Complementary DNA

In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template in a reaction catalysed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes.

Overview

The central dogma of molecular biology outlines that in synthesizing proteins, DNA is transcribed into mRNA, which is translated into protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes can contain introns (intervening sequences), which are not coding sequences, and must be spliced out of the RNA primary transcript before it becomes mRNA and can be translated into protein. Prokaryotic genes have no introns, so their RNA is not subject to splicing.

Often it is desirable to express eukaryotic genes in prokaryotic cells. A simplified method of doing so would include the addition of eukaryotic DNA to a prokaryotic host, which would transcribe the DNA to mRNA and then translate it to protein. However, as eukaryotic DNA has introns, and since prokaryotes lack the machinery to splice them, the splicing of eukaryotic DNA must be done prior to adding the eukaryotic DNA into the host. This spliced DNA is called complementary DNA. To obtain expression of the protein encoded by the eukaryotic cDNA, prokaryotic regulatory sequences would also be required (e.g. a promoter).

Synthesis

Though there are several methods for doing so, cDNA is most often synthesized from mature (fully spliced) mRNA using the enzyme reverse transcriptase. This enzyme operates on a single strand of mRNA, generating its complementary DNA based on the pairing of RNA base pairs (A, U, G and C) to their DNA complements (T, A, C and G respectively).

To obtain eukaryotic cDNA whose introns have been spliced:

1. A eukaryotic cell transcribes the DNA (from genes) into RNA (pre-mRNA).
2. The same cell processes the pre-mRNA strands by splicing out introns, and adding a poly-A tail and 5’ Methyl-Guanine cap.
3. This mixture of mature mRNA strands are extracted from the cell.
4. A poly-T oligonucleotide primer is hybridized onto the poly-A tail of the mature mRNA template, or random hexamer primers can be added which contain every possible 6 base single strand of DNA and can therefore hybridize anywhere on the RNA (Reverse transcriptase requires this double-stranded segment as a primer to start its operation.)
5. Reverse transcriptase is added, along with deoxynucleotide triphosphates (A, T, G, C).

The reverse transcriptase scans the mature mRNA and synthesizes a sequence of DNA that complements the mRNA template. This strand of DNA is complementary DNA.

Note that the central dogma of molecular biology is not broken in this process.

Applications

Complementary DNA is often used in gene cloning or as gene probes or in the creation of a cDNA library. Partial sequences of cDNAs are often obtained as expressed sequence tags.

Viruses

Some viruses also use cDNA to turn their viral RNA into mRNA (viral RNA → cDNA → mRNA). The mRNA is used to make viral proteins to take over the host cell.

http://en.wikipedia.org/wiki/Complementary_DNA

A-DNA

The A-DNA structure.

A-DNA is one of the many possible double helical structures of DNA.
It is a right-handed double helix fairly similar to the more common and well-known B-DNA form, but with a shorter more compact helical structure. A-DNA is thought to be one of three biologically active double helical structures along with B- and Z-DNA. It appears likely that it occurs only in dehydrated samples of DNA, such as those used in crystallographic experiments, and possibly in hybrid pairings of DNA and RNA strands.

Structure

A-DNA is fairly similar to B-DNA given that it is a right-handed double helix with major and minor grooves. However, as shown in the comparison table below, there is a slight increase in the number of base pairs per rotation (resulting in a tighter rotation angle), and smaller rise/turn. This results in a deepening of the major groove and a shallowing of the minor.

Predicting A-DNA structure

An algorithm for predicting the propensity of a sequence to flip from B-DNA to A-DNA was developed by Beth Basham, Gary Schroth, and P. Shing Ho at Oregon State University.^[1]

The abstract of their work describes this algorithm:

The ability to predict macromolecular conformations from sequence and thermodynamic principles has long been coveted but generally has not been achieved. We show that differences in the hydration of DNA surfaces can be used to distinguish between sequences that form A- and B-DNA. From this, a "triplet code" of A-DNA propensities was derived as energetic rules for predicting A-DNA formation. This code correctly predicted > 90% of A- and B-DNA sequences in crystals and correlates with A-DNA formation in solution. Thus, with our previous studies on Z-DNA, we now have a single method to predict the relative stability of sequences in the three standard DNA duplex conformations.^[1]

Comparison Geometries of the Most Common DNA Forms

Side view of A-, B-, and Z-DNA.

The helix axis of A-, B-, and Z-DNA.

