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The purpose of this chapter is to present the background to this project together with a critical review of the literature that is directly relevant to this project. This project aims to demonstrate the ability to print DNA that will have direct relevance to biomedical research adopting biomedical sensing as a potential application. Consequently the review will address the relevant essential aspects of DNA, printing technologies and the principles of sensor devices.
DNA structure and function
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".[Dahm R., 2008] In1919, Phoebus Levene identified the base, sugar and phosphate nucleotide unit.[levene P., 1919] 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.[Hershey A. and Chase M., 1952] 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.[Watson J.D. and Crick F.H.C., 1953] Chargaff's rules played a very important role in establishing double-helix configurations for B-DNA as well as A-DNA.
Nucleic acids not only serve as genetic material of living organisms including humans but also involved in the storage, transfer and expression of genetic information. They contain all the necessary information required for the formation of individual or organism and determines physical fitness of an individual to life, at the same time some nucleic acids acts as enzymes and coenzymes. Moreover DNA exhibits structural polymorphism. It assumes several forms depending on certain conditions. Several DNA variants are known. After Human Genome Project (HGP) is completed in 2000. It is useful for finding causes of several diseases whose causes are unknown till. It may also lead to development of new therapeutics as well as diagnostics.
Nucleic acid structure
Nucleic acid structure refers to the structure of nucleic acids such as DNA and RNA It is often divided into four different levels:
Primary structure-the raw sequence of nucleobases of each of the component DNA strands; Secondary structure-the set of interactions between bases, i.e., which parts of which strands are bound to each other; Tertiary structure-the locations of the atoms in three-dimensional space, taking into consideration geometrical and steric constraints; Quatemary structure-the higher-level organization of DNA in chromatin, or to the interactions between separate RNA units in the ribosome or spliceosome.
1.1.1 DNA structure
DNA is a linear, unbranched polymer in which the monomeric subunits are four chemically distinct nucleotides that can be linked together in any order in chains hundreds, thousands or even millions of units in length. Each nucleotide in a DNA polymer is made up of three components (Figure 1.1) This is the primary structure.
Figure 1.6. The structure of a nucleotide.
Figure 1.1 The structure of a nucleotide
(A) The general structure of a deoxyribonucleotide, the type of nucleotide found in DNA. (B) The four bases that occur in deoxyribonucleotides.
1. 2â€²-deoxyribose, which is a pentose, a type of sugar composed of five carbon atoms. These five carbons are numbered 1â€², 2â€², etc. The name '2â€²-deoxyribose' indicates that this particular sugar is a derivative of ribose, one in which the hydroxyl (-OH) group attached to the 2â€²-carbon of ribose has been replaced by a hydrogen (-H) group.
2. A nitrogenous base, one of cytosine, thymine (single-ring pyrimidines), adenine or guanine(double-ring purines). The base is attached to the 1â€²-carbon of the sugar by a Î²- N -glycosidic bond attached to nitrogen number 1 of the pyrimidine or number 9 of the purine.
3. A phosphate group, comprising one, two or three linked phosphate units attached to the 5â€²-carbon of the sugar. The phosphates are designated Î±, Î² and Î³, with the Î±-phosphate being the one directly attached to the sugar.
A molecule made up of just the sugar and base is called a nucleoside; addition of the phosphates converts this to a nucleotide. Although cells contain nucleotides with one, two or three phosphate groups, only the nucleoside triphosphates act as substrates for DNA synthesis. The full chemical names of the four nucleotides that polymerize to make DNA are:
The abbreviations of these four nucleotides are dATP, dCTP, dGTP and dTTP, respectively, or, when referring to a DNA sequence, A, C, G and T, respectively.
Figure 1.7. A short DNA polynucleotide showing the structure of the phosphodiester bond. Note that the two ends of the polynucleotide are chemically distinct.
Figure 1.2 A Short DNA polynucleotide showing the structure of the phosphodiester bond. Note that the two ends of the polynucleotide are chemically distinct
Figure 1.8. The polymerization reaction that results in synthesis of a DNA polynucleotide.
Figure 1.3 The polymerization reaction that results in synthesis of a DNA polynucleotide
Synthesis occurs in the 5â€²â†’3â€² direction, with the new nucleotide being added to the 3â€²-carbon at the end of the existing polynucleotide. The Î²- and Î³-phosphates of the nucleotide are removed as a pyrophosphate molecule.
