The mediator binds intimate y to the DNA, abstracts electrons quickly from guanines in the DNA macromolecule, diffuses freely to the electrode, and then delivers the electrons from the guanines to the electrode to produce a measurable current. If DNA is attached to an electrode, no current is obtained in the absence of a mediator; however, when the mediator is present, the current obtained reflects the quantity of guanine bases bound to the electrode. Detecting mismatches

A complication that arises when attempts are made to detect sequences by hybridization is that stable hybrids can form between two complementary strands where there is a single error in the Watson-Crick base pairing (that is, where either A is not bound to T or G is not bound to C).
Such an error is called a mismatch, and a single one of these errors in a DNA duplex is called a single-base mismatch. Duplexes that contain one single-base mismatch are only slightly less stable than duplexes that match perfectly. Therefore, in a hybridization assay it is not always possible to determine whether the hybridized DNA is a perfect match or contains a mismatch.

This problem is particularly frustrating because most genetic abnormalities of interest involve the mutation of a single base. The electrochemical detection scheme provides a means for distinguishing perfectly matched duplexes from strands that contain a mismatch at guanine. The current obtained from guanine oxidation by the mediator is a function of the rate at which the electron is abstracted from DNA.

It is believed that the rate of guanine in the GC pair is the slowest because the helix packs together best when there are no mismatches and the solvent accessibility of guanine is therefore the lowest. In the mismatches, the packing is not as good, which distorts the DNA and makes guanine more solvent accessible. Subtle changes in the driving force are not believed to be responsible for the change in rate, because the change in driving force required to effect the changes in rate seen are too large to reasonable. Electron-transfer reactions that occur over relatively long distances proceed by tunneling.

(Tunneling is the process whereby an electron can start in one place and end up somewhere else without ever being in between. Because of the uncertainty principle, there is a finite probability of the electron being either on guanine or on the mediator. Thus, the electron could be on guanine at one instant and on the next.)

The rates of electron transfer reactions that occur over relatively long distances vary exponentially with the distance between the two redox partners, in this case, Ru p 33 b, to the outside of DNA and guanine situated on 't is the double heat (he Ru(bpy)t binds to DNA groove, which precludes intimate contact with! t base. As a result, the electron must tunnel are distance.

When the guanine is perfectly matched it is most effectively protected from oxidation, s is maximal. However, if guanine is paired with base, the double helix does not pack together as the guanine is more exposed to the solvent and consequently is more accessible to the Ru(bpy)3 Power3+ oxidant.

As a result, the distance is shorter in the mismatch than in the GC pair, leading to a faster rate of electron transfer and a higher current. Studies of all of the possible mismatches show that the rates of oxidation increase in the order GC GT GG GA single-stranded G, which correlates with the size of the base opposite G.

Advantages Mediated electrochemical detection of DNA provides some important advantages over existing fluorescent and radiochemical methods. The first advantage is that labeling of the  target strand is not necessary. This may greatly simplify the analysis procedure. Second, the electrochemical method provides a means for distinguishing a single-base mismatch in duplex DNA that is maintained in its native structure.  Numerous breakthroughs in technology are required to integrate genetic analysis into the everyday activities of physicians and patients, and mediated electrochemical detection may be one such breakthrough.

DNA Replication and Repair Helicases
Deoxyribonucleic acid is a complex molecule composed four bases covalently attached to a phosphorylated deoxyribose sugar. Phosphodiester linkages between sugar form the backbone of the DNA molecule, while the unique order of repeating bases specifies genes which code for proteins. The most stable form of DNA is a double-strand molecule (dsDNA), composed of two complementary strands held together by interstrand hydrogen bonds between paired nucleotides. However, many aspects of DNA metabolism require the transient generation of localized regions of single-stranded DNA (ssDNA). The individual strands have a chemical and structural polarity defined by the orientation of the sugar; in double-stranded DNA the strands are antiparallel with one having a 5’ to 3’ polarity and the other having a 3’ to 5’ polarity

Each somatic cell in a human contains about 6 billion base pairs organized on 46 chromosomes; individual genes specify an estimated 50,000 proteins by first transcribing an RNA copy with the corresponding sequence of bases. The sequence of bases in the RNA is translated into a specific sequence of amino acids in the protein. Only a subset of genes is expressed at a particular time in any given cell type. Since each of these proteins is vital to the normal development and function of the nearly 60 trillion cells which constitute an adult human, it is important that chromosomes are stably maintained throughout an individual’ s life-span and both the maintenance of intact chromosomes and the correctness of each gene in the DNA.