Geometry attribute	A-form	B-form	Z-form
Helix sense	right-handed	right-handed	left-handed
Repeating unit	1 bp	1 bp	2 bp
Rotation/bp	33.6°	35.9°	60°/2
Mean bp/turn	10.7	10.0	12
Inclination of bp to axis	+19°	−1.2°	−9°
Rise/bp along axis	2.3 Å (0.23 nm)	3.32 Å (0.332 nm)	3.8 Å (0.38 nm)
Rise/turn of helix	24.6 Å (2.46 nm)	33.2 Å (3.32 nm)	45.6 Å (4.56 nm)
Mean propeller twist	+18°	+16°	0°
Glycosyl angle	anti	anti	pyrimidine: anti, purine: syn
Sugar pucker	C3'-endo	C2'-endo	C: C2'-endo, G: C2'-exo
Diameter	26 Å (2.6 nm)	20 Å (2.0 nm)	18 Å (1.8 nm)

http://en.wikipedia.org/wiki/A-DNA

DNA - History

Francis Crick

James Watson

DNA was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein".^[129] In 1919 this discovery was followed by Phoebus Levene's identification of the base, sugar and phosphate nucleotide unit.^[130] Levene suggested that DNA consisted of a string of nucleotide units linked together through the phosphate groups. However, Levene thought the chain was short and the bases repeated in a fixed order. In 1937 William Astbury produced the first X-ray diffraction patterns that showed that DNA had a regular structure.^[131]

In 1928, Frederick Griffith discovered that traits of the "smooth" form of the Pneumococcus could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form.^[132] This system provided the first clear suggestion that DNA carried genetic information, when Oswald Theodore Avery, along with coworkers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle in 1943.^[133] DNA's role in heredity was confirmed in 1953, when Alfred Hershey and Martha Chase in the Hershey-Chase experiment showed that DNA is the genetic material of the T2 phage.^[134]

In 1953, based on X-ray diffraction images^[135] taken by Rosalind Franklin and the information that the bases were paired, James D. Watson and Francis Crick suggested^[135] what is now accepted as the first accurate model of DNA structure in the journal Nature.^[5] Experimental evidence for Watson and Crick's model were published in a series of five articles in the same issue of Nature.^[136] Of these, Franklin and Raymond Gosling's paper was the first publication of X-ray diffraction data that supported the Watson and Crick model,^[137][138] this issue also contained an article on DNA structure by Maurice Wilkins and his colleagues.^[139] In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine.^[140] However, speculation continues on who should have received credit for the discovery, as it was based on Franklin's data.

In an influential presentation in 1957, Crick laid out the "Central Dogma" of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis".^[141] Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson-Stahl experiment.^[142] Further work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley and Marshall Warren Nirenberg to decipher the genetic code.^[143] These findings represent the birth of molecular biology.

http://en.wikipedia.org/wiki/DNA

DNA - Uses in technology

Genetic engineering

Modern biology and biochemistry make intensive use of recombinant DNA technology. Recombinant DNA is a man-made DNA sequence that has been assembled from other DNA sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector.^[109] The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research,^[110] or be grown in agriculture.^[111][112]

Forensics

Forensic scientists can use DNA in blood, semen, skin, saliva or hair at a crime scene to identify a perpetrator. This process is called genetic fingerprinting, or more accurately, DNA profiling. In DNA profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a criminal.^[113] However, identification can be complicated if the scene is contaminated with DNA from several people.^[114] DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys,^[115] and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.^[116] People convicted of certain types of crimes may be required to provide a sample of DNA for a database. This has helped investigators solve old cases where only a DNA sample was obtained from the scene. DNA profiling can also be used to identify victims of mass casualty incidents.^[117]

Bioinformatics

Bioinformatics involves the manipulation, searching, and data mining of DNA sequence data. The development of techniques to store and search DNA sequences have led to widely-applied advances in computer science, especially string searching algorithms, machine learning and database theory.^[118] String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides.^[119] In other applications such as text editors, even simple algorithms for this problem usually suffice, but DNA sequences cause these algorithms to exhibit near-worst-case behaviour due to their small number of distinct characters. The related problem of sequence alignment aims to identify homologous sequences and locate the specific mutations that make them distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function.^[120] Data sets representing entire genomes' worth of DNA sequences, such as those produced by the Human Genome Project, are difficult to use without annotations, which label the locations of genes and regulatory elements on each chromosome. Regions of DNA sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products in an organism even before they have been isolated experimentally.^[121]

