years,

1900-462: A viewpoint on the interdisciplinary relationships between molecular biology and other related fields. While researchers practice techniques specific to molecular biology, it is common to combine these with methods from genetics and biochemistry . Much of molecular biology is quantitative, and recently a significant amount of work has been done using computer science techniques such as bioinformatics and computational biology . Molecular genetics ,

2000-481: A wide variety of molecular genetics techniques including cloning , DNA screening and deleting sections of DNA in vitro . Restriction enzymes, like Eco RI, that generate sticky ends of DNA are often used to cut DNA prior to ligation , as sticky ends make the ligation reaction more efficient. One example of this use is in recombinant DNA production, when joining donor and vector DNA. Eco RI can exhibit non-site-specific cutting, known as star activity , depending on

2100-439: Is also a long tradition of studying biomolecules "from the ground up", or molecularly, in biophysics . Molecular cloning is used to isolate and then transfer a DNA sequence of interest into a plasmid vector. This recombinant DNA technology was first developed in the 1960s. In this technique, a DNA sequence coding for a protein of interest is cloned using polymerase chain reaction (PCR), and/or restriction enzymes , into

SECTION 20

#1732776360217

2200-575: Is also evidence of protein-DNA complexes forming Z-DNA structures. Other conformations are possible; A-DNA, B-DNA, C-DNA , E-DNA, L -DNA (the enantiomeric form of D -DNA), P-DNA, S-DNA, Z-DNA, etc. have been described so far. In fact, only the letters F, Q, U, V, and Y are now available to describe any new DNA structure that may appear in the future. However, most of these forms have been created synthetically and have not been observed in naturally occurring biological systems. There are also triple-stranded DNA forms and quadruplex forms such as

2300-439: Is becoming more affordable and used in many different scientific fields. This will drive the development of industries in developing nations and increase accessibility to individual researchers. Likewise, CRISPR-Cas9 gene editing experiments can now be conceived and implemented by individuals for under $ 10,000 in novel organisms, which will drive the development of industrial and medical applications. The following list describes

2400-413: Is called transfection . Several different transfection techniques are available, such as calcium phosphate transfection, electroporation , microinjection and liposome transfection . The plasmid may be integrated into the genome , resulting in a stable transfection, or may remain independent of the genome and expressed temporarily, called a transient transfection. DNA coding for a protein of interest

2500-410: Is centrifuged and the pellet which contains E.coli cells was checked and the supernatant was discarded. The E.coli cells showed radioactive phosphorus, which indicated that the transformed material was DNA not the protein coat. The transformed DNA gets attached to the DNA of E.coli and radioactivity is only seen onto the bacteriophage's DNA. This mutated DNA can be passed to the next generation and

2600-483: Is denatured, and so the intrinsic bend is lost. All DNA which bends anisotropically has, on average, a longer persistence length and greater axial stiffness. This increased rigidity is required to prevent random bending which would make the molecule act isotropically. DNA circularization depends on both the axial (bending) stiffness and torsional (rotational) stiffness of the molecule. For a DNA molecule to successfully circularize it must be long enough to easily bend into

2700-433: Is found in a cDNA library . PCR has many variations, like reverse transcription PCR ( RT-PCR ) for amplification of RNA, and, more recently, quantitative PCR which allow for quantitative measurement of DNA or RNA molecules. Gel electrophoresis is a technique which separates molecules by their size using an agarose or polyacrylamide gel. This technique is one of the principal tools of molecular biology. The basic principle

2800-477: Is in solution, it undergoes continuous structural variations due to the energy available in the thermal bath of the solvent. This is due to the thermal vibration of the molecule combined with continual collisions with water molecules. For entropic reasons, more compact relaxed states are thermally accessible than stretched out states, and so DNA molecules are almost universally found in a tangled relaxed layouts. For this reason, one molecule of DNA will stretch under