DNA replication, repair, and recombination are three processes essential for genomic stability. Prior to cell division, DNA polymerases use ssDNA as a template for the synthesis of new DNA molecules (replication), thus ensuring that each daughter cell contains the same genetic information as the parental cell. Cells continuously monitor DNA for the presence of inappropriate or damaged bases and, when found, these errors are corrected by DNA repair enzymes. Failure to repair such errors results in mutations, which are changes in the DNA base sequence that may alter the amino acid sequence and function of encoded proteins . Recombination is a mechanism for the exchange of genetic information and is a prerequisite for the normal segregation of chromosomes during meiosis. Errors in chromosome segregation can result in a condition such as Down syndrome.

Some type of DNA repair also Helicases Each of these metabolic pathways requires transient ssDNA templates or intermediates. The generation of ssDNA is mediated by DNA helicases, enzyme which bind both DNA and nucleoside 5’-triphophates (NTP) and couple the binding and hydrolysis of NTP to the unwinding a short region of ssDNA for binding, although some can bind to nicked DNA (dsDNA in which a Phosphodiester bond is cleaved in the deoxyribose-phosphate backbone of one strand of duplex DNA). The chemical energy from NTP hydrolysis is transduced into mechanical energy which fuels both the disruption of hydrogen bonds between base pairs and movement (translocation) of the helicase along the DNA molecule. This translocation occurs in one direction in nature, with the direction defined by the polarity of the DNA strand with which the helicase interacts.

Fig. 2 Mechanism of action for DNA helicases. This helicase requires a short ssDNA region for binding has 5' to 3'polarity, and uses the energy of ATP (adenosine 5'-triphosphate) Hydrolysis to translocate along the DNA molecule

Mechanism of action for DNA helicases.

Helicases are ubiquitous enzymes. Most organisms have multiple helicases which catalyze essentially the same biochemical reaction. At least 10 helicases have been identified in human cells. This apparent redundancy likely reflects specialization in terms of DNA structure and polarity, as well as protein-protein interactions, and serves to restrict a specific helicase activity to a particular subpathway of DNA metabolism.

Helicases were discovered in the 1970s, analyses grouped these enzymes into five related families with characteristic conserved amino acid sequence motifs separated by nonconserved spacer regions of variable amino acid lengths. Nucleotide binding and helix unwinding activities have been demonstrated for many of the family members, and it is presume that other proteins with these seven motifs (I, IA, and II-VI) are either DNA or ribonucleic acid (RNA) helicases. The presence of these conserved regions is not an absolute indication of helicase activity, as there are some helicases which have only domains I and II, identified as the NTP and magnesium ion (Mg2+) cofactor binding domains, and other proteins which have all seven domains but lack true helicase activity. Nonetheless, these domains have been useful predictors of helicase activity when identified in newly cloned genes of unknown function.

DNA damage , repair, and cancer Malignancies arise through an accumulation of mutations in genes which encode proteins, such as oncogenes or tumor suppressors. This mutation-causing damage may arise from normal, endogenous events (for example, oxygen metabolites generated by the cell), or may be caused by exogenous, environment factors (for example, solar ultraviolet irradiation or exposure t certain chemicals). Under normal circumstances (such as low levels of agenotoxic agents and functional responses to cellular damage), the agent is either eliminated or neutralized by detoxification pathways before the DNA is altered. In the event that the DNA is modified, there are mechanisms for the removal of DNA damage.

The most prevalent mechanism for damage removal is nucleotide excision repair, a complex pathway which removes damage in the form of short oligonucleotides and then uses the complementary, undamaged strand as a template for resynthesis of the excised oligomerFig. 3). Recent research has contributed much to understanding the mechanistic details of excision repair in humans, a process requiring as many as 20-25 polypeptides for events that are accomplished by Escherichia coli with just six proteins: UVR(A)BC excinuclease, helicase II, DNA polymerase, and ligase.