DNA nanotechnology

The DNA structure at left (schematic shown) will self-assemble into the structure visualized by atomic force microscopy at right. DNA nanotechnology is the field which seeks to design nanoscale structures using the molecular recognition properties of DNA molecules. Image from Strong, 2004. [1]

DNA nanotechnology uses the unique molecular recognition properties of DNA and other nucleic acids to create self-assembing branched DNA complexes with useful properties. DNA is thus used as a structural material rather than as a carrier of biological information. This has lead to the creation of two-dimensional periodic lattices (both tile-based as well as using the "DNA origami" method) as well as three-dimensional structures in the shapes of polyhedra. Nanomechanical devices and algorithmic self-assembly have also been demonstrated, and these DNA structures have been used to template the arrangement of other molecules such as gold nanoparticles and streptavidin proteins.

DNA and computation

DNA was first used in computing to solve a small version of the directed Hamiltonian path problem, an NP-complete problem.^[122] DNA computing is advantageous over electronic computers in power use, space use, and efficiency, due to its ability to compute in a highly parallel fashion (see parallel computing). A number of other problems, including simulation of various abstract machines, the boolean satisfiability problem, and the bounded version of the travelling salesman problem, have since been analysed using DNA computing.^[123] Due to its compactness, DNA also has a theoretical role in cryptography, where in particular it allows unbreakable one-time pads to be efficiently constructed and used.^[124]

History and anthropology

Because DNA collects mutations over time, which are then inherited, it contains historical information and by comparing DNA sequences, geneticists can infer the evolutionary history of organisms, their phylogeny.^[125] This field of phylogenetics is a powerful tool in evolutionary biology. If DNA sequences within a species are compared, population geneticists can learn the history of particular populations. This can be used in studies ranging from ecological genetics to anthropology; for example, DNA evidence is being used to try to identify the Ten Lost Tribes of Israel.^[126][127]

DNA has also been used to look at modern family relationships, such as establishing family relationships between the descendants of Sally Hemings and Thomas Jefferson. This usage is closely related to the use of DNA in criminal investigations detailed above. Indeed, some criminal investigations have been solved when DNA from crime scenes has matched relatives of the guilty individual.^[128]

http://en.wikipedia.org/wiki/DNA

Evolution of DNA metabolism

DNA contains the genetic information that allows all modern living things to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life DNA has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material.^[90][102] RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes.^[103] This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur since the number of unique bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes.^[104]

Unfortunately, there is no direct evidence of ancient genetic systems, as recovery of DNA from most fossils is impossible. This is because DNA will survive in the environment for less than one million years and slowly degrades into short fragments in solution.^[105] Although claims for older DNA have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250-million years old,^[106] these claims are controversial and have been disputed.^[107][108]

http://en.wikipedia.org/wiki/DNA

DNA - Genetic recombination

Structure of the Holliday junction intermediate in genetic recombination. The four separate DNA strands are coloured red, blue, green and yellow.^[95]

Recombination involves the breakage and rejoining of two chromosomes (M and F) to produce two re-arranged chromosomes (C1 and C2).

A DNA helix usually does not interact with other segments of DNA, and in human cells the different chromosomes even occupy separate areas in the nucleus called "chromosome territories".^[96] This physical separation of different chromosomes is important for the ability of DNA to function as a stable repository for information, as one of the few times chromosomes interact is during chromosomal crossover when they recombine. Chromosomal crossover is when two DNA helices break, swap a section and then rejoin.

Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of new proteins.^[97] Genetic recombination can also be involved in DNA repair, particularly in the cell's response to double-strand breaks.^[98]

The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51.^[99] The first step in recombination is a double-stranded break either caused by an endonuclease or damage to the DNA.^[100] A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA.^[101]

http://en.wikipedia.org/wiki/Genetic_recombination

HD Jargon Buster

Don’t let ‘technical jargon’ stop you joining the HDTV revolution! Our no-nonsense guide to the common terms used will help you to understand all you need to know to enjoy an intense viewing experience.

720-line HD

The 720-line system is the most common format for the launch of HD television. The 720-line system is 1280 pixels across, so the resolution is 1280 x 720, giving just over twice the resolution of a 625-line standard definition picture.

1080-line HD

The 1080-line system is the other common HD format. A 1080-line system has 1080 vertical pixels and 1920 horizontal pixels, with up to five times the resolution of a standard definition picture.