2900-454: Is known as horizontal gene transfer (HGT). This phenomenon is now referred to as genetic transformation. Griffith's experiment addressed the pneumococcus bacteria, which had two different strains, one virulent and smooth and one avirulent and rough. The smooth strain had glistering appearance owing to the presence of a type of specific polysaccharide – a polymer of glucose and glucuronic acid capsule. Due to this polysaccharide layer of bacteria,

3000-399: Is nonetheless overall preserved (AT-rich). Periodic fracture of the base-pair stack with a break occurring once per three bp (therefore one out of every three bp-bp steps) has been proposed as a regular structure which preserves planarity of the base-stacking and releases the appropriate amount of extension, with the term "Σ-DNA" introduced as a mnemonic, with the three right-facing points of

3100-472: Is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in

EcoRI - Misplaced Pages Continue

3200-456: Is occurring by measuring how much of that RNA is present in different samples, assuming that no post-transcriptional regulation occurs and that the levels of mRNA reflect proportional levels of the corresponding protein being produced. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues. A western blot is a technique by which specific proteins can be detected from

3300-642: Is referred to, respectively, as positively or negatively supercoiled . DNA in vivo is typically negatively supercoiled, which facilitates the unwinding (melting) of the double-helix required for RNA transcription . Within the cell most DNA is topologically restricted. DNA is typically found in closed loops (such as plasmids in prokaryotes) which are topologically closed, or as very long molecules whose diffusion coefficients produce effectively topologically closed domains. Linear sections of DNA are also commonly bound to proteins or physical structures (such as membranes) to form closed topological loops. Francis Crick

3400-404: Is subsequently increased or decreased by supercoiling then the writhe will be appropriately altered, making the molecule undergo plectonemic or toroidal superhelical coiling. When the ends of a piece of double stranded helical DNA are joined so that it forms a circle the strands are topologically knotted . This means the single strands cannot be separated any process that does not involve breaking

3500-484: Is susceptible to influence by strong alkaline buffering agents, such as sodium dodecyl sulfate (SDS). The terms northern , western and eastern blotting are derived from what initially was a molecular biology joke that played on the term Southern blotting , after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as

3600-400: Is that DNA fragments can be separated by applying an electric current across the gel - because the DNA backbone contains negatively charged phosphate groups, the DNA will migrate through the agarose gel towards the positive end of the current. Proteins can also be separated on the basis of size using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as

3700-412: Is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR , to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number in transgenic mice or in

3800-520: Is used to detect post-translational modification of proteins. Proteins blotted on to the PVDF or nitrocellulose membrane are probed for modifications using specific substrates. A DNA microarray is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragments. Arrays make it possible to put down large quantities of very small (100 micrometre diameter) spots on

3900-460: The G-quadruplex and the i-motif . Twin helical strands form the DNA backbone. Another double helix may be found by tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site . As the strands are not directly opposite each other, the grooves are unequally sized. One groove, the major groove, is 22 Å wide and the other,

4000-565: The Medical Research Council Unit, Cavendish Laboratory , were the first to describe the double helix model for the chemical structure of deoxyribonucleic acid (DNA), which is often considered a landmark event for the nascent field because it provided a physico-chemical basis by which to understand the previously nebulous idea of nucleic acids as the primary substance of biological inheritance. They proposed this structure based on previous research done by Franklin, which

4100-425: The genetic code is a triplet code, where each triplet (called a codon ) specifies a particular amino acid. Furthermore, it was shown that the codons do not overlap with each other in the DNA sequence encoding a protein, and that each sequence is read from a fixed starting point. During 1962–1964, through the use of conditional lethal mutants of a bacterial virus, fundamental advances were made in our understanding of

EcoRI - Misplaced Pages Continue

4200-496: The molecular basis of biological activity in and between cells , including biomolecular synthesis, modification, mechanisms, and interactions. Though cells and other microscopic structures had been observed in living organisms as early as the 18th century, a detailed understanding of the mechanisms and interactions governing their behavior did not emerge until the 20th century, when technologies used in physics and chemistry had advanced sufficiently to permit their application in