In a polynucleotide, individual nucleotides are linked together by phosphodiester bonds between their 5â€²- and 3â€²-carbons (Figure 1.2). From the structure of this linkage we can see that the polymerization reaction (Figure 1.3) involves removal of the two outer phosphates (the Î²- and Î³-phosphates) from one nucleotide and replacement of the hydroxyl group attached to the 3â€²-carbon of the second nucleotide. Note that the two ends of the polynucleotide are chemically distinct, one having an unreacted triphosphate group attached to the 5â€²-carbon (the 5â€² or 5â€²-P terminus) and the other having an unreacted hydroxyl attached to the 3â€²-carbon (the 3â€² or 3â€²-OH terminus). This means that the polynucleotide has a chemical direction, expressed as either 5â€²â†’3â€² (down in Figure 1.3) or 3â€²â†’5â€² (up in Figure 1.3). An important consequence of the polarity of the phosphodiester bond is that the chemical reaction needed to extend a DNA polymer in the 5â€²â†’3â€² direction is different to that needed to make a 3â€²â†’5â€² extension. All natural DNA polymerase enzymes are only able to carry out 5â€²â†’3â€² synthesis, which adds significant complications to the process by which double-stranded DNA is replicated. Then the secondary structure is described as follows.
1.1.2 DNA double helix
The names of James Watson and Francis Crick are so closely linked with DNA that it is easy to forget that, when they began their collaboration in Cambridge, England in October 1951, the detailed structure of the DNA polymer was already known. Their contribution was not to determine the structure of DNA per se, but to show that in living cells two DNA chains are intertwined to form the double helix.
In the years before 1950, various lines of evidence had shown that cellular DNA molecules are comprised of two or more polynucleotides assembled together in some way. The possibility that unraveling the nature of this assembly might provide insights into how genes work prompted Watson and Crick, among others, to try to solve the structure. According to Watson in his book The Double Helix (see Further Reading), their work was a desperate race against the famous American biochemist, Linus Pauling, who initially proposed an incorrect triple helix model, giving Watson and Crick the time they needed to complete the double helix structure (Watson and Crick, 1953). It is now difficult to separate fact from fiction, especially regarding the part played by Rosalind Franklin, whose X-ray diffraction studies provided the bulk of the experimental data in support of the double helix and who was herself very close to solving the structure. The one thing that is clear is that the double helix, discovered by Watson and Crick on Saturday 7 March 1953, was the single most important breakthrough in biology during the 20th century.
Figure 1.11. The double helix structure of DNA.
Figure 1.4 The double helix structure of DNA
(A) Two representations of the double helix. On the left the structure is shown with the sugar-phosphate 'backbones' of each polynucleotide drawn as a red ribbon with the base pairs in black. On the right the chemical structure for three base pairs is given. (B) A base-pairs with T, and G base-pairs with C. The bases are drawn in outline, with the hydrogen bonding indicated by dotted lines. Note that a G-C base pair has three hydrogen bonds whereas an A-T base pair has just two. The structures in part (A) are redrawn from [Turner et al., 1997] (left) and [Strachan and Read, 1999] (right).
The double helix is right-handed, which means that if it were a spiral staircase and you were climbing upwards then the rail on the outside of the staircase would be on your right-hand side. The two strands run in opposite directions (Figure 1.4A). The helix is stabilized by two types of chemical interaction:
Base-pairing between the two strands involves the formation of hydrogen bonds between an adenine on one strand and a thymine on the other strand, or between a cytosine and a guanine (Figure 1.4B).Hydrogen bonds are weak electrostatic attractions between an electronegative atom (such as oxygen or nitrogen) and a hydrogen atom attached to a second electronegative atom. Hydrogen bonds are longer than covalent bonds and are much weaker, typical bond energies being 1-10 kcal mol-1 at 25 °C, compared with up to 90 kcal mol-1 for a covalent bond. As well as their role in the DNA double helix, hydrogen bonds stabilize protein secondary structures. The two base-pair combinations - A base-paired with T, and G base-paired with C - explain the base ratios discovered by Chargaff. These are the only pairs that are permissible, partly because of the geometries of the nucleotide bases and the relative positions of the groups that are able to participate in hydrogen bonds, and partly because the pair must be between a purine and a pyrimidine; a purine-purine pair would be too big to fit within the helix, and a pyrimidine-pyrimidine pair would be too small.
