REPLICATION DNA: 02/27/11

Sunday, February 27, 2011

Microarrays Application

DNA Microarray technology helps in the identification of new genes, know about their functioning and expression levels under different conditions. Disease diagnosis of DNA Microarray technology helps researchers learn more about different diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer. Until recently, different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in tumor cells. This will tremendously help the pharmaceutical community to develop more effective drugs as the treatment strategies will be targeted directly to the specific type of cancer.

Drug discovery: Microarray technology has extensive application in Pharmacogenomics. Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the genetic profiles of the patients. Comparative analysis of the genes from a diseased and normal cell will help the identification of the biochemical constitution of the proteins synthesized by the diseased genes. The researchers can use this information to synthesize drugs which combat with these proteins and reduce their effect.

Toxicological research: Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and changes in the genetic profiles of the cells exposed to such toxicants

RNA data Sequences to DNA

Proteins are not the only substances that are synthesized directly from data within the DNA. Some forms of RNA are specialized and also have their formula encoded directly in digital DNA formulae. Not all types of RNA are temporary intermediate forms with their form depending on whatever DNA they are copying. There are certain forms of RNA that have a particular form that is the same across all individuals. Some of these special-purpose RNA forms are,

tRNA - transfer RNA
rRNA - ribosome RNA

There are exactly 20 forms of tRNA one each transfer a particular amino acid. tRNA molecules contain about 75-80 bases. tRNA recognizes one of the 64 triplets and matches it one of the 20 amino acids. Since there are 20 tRNA types, and not 64, each tRNA molecule has to recognize more than one triplet ordering as a match. The DNA code contains multiple repetitions of codes for tRNA and rRNA. About 280 copies are spread over 5 chromosomes. Presumably, this allows each cell to make multiple copies of tRNA and rRNA molecules at once from its single copy of the DNA.

Grooves in the DNA

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, 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 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.

DNA Base Pairing

Each type of base on one strand forms a bond with just one type of base on the other strand.This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines with A bonding only to T and C bonding only to G. This arrangement of two nucleotides binding together across the double helix is called a base pair. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can therefore be pulled apart like a zipper, either by a mechanical force or high temperature. As a result of this complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. Indeed, this reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in living organisms.

GC DNA base pair-svg
AT DNA base pair-svg

Top a GC base pair with three hydrogen bonds. Bottom, an AT base pair with two hydrogen bonds. Non-covalent hydrogen bonds between the pairs are shown as dashes lines.

The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds (see figures, left). DNA with high GC-content is more stable than DNA with low GC-content, but contrary to popular belief, this is not due to the extra hydrogen bond of a GC base pair but rather the contribution of stacking interactions (hydrogen bonding merely provides specificity of the pairing, not stability). As a result, it is both the percentage of GC base pairs and the overall length of a DNA double helix that determine the strength of the association between the two strands of DNA. Long DNA helices with a high GC content have stronger-interacting strands, while short helices with high AT content have weaker-interacting strands. In biology, parts of the DNA double helix that need to separate easily, such as the TATAAT Pribnow box in some promoters, tend to have a high AT content, making the strands easier to pull apart. In the laboratory, the strength of this interaction can be measured by finding the temperature required to break the hydrogen bonds, their melting temperature (also called Tm value). When all the base pairs in a DNA double helix melt the strands separate and exist in solution as two entirely independent molecules. These single-stranded DNA molecules have no single common shape, but some conformations are more stable than others.

Sense and Antisense

A DNA sequence is called sense if its sequence is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is called the antisense sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences). In both prokaryotes and eukaryotes, anti sense RNA sequences are produced, but the functions of these RNAs are not entirely clear. One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.

A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes. In these cases some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription, while in viruses overlapping genes increase the amount of information that can be encoded within the small viral genome.

Alternate structures of DNA

DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms although, only B-DNA and Z-DNA have been directly observed in functional organisms. The conformation that DNA adopts depends on the hydration level DNA sequence, the amount and direction of supercooling chemical modifications of the bases, the type and concentration of metal ions, as well as the presence of polyamines in solution.

The first published reports of A-DNA X-ray diffraction patterns and also B-DNA used analysis based on Patterson transforms that provided only a limited amount of structural information for oriented fibers of DNA. An alternate analysis was then proposed by Wilkins act. all in 1953, for the in vivo B-DNA X-ray diffraction/scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions. In the same journal, Watson and Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double helix.

Although the B-DNA form is most common under the conditions found in cells, it is not a well defined conformation but a family of related DNA conformations that occur at the high hydration levels present in living cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder.

Compared to B-DNA the A-DNA form is a wider right handed spiral with a shallow, wide minor groove and a narrower, deeper major groove. The A form occurs under non-physiological conditions in partially dehydrated samples of DNA while in the cell it may be produced in hybrid pairings of DNA and RNA strands as well as in enzyme-DNA complexes. Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form. Here, the strands turn about the helical axis in a left-handed spiral, the opposite of the more common B form. These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription

Quadruplex structutrs of DNA

At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase as the enzymes that normally replicate DNA cannot copy the extreme 3 ends of chromosomes. These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected. In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence.

These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases form a flat plate and these flat four-base units then stack on top of each other to form a stable G-quadruplex structure. These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit. Other structures can also be formed with the central set of four bases coming from either a single strand folded around the bases or several different parallel strands each contributing one base to the central structure.

In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single stranded DNA curls around in a long circle stabilized by telomere-binding proteins. At the very end of the T-loop the single stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop.