4300-422: The northern blot , actually did not use the term. Named after its inventor, biologist Edwin Southern , the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme (restriction endonuclease) digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action . The membrane

4400-407: The nucleosome core particle , and the discovery of topoisomerases . Also, the non-double-helical models are not currently accepted by the mainstream scientific community. DNA is a relatively rigid polymer, typically modelled as a worm-like chain . It has three significant degrees of freedom; bending, twisting, and compression, each of which cause certain limits on what is possible with DNA within

4500-463: The worm-like chain model is the observation that bending DNA is also described by Hooke's law at very small (sub- piconewton ) forces. For DNA segments less than the persistence length, the bending force is approximately constant and behaviour deviates from the worm-like chain predictions. This effect results in unusual ease in circularising small DNA molecules and a higher probability of finding highly bent sections of DNA. DNA molecules often have

4600-402: The 300 enzymes with more than 200 different sequence-specificities, which has transformed molecular biology and medicine . EcoRI, discovered in 1970, was isolated by PhD student Robert Yoshimori who investigated clinical E. coli isolates that contained restriction systems presented on its plasmids . The purified isolates became known as EcoRI that is used to cleave G’AATTC. Eco RI contains

4700-640: The Bradford assay can then be measured using a visible light spectrophotometer , and therefore does not require extensive equipment. This method was developed in 1975 by Marion M. Bradford , and has enabled significantly faster, more accurate protein quantitation compared to previous methods: the Lowry procedure and the biuret assay. Unlike the previous methods, the Bradford assay is not susceptible to interference by several non-protein molecules, including ethanol, sodium chloride, and magnesium chloride. However, it

4800-461: The DNA model was Phoebus Levene , who proposed the "polynucleotide model" of DNA in 1919 as a result of his biochemical experiments on yeast. In 1950, Erwin Chargaff expanded on the work of Levene and elucidated a few critical properties of nucleic acids: first, the sequence of nucleic acids varies across species. Second, the total concentration of purines (adenine and guanine) is always equal to

4900-617: The DNA will preferentially bend away from that direction. As bend angle increases then steric hindrances and ability to roll the residues relative to each other also play a role, especially in the minor groove. A and T residues will be preferentially be found in the minor grooves on the inside of bends. This effect is particularly seen in DNA-protein binding where tight DNA bending is induced, such as in nucleosome particles. See base step distortions above. DNA molecules with exceptional bending preference can become intrinsically bent. This

5000-454: The PD..D/EXK motif within its active site like many restriction endonucleases . The enzyme is a homodimer of a 31 kilodalton subunit consisting of one globular domain of the α/β architecture. Each subunit contains a loop which sticks out from the globular domain and wraps around the DNA when bound. Eco RI has been cocrystallized with the sequence it normally cuts. This crystal was used to solve

5100-549: The Sigma character serving as a reminder of the three grouped base pairs. The Σ form has been shown to have a sequence preference for GNC motifs which are believed under the GNC hypothesis to be of evolutionary importance. The B form of the DNA helix twists 360° per 10.4-10.5 bp in the absence of torsional strain. But many molecular biological processes can induce torsional strain. A DNA segment with excess or insufficient helical twisting

SECTION 50

#1732776360217

5200-437: The array and visualization of the hybridization can be done. Since multiple arrays can be made with exactly the same position of fragments, they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors. There are many different ways to fabricate microarrays;

5300-473: The atomic level. Molecular biologists today have access to increasingly affordable sequencing data at increasingly higher depths, facilitating the development of novel genetic manipulation methods in new non-model organisms. Likewise, synthetic molecular biologists will drive the industrial production of small and macro molecules through the introduction of exogenous metabolic pathways in various prokaryotic and eukaryotic cell lines. Horizontally, sequencing data

5400-400: The average persistence length has been found to be of around 50 nm (or 150 base pairs). More broadly, it has been observed to be between 45 and 60 nm or 132–176 base pairs (the diameter of DNA is 2 nm) This can vary significantly due to variations in temperature, aqueous solution conditions and DNA length. This makes DNA a moderately stiff molecule. The persistence length of