One of the more intriguing findings of recent years has been the discovery of transcription-coupled repair, a phenomenon in which actively transcribed genes are preferentially repaired. Although the process was first identified in mammalian cells, the molecular details have been reported only for bacterial cells. A transcription-repair coupling factor (TRCF) recognizes and interacts with RNA polymerase stalled at a lesion, releases the polymerase, and recruits the E. coil repair enzymes to the damage site. Interestingly, this factor has the seven helicase domains, but the protein lacks true helicase activity

Fig. 3 Model for DNA excision repari (a) DNA damaged by ultraviolet irradiation or chemicals is a substrate for nucleotide excision repair. (b) DNA damage is recognized and bound by specific proteins. (c) Damage recognition proteins recruit other subunits, including those with helicase activity, to the damaged region, which is locally unwound. (d) Dual incisions are made both 3'and 5' to the damage, a 27-30-nucleotide oligomer is excised, and this short piece of DNA and some proteins of the excision complex are released from the DNA; release may be mediated by helicases. (e) Replication proteins are recruited to the resulting gap, which is filled by using the undamaged complementary strand as a template and remaining repari proteins are dissociated

Associated human disorders. Several rare human diseases are associated with an inherited condition of chromosomal instability and, in some cases, with a predisposition to cancer. These include xeroderma pigmentosum, Cockayne's syndrome, Bloom's syndrome, hereditary nonpolyposis colorectal cancer, Bloom’s syndrome hereditary nonpolyposis colorectal cancer, ataxia telangiectasia, Fanconi's anemia, and Werner's syndrome.

It was once hypothesized that each condition resulted from a defect in processing DNA damage. However it is now believed that only the xeroderma pigmentosum syndrome is directly due to a defect in basal nucleotide excision repair, while Cockayne's syndrome results when transcription-coupled repair is impaired; nonpolyposis colorectal cancer is correlated with a defect in mismatch repair. Xeroderma pigmentosum. The underlying cause of xeroderma pigmentosum is a defect in one of seven genes involved in nucleotide excision repair. Clinical manifestation of xeroderma pigmentosum is due to a combination of a genetic defect and environmental factors (for example, exposure to solar irradiation), and includes hypersensitivity to sunlight, an increased frequency of skin cancers or internal cancers and, in some cases, neurologic abnormalities.

Two of the proteins associated with xeroderma pigmentosum have the seven helicase motifs and NTP-­dependent helix-unwinding activity. Xeroderma pigmentosum group B (XPB), xeroderma pigmentosum group D (XPD), and five other polypeptides make a multiprotein complex called transcription factor IIH (TFIIH) which s a general transcription factor and a repair factor, but only the helicase activity of XPB is essential for transcription. It is not known why TFIIH contains two helicases or even if both of the function as helicases during repair. The precise roles of XPB and XPD in repair are unknown, but two suggestions are localized unwinding of the duplex to permit access of other repair proteins, and dissociation of other proteins following removal of damage.

Cockayne's syndrome. Cockayne's syndrome is Characterized by sun sensitivity and progressive neurologic abnormalities, ut without the increased frequency of skin cancers. The underlying defect in Cockayne's syndrome is due to a mutation in one of two genes, Cockayne's syndrome group A and group B (CSA and CSB), both of which  have been implicated in transcription-coupled repair. Sequence analysis of CSB revealed the seven helicase domains, although DNA unwinding activity has not been reported for the purified protein. Bloom's and Werner's syndromes Individuals with Bloom's syndrome exhibit extreme genomic instability resulting in a spectrum of clinical features including sunlight hypersensitivity, immunodeficiency, fertility problems, and a predisposition to cancer. Some cultured cells show an abnormal response to DNA-damage agents, although no defect in a specific repair pathway has been found. Rather, it is believed that cells associated with Bloom’s syndrome are unable to efficiently complete certain, as yet unidentified DNA metabolic events.