Base-stacking, sometimes called Ï€-Ï€ interactions, involves hydrophobic interactions between adjacent base pairs and adds stability to the double helix once the strands have been brought together by base-pairing. These hydrophobic interactions arise because the hydrogen-bonded structure of water forces hydrophobic groups into the internal parts of a molecule.
Both base-pairing and base-stacking are important in holding the two polynucleotides together, but base-pairing has added significance because of its biological implications. The limitation that A can only base-pair with T, and G can only base-pair with C, means that DNA replication can result in perfect copies of a parent molecule through the simple expedient of using the sequences of the pre-existing strands to dictate the sequences of the new strands. This is template-dependent DNA synthesis and it is the system used by all cellular DNA polymerases
Figure 1.12. Computer-generated images of B-DNA (left), A-DNA (center) and Z-DNA (right).
Figure 1.5 Computer-generated images of B-DNA (left), A-DNA (center) and Z-DNA (right)
Reprinted with permission from [Kendrew A 1994]
The double helix has structural flexibility
The double helix described by Watson and Crick, and shown in Figure 1.4A, is called the B-form of DNA. Its characteristic features lie in its dimensions: a helical diameter of 2.37 nm, a rise of 0.34 nm per base pair, and a pitch (i.e. distance taken up by a complete turn of the helix) of 3.4 nm, this corresponding to ten base pairs per turn. The DNA in living cells is thought to be predominantly in this B-form, but it is now clear that genomic DNA molecules are not entirely uniform in structure. This is mainly because each nucleotide in the helix has the flexibility to take up slightly different molecular shapes. To adopt these different conformations, the relative positions of the atoms in the nucleotide must change slightly. There are a number of possibilities but the most important conformational changes involve rotation around the Î²-N-glycosidic bond, changing the orientation of the base relative to the sugar, and rotation around the bond between the 3â€²- and 4â€²-carbons. Both rotations have a significant effect on the double helix: changing the base orientation influences the relative positioning of the two polynucleotides, and rotation around the 3â€²-4â€² bond affects the conformation of the sugar-phosphate backbone.
Rotations within individual nucleotides therefore lead to major changes in the overall structure of the helix. It has been recognized since the 1950s that changes in the dimensions of the double helix occur when fibers containing DNA molecules are exposed to different relative humidities. For example, the modified version of the double helix called the A-form (Figure 1.5) has a diameter of 2.55 nm, a rise of 0.29 nm per base pair and a pitch of 3.2 nm, corresponding to 11 base pairs per turn. Other variations include Bâ€²-, C-, Câ€²-, Câ€²â€²-, D-, E- and T-DNAs. All these are right-handed helices like the B-form. A more drastic reorganization is also possible, leading to the left-handed Z-DNA (Figure 1.5), a slimmer version of the double helix with a diameter of only 1.84 nm.
DNA is the genetic material of living systems. It is super chip ever made by man present in living systems. It contains all the information required for the formation of an individual or organism. The genetic information in DNA is converted to characteristic features of living organisms like colour of the skin and eye, height, intelligence, ability to metabolize particular substance, ability to with stand stress, susceptibility to disease and unable to produce or synthesize certain substances etc. All the above phenotype characters of living organisms are intimately related to functions of proteins. Thus, DNA is the source of information for the synthesis of all cellular proteins. The segment of DNA that contains information for a protein is known as gene. DNA is transmitted from parent to off spring and hence DNA flows from one generation to other in a given species. Further, DNA provides information inherited by daughter cells from parent cells. The amount of DNA per cell is proportional to the complexity of the organism and hence to the amount of genetic information. The amount of DNA in mammalian cell is 1000 times more than bacteria. Likewise, bacteria contains more DNA than virus and plasmids. The amount of DNA in any given species or cell is constant and is not affected by nutritional or metabolic states. The tertiary and quatemary structure are described in the variants.