5500-403: The bacteriophage's protein coat with radioactive sulphur and DNA with radioactive phosphorus, into two different test tubes respectively. After mixing bacteriophage and E.coli into the test tube, the incubation period starts in which phage transforms the genetic material in the E.coli cells. Then the mixture is blended or agitated, which separates the phage from E.coli cells. The whole mixture

5600-498: The bases splaying outwards and the phosphates moving to the middle. This proposed structure for overstretched DNA has been called P-form DNA , in honor of Linus Pauling who originally presented it as a possible structure of DNA. Evidence from mechanical stretching of DNA in the absence of imposed torque points to a transition or transitions leading to further structures which are generally referred to as S-form DNA . These structures have not yet been definitively characterised due to

5700-553: The bending stiffness of DNA is measured by the persistence length, defined as: Bending flexibility of a polymer is conventionally quantified in terms of its persistence length, Lp, a length scale below which the polymer behaves more or less like a rigid rod. Specifically, Lp is defined as length of the polymer segment over which the time-averaged orientation of the polymer becomes uncorrelated... This value may be directly measured using an atomic force microscope to directly image DNA molecules of various lengths. In an aqueous solution,

5800-678: The biological sciences. The term 'molecular biology' was first used in 1945 by the English physicist William Astbury , who described it as an approach focused on discerning the underpinnings of biological phenomena—i.e. uncovering the physical and chemical structures and properties of biological molecules, as well as their interactions with other molecules and how these interactions explain observations of so-called classical biology, which instead studies biological processes at larger scales and higher levels of organization. In 1953, Francis Crick , James Watson , Rosalind Franklin , and their colleagues at

5900-410: The conditions present in the reaction. Conditions that can induce star activity when using Eco RI include low salt concentration, high glycerol concentration, excessive amounts of enzyme present in the reaction, high pH and contamination with certain organic solvents. Molecular biology Molecular biology / m ə ˈ l ɛ k j ʊ l ər / is a branch of biology that seeks to understand

6000-493: The development of new technologies and their optimization. Molecular biology has been elucidated by the work of many scientists, and thus the history of the field depends on an understanding of these scientists and their experiments. The field of genetics arose from attempts to understand the set of rules underlying reproduction and heredity , and the nature of the hypothetical units of heredity known as genes . Gregor Mendel pioneered this work in 1866, when he first described

6100-422: The difficulty of carrying out atomic-resolution imaging in solution while under applied force although many computer simulation studies have been made (for example, ). Proposed S-DNA structures include those which preserve base-pair stacking and hydrogen bonding (GC-rich), while releasing extension by tilting, as well as structures in which partial melting of the base-stack takes place, while base-base association

SECTION 60

#1732776360217

6200-621: The discovery. Hybridization is the process of complementary base pairs binding to form a double helix. Melting is the process by which the interactions between the strands of the double helix are broken, separating the two nucleic acid strands. These bonds are weak, easily separated by gentle heating, enzymes , or mechanical force. Melting occurs preferentially at certain points in the nucleic acid. T and A rich regions are more easily melted than C and G rich regions. Some base steps (pairs) are also susceptible to DNA melting, such as T A and T G . These mechanical features are reflected by

6300-401: The engineering of gene knockout embryonic stem cell lines . The northern blot is used to study the presence of specific RNA molecules as relative comparison among a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis , and a blot . In this process RNA is separated based on size and is then transferred to a membrane that is then probed with

6400-412: The experiment involved growing E. coli bacteria in a medium containing heavy isotope of nitrogen ( N) for several generations. This caused all the newly synthesized bacterial DNA to be incorporated with the heavy isotope. After allowing the bacteria to replicate in a medium containing normal nitrogen ( N), samples were taken at various time points. These samples were then subjected to centrifugation in

6500-399: The extract. They discovered that when they digested the DNA in the extract with DNase , transformation of harmless bacteria into virulent ones was lost. This provided strong evidence that DNA was the genetic material, challenging the prevailing belief that proteins were responsible. It laid the basis for the subsequent discovery of its structure by Watson and Crick. Confirmation that DNA is