Aspect Ratio

This refers to the ratio of a pictures width relative to it’s height. The aspect ratio of a standard television is 4:3, whereas HDTV has an aspect ratio of 16:9, for a more intense viewing experience. The more common names for aspect ratio are ‘Widescreen’ or ‘Letter-box’.

AC-3

Also known as ‘Dolby Digital’ this is the 5.1-channel sound system specified in the Standard for Digital HDTV, delivering CD quality digital audio from six speakers, front left, right and centre (where most of the ‘voice’ comes from), rear left and right plus a subwoofer for depth, to produce a cinematic sound! True 5.1-channel sound is only available via a home cinema system

Bit Rate

‘Bits per second’ or bps, expresses the rate at which data is transmitted. Generally, the higher the bit rate, the better the image and sound quality.

Brightness

Expressed as candelas per square metre (cd/m2) brightness simply indicates how much light is emitted by the screen. A higher candela means a brighter picture.

Component Video

Three connectors (usually red, green and blue RCA jacks) that transmit and receive component video signals. The combination of these signals conveys all the picture information.

Contrast Ratio

Essentially contrast ratio is a comparison of a screens blackest black and whitest white.A higher contast ratio indicates that on screen colours will be richer.

DVI

Digital Video Interface. DVI is a type of cable connector which provides a high-bandwidth connection between a video source and a display device.

EPG

Electronic Programme Guide. An onscreen display of channels and programme data.

HDMI

High Definition Multimedia Interface is a digital connection for video/audio data. It ensures a high-quality video signal is delivered to your display via a single cable.

HDCP

This is a copyright protection system that is incorporated into HD receivers and displays. It stands for High definition Digital Content Protection and prevents unauthorised use of content which is copyrighted.

LCD TV

Liquid Crystal Displays are flat-panel televisions designed to offer superior images. A liquid crystal solution is sandwiched between two panels and electrified. This causes the crystals to act as ‘shutters’, some allowing light to pass through, other blocking light out. These ‘shutters’ on the electrified crystals form the image on the LCD TV.

Pixels

A pixel is literally a single dot on the screen and the pixels form the image on your display. The more pixels, the better the picture. With HDTV there are many more pixels (typically 1920 x 1080 or 1280 x 720) than with Standard Definition TV (720 x 576), giving a crisper, clearer and sharper picture.

Plasma Display

A compatible plasma TV is one way to display HDTV. The image is created by hundreds of thousands of tiny cells filled by ionized gas in a plasma state.

Resolution

The measure of the amount of detail an image can show. HD has a maximum resolution of 1920 x 1080 which equates to 2,073,000 pixels whereas standard definition has a resolution of 720 x 576. The higher the resolution – the better the resulting image.

Standard Definition (SDTV)

This is the traditional definition television system, currently used. A standard definition picture is 720 x 576 pixels.

Viewing Angle

LCDs were originally designed as computer monitors, and as such were designed for head on viewing. Viewed at an angle these early screens lost much of their contrast and brightness. In response to this manufacturers are continually increasing viewing angles for LCDs where the quality is retained. Viewing angles as high as 176 degrees are now being achieved.

http://www.hdtvorg.co.uk/guide/hdtv/jargon.htm

Home Cinema Sound Formats

Dolby Digital 5.1 Digital surround sound available on all home cinema packages. Uses five discrete channels of digital sound and a separate subwoofer channel (the ‘.1’ in 5.1)

Dolby Digital EX Updated version of Dolby Digital 5.1. Includes a third non-discrete rear-speaker channel to enable you to add one or two extra rear ‘centre’ speakers.

DTS 5.1 Rival to Dolby Digital 5.1, now included on many DVD films. More musical than Dolby Digital

DTS ES Matrix 6.1 DTS rival to Dolby EX

DTS ES Discrete 6.1 Ups the ante on Matrix 6.1 and Dolby Digital EX by including a discrete channel for centre-rear effects. In other words, this is true 6.1- channel surround

Dolby Pro-Logic Analogue surround processing that takes Dolby Surround-encoded stereo signal from TV broadcasts or VHS videos, and turns it into surround sound. Only the front stereo speakers boast discrete surround sound here, with centre speaker and mono rears being derived from the stereo signal

Dolby Pro-Logic II A soupedup version of Pro-Logic, this uses processing jiggery-pokery to make the non-discrete analogue surround sound more like Dolby Digital 5.1

Dolby Surround Most television programmes carry this additional, analogue surroundsound signal, which can be decoded by home cinema systems into Pro-Logic I or II

http://www.hdtvorg.co.uk/technology/home_cinema_sound_formats.htm

Home Cinema Speaker Placement

Square Room

The ideal room and speaker placement for any home cinema setup is a square room with the TV directly facing the main viewing position. The front speakers should be placed either side of the TV screen, around one metre away from the side of the screen. The centre speaker can be positioned above or below the screen, but should be as close to the screen as possible. The surround sound speakers are ideally placed on axis at either side of the main listening position (usually a sofa), or behind.