In 1995 the cloning and sequence analysis of BLM, the gene responsible for Bloom’s syndrome, indicated that the gene is a putative helicase with homology to the E. coli RecQ protein, which is known to be involved in recombination. In 1996 the gene (WRN) that causes Werner’s syndrome was cloned. Although sequence analysis of WRN revealed seven conserved helicase domains, an enzymatic activity has not been demonstrated. Werner's syndrome is a condition diagnosed in postadolescent individuals with premature signs of aging including hair graying, balding, arteriosclerosis, cataracts, wasting of musculature, and tumors. A specific DNA repair defect has not been hypothesized that a consequence of aberrant DNA metabolism may be the early accumulation of mutations in proteins associated with age-related diseases or cancer.

DNA Modified to Mimic RNA Function Many microorganisms produce biochemicals that are natural antibiotics, toxic to competitors within the environment niche occupied by the organism. Some antibiotics have become part of the physician’s conventional reperatoire use of antibiotics microorganisms have evolved mechanisms to evade the effects of antibiotics. Many antibiotics target the protein-synthesizing machinery, the ribosome, of bacteria and fungi. Microorganisms that are resistant to an antibiotic have ribosomes that are mutated at specific locations and thus are no longer affected by the drug. The ribosome decoding site is a location on the ribosome where the genetic information for a protein is translated into that protein's sequence of amino acids. A nucleic acid has been designed to inhibit protein synthesis at the ribosomal decoding site, and it has antibacterial activity.

Ribosome. Living organisms contain two types of nucleic acid molecules: deoxyribonucleic acid and ribonucleic acid (RNA). Ribosomes are composed of RNA and proteins. There is a clear line of demarcation between the biological functions of DNA and those of the different RNA. DNA, the genetic material of almost all organisms; including viruses, is the inherited blueprint for the required biochemistry, development, and maturation of the organism. For all organisms, RNA is the irreplaceable translator of genetic information into proteins. It is the genetic material for a few mammalian and bacterial viruses. However, the two nucleic acids are very similar.

They are polymers composed of monomers called nucleosides. The majority of nucleosides have a single base: adenine (A), guanine (G), cytosine (C), or thymine (T) for DNA, and uracil (U) for RNA. These (nucleosides are attached to a five-carbon sugar: deoxyribose for DNA and ribose for RNA. In the polymer, the sugar of each major nucleoside is attached to an adjacent nucleoside by a phosphate. Three types of RNA are directly involved in proem synthesis: messenger RNA (mRNA), ribosomal RNA (RNA), and transfer RNA (tRNA). They have many modified' nucleosides in addition to the four major nucleosides. Currently, there are over 90 known naturally occurring modified nucleosides in mRNA, RNA, and tRNA. Additional modified nucleosides have been devised by researchers, including the acquired immune deficiency syndrome (AIDS) drug azidothymidine (AZT). Naturally occurring modified nucleosides alter the chemistry and structure of RNA, and so enhance and modify the biological function of RNA.

Much of the chemistry contributed to RNA by modified nucleosides has its exact counterpart in the contribution of amino acids to proteins.Some modified nucleosides' are hydrophobic and thereby contribute the lipidlike characteristic of being unable to associate with water at a site at which this nucleoside occurs in the RNA. Others are hydrophilic and polar, or hydrophilic and negatively or positively charged. Modified nucleosides have been used to design and produce a mimic to tRNA function. Transfer RNA.

Transfer RNA is responsible for bringing individual amino acids to the ribosome in response to the sequence of three-base codes (codons) in mRNA. Messenger RNA is" a copy of the genetic information for a specific protein. Individual amino acids are added to the nascent protein at the ribosome by formation of peptide bonds in the order prescribed by the sequence of codons in the mRNA. At the ribosome's decoding site, tRNA read the codons correctly at the ribosome by having a complementary set of three bases, an antic odon that is specific for each amino acid. The anticodon recognizes the codon by virtue of Watson-Crick base pairing (as in DNA). The anticodon GAA for the amino acid phenylalanine binds the codon UUC, with phosphate backbones GAA and CUU running in opposite directions. The reading of the codons is such a fundamental part of protein synthesis that it is considered a virtually immutable function. Thus, this function of tRNA could be' a target for the production of a mimic that 4IDibits protein synthesis.