6600-409: The full circle and must have the correct number of bases so the ends are in the correct rotation to allow bonding to occur. The optimum length for circularization of DNA is around 400 base pairs (136 nm) , with an integral number of turns of the DNA helix, i.e., multiples of 10.4 base pairs. Having a non integral number of turns presents a significant energy barrier for circularization, for example

6700-504: The functions and interactions of the proteins employed in the machinery of DNA replication , DNA repair , DNA recombination , and in the assembly of molecular structures. In 1928, Frederick Griffith , encountered a virulence property in pneumococcus bacteria, which was killing lab rats. According to Mendel, prevalent at that time, gene transfer could occur only from parent to daughter cells. Griffith advanced another theory, stating that gene transfer occurring in member of same generation

6800-526: The genetic material which is cause of infection came from the Hershey–Chase experiment . They used E.coli and bacteriophage for the experiment. This experiment is also known as blender experiment, as kitchen blender was used as a major piece of apparatus. Alfred Hershey and Martha Chase demonstrated that the DNA injected by a phage particle into a bacterium contains all information required to synthesize progeny phage particles. They used radioactivity to tag

6900-515: The handedness and pitch of the helix. The other coordinates, by contrast, can be zero. Slide and shift are typically small in B-DNA, but are substantial in A- and Z-DNA. Roll and tilt make successive base pairs less parallel, and are typically small. "Tilt" has often been used differently in the scientific literature, referring to the deviation of the first, inter-strand base-pair axis from perpendicularity to

7000-425: The helical pitch ) depends largely on stacking forces that each base exerts on its neighbours in the chain. The absolute configuration of the bases determines the direction of the helical curve for a given conformation. A-DNA and Z-DNA differ significantly in their geometry and dimensions to B-DNA, although still form helical structures. It was long thought that the A form only occurs in dehydrated samples of DNA in

7100-533: The helix axis. This corresponds to slide between a succession of base pairs, and in helix-based coordinates is properly termed "inclination". At least three DNA conformations are believed to be found in nature, A-DNA , B-DNA , and Z-DNA . The B form described by James Watson and Francis Crick is believed to predominate in cells. It is 23.7 Å wide and extends 34 Å per 10 bp of sequence. The double helix makes one complete turn about its axis every 10.4–10.5 base pairs in solution. This frequency of twist (termed

7200-463: The implications of this unique structure for possible mechanisms of DNA replication. Watson and Crick were awarded the Nobel Prize in Physiology or Medicine in 1962, along with Wilkins, for proposing a model of the structure of DNA. In 1961, it was demonstrated that when a gene encodes a protein , three sequential bases of a gene's DNA specify each successive amino acid of the protein. Thus

7300-402: The laboratory, such as those used in crystallographic experiments, and in hybrid pairings of DNA and RNA strands, but DNA dehydration does occur in vivo , and A-DNA is now known to have biological functions . Segments of DNA that cells have methylated for regulatory purposes may adopt the Z geometry, in which the strands turn about the helical axis the opposite way to A-DNA and B-DNA. There

7400-434: The laws of inheritance he observed in his studies of mating crosses in pea plants. One such law of genetic inheritance is the law of segregation , which states that diploid individuals with two alleles for a particular gene will pass one of these alleles to their offspring. Because of his critical work, the study of genetic inheritance is commonly referred to as Mendelian genetics . A major milestone in molecular biology

7500-535: The major and minor grooves are always named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form. Alternative non-helical models were briefly considered in the late 1970s as a potential solution to problems in DNA replication in plasmids and chromatin . However, the models were set aside in favor of the double-helical model due to subsequent experimental advances such as X-ray crystallography of DNA duplexes and later

7600-424: The minor groove, is 12 Å wide. The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove. This situation varies in unusual conformations of DNA within the cell (see below) , but