Subwoofer placement can be critical, and is system and room-dependent. Our preferred placement is between the front stereo speakers, for the most integrated sound performance between subwoofer and satellites, or to the side of the room. Experimentation is the key here. Try it in several different positions to see which sounds best with your particular system and room.

Rectangular Room

Most domestic living rooms place the TV in the corner of the room. This is not ideal for getting the most evenly distributed sound performance for a home cinema system, but effective results can still be achieved.

Place the speakers either side of the TV, and point them directly at the main seating area. The centre speaker should be positioned as close to the screen as possible, and the subwoofer placed close to the front soundstage. Position the rear speakers across the opposite corner facing the TV, and point them towards the seating area to cover as broad a listening area as possible.

L-Shaped Room

L-shaped areas can deaden your home cinema soundstage, sucking that all-important subwoofer bass into the area on the left of the picture. Position your front stereo speakers close to your screen, aimed tightly towards your seating area. The sub/sat speakers that come with all-in-one systems maintain bass well in rooms like these, spreading it between all of the speakers.

Consider moving your furniture around to improve speaker direction and sound performance, but don’t panic! Home cinema is not an exact science.

http://www.hdtvorg.co.uk/technology/home_cinema_speaker_placement.htm

Home Cinema Sound

We hear a lot about 5.1 surround sound on DVDs. Basically, all one-box home cinema systems with five speakers and a subwoofer can handle 5.1 soundtracks. 5.1 refers to a multichannel soundtrack that is split into five discrete channels of digital sound information.

Each of the five individual channels provide sound to the front left, front right, centre, surround left and surround right speakers. A sixth channel is reserved for the subwoofer, and is referred to as the .1 in a 5.1 soundtrack.

Prerecorded DVDs contain a variety of digital soundtracks. The most popular soundtrack format is Dolby Digital 5.1. It’s found on most DVD blockbuster movies, and all home cinema systems can handle Dolby Digital 5.1 surround sound. DTS 5.1 is the direct rival to Dolby Digital 5.1, and is found on an increasing number of DVD soundtracks. DTS is generally considered to be of superior sound quality, but not all systems are compatible with it so it is worth checking before you buy.

Newer formats called Dolby Digital EX and DTS ES are emerging with a 6.1 multichannel soundtrack. These utilise a rear-centre channel for more effective wraparound effects, and will appeal to anyone who owns a home cinema system with six surround speakers.

Just around the corner are high definition formats called Dolby Digital Plus and Dolby TrueHD. Dolby Digital Plus was designed as a bridge between existing hardware and high definition formats, including HD DVD, Blu-Ray and HDTV broadcasting. The format is based on Dolby Digital but enjoys more efficient coding. It works with multichannel audio programs of up to 7.1 channels. Dolby TrueHD is designed for HD DVD and Blu-Ray and delivers sound that is identical to the studio master.

Dolby Digital or DTS?

Consumers probably don't care if a DVD movie release uses Dolby Digital or DTS for surround sound because, by the time the product reaches them, the decision to encode the audio with Dolby or DTS has been made by the studio.

Most home cinema playback systems support both Dolby Digital and DTS decoding and many titles are issued with Dolby Digital and DTS encoded tracks on the same DVD.

Both systems provide high-quality 5.1 digital audio that can be played through the same amplifiers and loudspeakers. On dual standard titles, the consumer can select the Dolby Digital or the DTS soundtrack from the audio submenu.

http://www.hdtvorg.co.uk/technology/home_cinema_sound.htm

Home Cinema Build Your Own

The most long-winded way of realising your home cinema dreams is also the most flexible. Separate components allow you to mix and match the best products from your favourite manufacturers, updating and upgrading when the mood takes you. And if you already own a DVD player, you’re a third of the way there!

There are loads of integrated home cinema amplifiers (or receivers) on sale. These combine amplification and surround sound processing in one box and are incredible value or money. Add 5.1-channel speakers and a DVD player to complete the setup.

http://www.hdtvorg.co.uk/technology/home_cinema_build.htm