Boilogical Funtions Of DNA

DNA usually occurs as linear chromosomes in eukaryotes and circular chromosomes in prokaryotes. The set of chromosomes in a cell makes up its genome. the human genome has approximately 3 billion base pairs of DNA arranged into 46 chromosomes. The information carried by DNA is held in the sequence of pieces of DNA called genes. Transmission of genetic information in genes is achieved via complementary base pairing. For example in transcription, when a cell uses the information in a gene, the DNA sequence is copied into a complementary RNA sequence through the attraction between the DNA and the correct RNA nucleotides. Usually this RNA copy is then used to make a matching protein sequence in a process called translation which depends on the same interaction between RNA nucleotides. Alternatively, a cell may simply copy its genetic information in a process called DNA replication. The details of these functions are covered in other articles here we focus on the interactions between DNA and other molecules that mediate the function of the genome.

Genomes and Genes

Genomic DNA is located in the cell nucleus of eukaryotes as well as small amounts in mitochondria and chloroplasts. In prokaryotes the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid. The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, as well as regulatory sequences such as promoters and enhancers, which control the transcription of the open reading frame.

In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein coding exons with over 50% of human DNA consisting of non-coding repetitive sequences. The reasons for the presence of so much non-coding DNA in eukaryotic genomes and the extraordinary differences in genome size or C-value, among species represent a long-standing puzzle known as the C-value enigma. However, DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.
T7 RNA polymerase (blue) producing a mRNA (green) from a DNA template (orange)

Some non-coding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes but are important for the function and stability of chromosomes. An abundant form of non-coding DNA in humans are pseudogenes which are copies of genes that have been disabled by mutation. These sequences are usually just molecular fossils although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence.

DNA Binding Proteins

Structural proteins that bind DNA are well-understood examples of non-specific DNA-protein interactions. Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome which contains two complete turns of double-stranded DNA wrapped around its surface. These non-specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are therefore largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation phosphorylation and acetylation. These chemical changes alter the strength of the interaction between the DNA and the histones, making the DNA more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA. These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes.

A distinct group of DNA-binding proteins are the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best understood member of this family and is used in processes where the double helix is separated, including DNA replication recombination and DNA repair. These binding proteins seem to stabilize single-stranded DNA and protect it from forming stem-loops or being degraded by nucleases.
The lambda repressor helix-turn-helix transcription factor bound to its DNA target

In contrast other proteins have evolved to bind to particular DNA sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of DNA sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription. Alternatively, transcription factors can bind enzymes that modify the histones at the promoter this will change the accessibility of the DNA template to the polymerase.

As these DNA targets can occur throughout an organism genome changes in the activity of one type of transcription factor can affect thousands of genes. Consequently these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with DNA come from the proteins making multiple contacts to the edges of the DNA bases allowing them to"read the DNA sequence. Most of these base-interactions are made in the major groove where the bases are most accessible.

Translation and Transcription of DNA

A gene is a sequence of DNA that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a DNA strand defines a messenger RNA sequence which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code. The genetic code consists of three letter words called codons formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT).

In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3 letter combinations there are 64 possible codons (43 combinations). These encode the twenty standard amino acids giving most amino acids more than one possible codon. There are also three stop or nonsense codons signifying the end of the coding region; these are the TAA, TGA and TAG codons.

Genatic Enginearing

Methods have been developed to purify DNA from organisms, such as phenol-chloroform extraction and manipulate it in the laboratory such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in 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. The genetically modified organisms produced can be used to produce products such as recombinant proteins used in medical research or be grown in agriculture.

Forensics of DNA

Forensic scientists can use DNA in blood, semen, skin, saliva or hair found at a crime scene to identify a matching DNA of an individual, such as 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 matching DNA. However identification can be complicated if the scene is contaminated with DNA from several people. DNA profiling was developed in 1984 by British geneticist Sir Alec Jeffreys and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case.

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. On the other hand many convicted people have been released from prison on the basis of DNA techniques, which were not available when a crime had originally been committed.

Bioinformatics of DNA

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. 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. 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. 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.

Research Histry of DNA

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". In 1919, Phoebus Levene identified the base sugar and phosphate nucleotide unit. 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.

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. This system provided the first clear suggestion that DNA carried genetic information—the Avery-MacLeod-McCarty experiment when Oswald Avery, along with coworkers Colin MacLeod and Maclyn McCarty, identified DNA as the transforming principle in 1943. DNA's role in heredity was confirmed in 1952, when Alfred Hershey and Martha Chase in the Hershey-Chase experiment showed that DNA is the genetic material of the T2 phage.

In 1953 James D. Watson and Francis Crick suggested what is now accepted as the first correct double-helix model of DNA structure in the journal Nature. Their double-helix, molecular model of DNA was then based on a single X-ray diffraction image taken by Rosalind Franklin and Raymond Gosling in May 1952, as well as the information that the DNA bases were paired—also obtained through private communications from Erwin Chargaff in the previous years. Chargaff's rules played a very important role in establishing double-helix configurations for B-DNA as well as A-DNA.

Experimental evidence supporting the Watson and Crick model were published in a series of five articles in the same issue of Nature. Of these, Franklin and Gosling's paper was the first publication of their own X-ray diffraction data and original analysis method that partially supported the Watson and Crick mode; this issue also contained an article on DNA structure by Maurice Wilkins and two of his colleagues, whose analysis and in vivo B-DNA X-ray patterns also supported the presence in vivo of the double-helical DNA configurations as proposed by Crick and Watson for their double-helix molecular model of DNA in the previous two pages of Nature. In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine. Unfortunately, Nobel rules of the time allowed only living recipients, but a vigorous debate continues on who should receive credit for the discovery.

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". Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson-Stahl experiment. 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. These findings represent the birth of molecular biology.