7700-415: The most common are silicon chips, microscope slides with spots of ~100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array. Arrays can also be made with molecules other than DNA. Allele-specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without

7800-399: The need for PCR or gel electrophoresis. Short (20–25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA, hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the unhybridized probes are removed. The target DNA is then analyzed for the presence of

7900-463: The pharmaceutical industry, the activity of new drugs against the protein can be studied. Polymerase chain reaction (PCR) is an extremely versatile technique for copying DNA. In brief, PCR allows a specific DNA sequence to be copied or modified in predetermined ways. The reaction is extremely powerful and under perfect conditions could amplify one DNA molecule to become 1.07 billion molecules in less than two hours. PCR has many applications, including

8000-421: The phosphate backbone of one of the strands so that it can swivel around the other. Helicases unwind the strands to facilitate the advance of sequence-reading enzymes such as DNA polymerase . The geometry of a base, or base pair step can be characterized by 6 coordinates: shift, slide, rise, tilt, roll, and twist. These values precisely define the location and orientation in space of every base or base pair in

8100-405: The probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation. In molecular biology, procedures and technologies are continually being developed and older technologies abandoned. For example, before the advent of DNA gel electrophoresis ( agarose or polyacrylamide ), the size of DNA molecules

8200-530: The protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence , or autoradiography . Often, the antibodies are labeled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis. Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. The eastern blotting technique

8300-471: The structure of the complex ( 1QPS ​). The solved crystal structure shows that the subunits of the enzyme homodimer interact with the DNA symmetrically. In the complex, two α-helices from each subunit come together to form a four-helix bundle. On the interacting helices are residues Glu144 and Arg145, which interact together, forming a crosstalk ring that is believed to allow the enzyme's two active sites to communicate. Restriction enzymes are used in

8400-421: The study of gene expression, the detection of pathogenic microorganisms, the detection of genetic mutations, and the introduction of mutations to DNA. The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA, the latter is a method referred to as site-directed mutagenesis . PCR can also be used to determine whether a particular DNA fragment

8500-532: The study of gene structure and function, has been among the most prominent sub-fields of molecular biology since the early 2000s. Other branches of biology are informed by molecular biology, by either directly studying the interactions of molecules in their own right such as in cell biology and developmental biology , or indirectly, where molecular techniques are used to infer historical attributes of populations or species , as in fields in evolutionary biology such as population genetics and phylogenetics . There

8600-554: The synthesis of a new complementary strand, resulting in two daughter DNA molecules, each consisting of one parental and one newly synthesized strand. The Meselson-Stahl experiment provided compelling evidence for the semiconservative replication of DNA, which is fundamental to the understanding of genetics and molecular biology. In the early 2020s, molecular biology entered a golden age defined by both vertical and horizontal technical development. Vertically, novel technologies are allowing for real-time monitoring of biological processes at

8700-450: The term double helix refers to the structure formed by double-stranded molecules of nucleic acids such as DNA . The double helical structure of a nucleic acid complex arises as a consequence of its secondary structure , and is a fundamental component in determining its tertiary structure . The structure was discovered by Maurice Wilkins , Rosalind Franklin , her student Raymond Gosling , James Watson , and Francis Crick , while

8800-586: The term "double helix" entered popular culture with the 1968 publication of Watson's The Double Helix: A Personal Account of the Discovery of the Structure of DNA . The DNA double helix biopolymer of nucleic acid is 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. The double helix structure of DNA contains

8900-486: The theory of Transduction came into existence. Transduction is a process in which the bacterial DNA carry the fragment of bacteriophages and pass it on the next generation. This is also a type of horizontal gene transfer. The Meselson-Stahl experiment was a landmark experiment in molecular biology that provided evidence for the semiconservative replication of DNA. Conducted in 1958 by Matthew Meselson and Franklin Stahl ,

9000-451: The total concentration of pyrimidines (cysteine and thymine). This is now known as Chargaff's rule. In 1953, James Watson and Francis Crick published the double helical structure of DNA, based on the X-ray crystallography work done by Rosalind Franklin which was conveyed to them by Maurice Wilkins and Max Perutz . Watson and Crick described the structure of DNA and conjectured about