1.1.4 DNA variants
DNA structural polymorphism or DNA variants
Thus DNA molecule has chameleon like property assumes various forms depending on environment. [N. Mallikarjuna Rao, 2006]
Most of the DNA in the genome is in B-form. Other forms of DNA are A-DNA and Z-DNA. When DNA fibre is dehydrated it acquires another form. It is known as A-DNA. It is aborter than B-DNA. The base pairs are not perpendicular to the axis they are tilted by 19°. In A and B forms, glycosidic bonds are in 'anti' conformation. Z-DNA which is left handed double helix. A small stretch of Z-DNA can occur in B-DNA. Z-DNA is due to the presence of dinucleotides like CG CG CG containing alternate purine and pyrimidine bases. In Z-DNA, glycosidic bonds are in syn conformation.
Recent studies have established existence of several forms of DNA structures not just A, B and Z as mentioned earlier. The helical structure of DNA assumes various forms depending on conditions. Some DNA structures show minor differences from Watson-Crick model while many of them are completely different in essential features such as handedness, base pairing and number of strands. DNA variants are identified by one letter code and currently there are polymorphic DNA structures associated with 21 of 26 letters of English alphabet. Only, F, Q, U, V and Y are not used.
Few unusual and interesting DNA structures are H-DNA which is an intramolecular triple helical structure of DNA. It is made up of three strands. It is formed at low pH conditions. This type of structure is formed in DNA containing long stretches of polyurine and polypyrimidine sequences. The pyrimidine rich strand dissociates from complementary strand and folds back on itself to lie in the major groove and hydrogen bonded to purine rich strand. This type of structures plays role in transcriptional control of gene expression. G-Quadruplex structure which is made up of four strands. Several four-stranded quadruplex DNA structures occurs in G-rich DNA sequences. They are also known as G-tetrads. In these DNA structures, the four strands are parallel arranged. They are found in telomeric regions of chromosomes. And holliday junction which forms during genetic recombination. One of the strand from each of the duplex DNA molecules exchange to form four ways junction, which is known as Holliday junction.
Different DNA resources introduced as follows:
Eukaryotic DNA: in non-dividing eukaryotic cell DNA exist as nucleoprotein called chromatin. Chromatin consist of DNA and basic proteins histones. This organizes into 23 pairs of chromosomes before cell division. Each chromosome represents one DNA molecule. The chromosomal DNA has length of about 30-60 mm. Such long molecule is present in nucleus whole dimension is less than 5 microns (5 u) (1u= 10-3mm). So, DNA molecule is tightly packed such that it can be accommodated within nuclear limit. Histones are used for packing of DNA. Five types of histones are used for packing of DNA. They are H1, H2A, H2B, H3 and H4.
Nucleosome: whole DNA is not packed as single coil instead it is present as small coils known as nucleosomes. Each nucleosome consist of histone octamer, which is made up of two units of H2A, H2B, H3 and H4 histones and DNA. Usually DNA is coiled around octamer, and approximately it takes two turns around histone octamer. Each nucleosome is joined by linker DNA and HI type of histones. The nucleosomes along with linker DNAs appears as beads on a string under electron microscope. Further coiling of nucleosomes forms chromatin fibre, Thus, long thread like DNA molecule is folded into chromosomes.
Mitochondrial DNA: eukaryotic mitochondria contains DNA. It is different from DNA present in nucleus. It account for 1% of cellular DNA. Base composition of mitochondrial DNA is different from nuclear DNA. Mitochondrial DNA is double stranded and circular.
Bacterial DNA: Bacteria like E. Coli contains single molecule of double stranded DNA. E. Coli DNA is 1.4 mm long which is 700 times bigger than the size of bacteria. Hence in bacteria also DNA is tightly packed or folded. In E. Coli the two ends of DNA are joined to form circular DNA. Histones are not used for packing of bacterial DNA because they are absent in bacteria. Super coiling of circular DNA allows its containment with in nuclear zone. Super-coiled DNA may be in association with some proteins, which stabilizes super coil.
Viral DNA: viruses are extremely small particles. They are composed of a piece of DNA, which is surrounded by protein cost called capsid. Viral DNA may be single stranded or double stranded. Adeno virus (cold virus), Herpes virus and Pox virus are examples for double stranded viruses. Parvo virus is a example for single strand DNA virus.
Plasmids: They exist in bacteria as circular DNA molecules. Plasmid DNA is different from bacterial DNA. They are present in anti-biotic resistant bacteria. They contain genes for inactivation of anti-biotics. pBR322 of E. Coli is an example for plasmid. Plasmids are used as vectors in genetic engineering.