9100-443: The use of molecular biology or molecular cell biology in medicine is now referred to as molecular medicine . Molecular biology sits at the intersection of biochemistry and genetics ; as these scientific disciplines emerged and evolved in the 20th century, it became clear that they both sought to determine the molecular mechanisms which underlie vital cellular functions. Advances in molecular biology have been closely related to

9200-550: The use of sequences such as TATA at the start of many genes to assist RNA polymerase in melting the DNA for transcription. Strand separation by gentle heating, as used in polymerase chain reaction (PCR), is simple, providing the molecules have fewer than about 10,000 base pairs (10 kilobase pairs, or 10 kbp). The intertwining of the DNA strands makes long segments difficult to separate. The cell avoids this problem by allowing its DNA-melting enzymes ( helicases ) to work concurrently with topoisomerases , which can chemically cleave

9300-500: The work of Rosalind Franklin and her student Raymond Gosling , who took the crucial X-ray diffraction image of DNA labeled as " Photo 51 ", and Maurice Wilkins , Alexander Stokes , and Herbert Wilson , and base-pairing chemical and biochemical information by Erwin Chargaff . Before this, Linus Pauling —who had already accurately characterised the conformation of protein secondary structure motifs—and his collaborator Robert Corey had posited, erroneously, that DNA would adopt

9400-416: The writhing number of a closed curve. Some simple examples are given, some of which may be relevant to the structure of chromatin. Analysis of DNA topology uses three values: Any change of T in a closed topological domain must be balanced by a change in W, and vice versa. This results in higher order structure of DNA. A circular DNA molecule with a writhe of 0 will be circular. If the twist of this molecule

9500-474: Was a landmark study conducted in 1944 that demonstrated that DNA, not protein as previously thought, carries genetic information in bacteria. Oswald Avery , Colin Munro MacLeod , and Maclyn McCarty used an extract from a strain of pneumococcus that could cause pneumonia in mice. They showed that genetic transformation in the bacteria could be accomplished by injecting them with purified DNA from

9600-495: Was conveyed to them by Maurice Wilkins and Max Perutz . Their work led to the discovery of DNA in other microorganisms, plants, and animals. The field of molecular biology includes techniques which enable scientists to learn about molecular processes. These techniques are used to efficiently target new drugs, diagnose disease, and better understand cell physiology. Some clinical research and medical therapies arising from molecular biology are covered under gene therapy , whereas

9700-491: Was first observed in trypanosomatid kinetoplast DNA. Typical sequences which cause this contain stretches of 4-6 T and A residues separated by G and C rich sections which keep the A and T residues in phase with the minor groove on one side of the molecule. For example: The intrinsically bent structure is induced by the 'propeller twist' of base pairs relative to each other allowing unusual bifurcated Hydrogen-bonds between base steps. At higher temperatures this structure

9800-401: Was one of the first to propose the importance of linking numbers when considering DNA supercoils. In a paper published in 1976, Crick outlined the problem as follows: In considering supercoils formed by closed double-stranded molecules of DNA certain mathematical concepts, such as the linking number and the twist, are needed. The meaning of these for a closed ribbon is explained and also that of

9900-414: Was the discovery of the structure of DNA . This work began in 1869 by Friedrich Miescher , a Swiss biochemist who first proposed a structure called nuclein , which we now know to be (deoxyribonucleic acid), or DNA. He discovered this unique substance by studying the components of pus-filled bandages, and noting the unique properties of the "phosphorus-containing substances". Another notable contributor to

10000-443: Was typically determined by rate sedimentation in sucrose gradients , a slow and labor-intensive technique requiring expensive instrumentation; prior to sucrose gradients, viscometry was used. Aside from their historical interest, it is often worth knowing about older technology, as it is occasionally useful to solve another new problem for which the newer technique is inappropriate. Double helix In molecular biology ,

#216783