Microchips

                        Microchip analysis has become an attractive option in CE for clinical applications, not only because these kinds of analysis require usage of complex samples that are often limited in quantity but also because this system could allow clinicians to make quicker treatment and drug therapy decisions. 

                        The use of CE in the microchip format allows high-speed separations of very small samples, and multiple channel systems have great potential for high-throughput analysis. The first report of DNA separation on a microchip device was published by Woolley and Mathies in 1994 (103). Since then, great progress has been made in the design of these devices, going from single-channel to multiple-channel chips, allowing analysis of several DNA samples. 

                         The design of these systems was reviewed by Gao et al. in 2001 (104). A typical microchip device consists of one or several separation channels of 3–10 cm in length and 10–100 μm in diameter. There are also buffer, waste, and sample reservoirs. High voltages of 2–30 kV can be applied to the reservoirs by platinum electrodes because these systems disperse the heat very efficiently. 

                          The small samples used in the CE microchips require extremely sensitive detection  techniques. LIF is the most commonly used detection method in CE microchips, although electrochemical, ultraviolet (UV), chemiluminiscence, and indirect fluorescence detection methods have been combined with these systems. 

Sequencing

                         DNA sequencing is the determination of the order of nucleotides in a DNA molecule. The most popular method for doing this is called the Sanger sequencing method or chain-termination method. This method uses  synthetic nucleotides that lack the 3′ hydroxyl group and are unable to form the 3′–5′ phosphodiester bond necessary for chain elongation. These nucleotides are called dideoxynucleotides (ddNTPs). 

                          The sequencing reaction consists in a single-stranded template DNA to which a short complementary primer is annealed and extended by a DNA polymerase. The sequencing reaction contains a low concentration of ddNTPs, each labeled with a different fluorochrome, in addition to the normal deoxynucleotides. 

                           Once a ddNTP is incorporated in the elongating chain, it blocks further chain extension; as a result, a mixture of chains of lengths determined by the template sequence is accumulated. This mixture can then be resolved by CGE. CGE resolution allows separation of chains that differ in length by only one nucleotide. 

Analysis of Methylation Status

                         DNA methylation refers to the addition of a methyl group to the 5-position of cytosine residues that are followed immediately by a guanine (so-called CpG dinucleotides). Small stretches of DNA known as CpG islands are rich in CpG dinucleotides. 

                         These CpG islands are frequently located within the promoter regions of human genes, and methylation within the islands has been shown to be associated with transcriptional inactivation of the corresponding gene. Changes in DNA methylation profiles are common features of development, and DNA methylation is also involved in X-chromosome inactivation and allele-specific silencing of imprinted genes. Aberrant changes in methylation profiles are associated with many serious pathological consequences such as tumorigenesis. 

                           Methylation of CpG islands has been shown to be important in transcriptional repression of numerous genes that function to prevent tumor growth or development. This phenomenon includes both genome-wide hypomethylation and genespecific hypermethylation. Capillary electrophoresis has been applied to the evaluation of the relative methylation degree of genomic DNA methylation. 

                            In this approach, genomic DNA is enzymatically hydrolyzed to single nucleotides. Single nucleotides are then labeled with a fluorescent marker and separated by CE to identify cytosine and 5-methyl cytosine residues.  

Quantification of DNA Damage

                         Damage to cellular DNA is implicated in the early stages of carcinogenesis and in the cytotoxicity of many anticancer agents, including ionizing radiation. CE is a sensitive technique for measuring cellular levels of DNA damage through the detection of specific DNA lesions by specific monoclonal  antibodies. 

                           The detection is done by the use of a secondary antibody labeled with a fluorochrome. This method has been applied to the determination of thymine glycol residues in DNA generated by irradiation of cells during radiation therapy. The sensitivity of detection is in the range of zeptomoles. 

                            Another way to detect DNA damage is the evaluation of DNA laddering during apoptosis. A key step in the onset of apoptosis is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosome-sized fragments of DNA that give rise to a characteristic DNA laddering pattern. 

                             A fluorescent intercalating dye is used to label the DNA molecules that will be resolved by CGE. Also, the use of a DNA standard curve  allows quantification of the apoptotic DNA. The use of CGE with LIF detection permits analysis of DNA laddering with improved automation and much greater sensitivity. 

Temperature Gradient Capillary Electrophoresis

                         In temperature gradient CE (TGCE), a continuous, gradually decreasing temperature gradient is established during the electrophoresis. This approach can be applied to detection of DNA mutations based on thermodynamic stability and mobility shift during electrophoresis. 

                          By spanning a wide temperature range, it is possible to perform simultaneous heteroduplex analysis for various mutation types that have different melting temperatures. Genomic DNA also can be amplified with one fluorescence-labeled primer and one GC-clamped primer and analyzed by TGCE under denaturing temperature conditions. 

                         This tactic has been applied to the detection of mutations in exon 8 of the p53 gene from tumor samples and controls. 

Analysis of DNA-Based Drugs

                        Antisense oligonucleotides are synthetic oligomers that bind a target mRNA or DNA, forming duplexes and thereby blocking its translation into a peptide or triggering its degradation. 

                       Among several DNA analogs proposed as antisense DNA drugs, antisense DNA in which the backbone is modified into phosphorothioate is the most promising for therapeutic application because of its resistance to nuclease degradation. 

                       This methodology is a useful tool for synthesis control and analysis of phosphorothioate analogs and for the determination of their subcellular distribution,  in vitro stability,  tissue half-life, purity,  and metabolism. 

Peptide Nucleic Acid Probes

                         Peptide nucleic acids are molecules in which the bases are conjugated to a polyamide back-bone. The distance between bases and the complementarity are the same as in a DNA or RNA molecule. 

                          The PNA probe is hybridized to a DNA sample amplified by PCR, denatured at low ionic strength, and resolved by CE. The neutral backbone of PNA ensures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA. 

                            DNA strands fully complementary to the target PNA are retarded compared to single-nucleotide mismatched strands. 

                           This method can be used to perform multiplex analyses on several mutations simultaneously when using different amplicon lengths and a set of mutation-specific PNAs. Each targeted mutation can be identified by the size of its corresponding amplicon. Its genotype is further characterized by its interaction with a specific PNA that can differentiate between wild-type and mutant alleles. 

                            This approach has been applied to the detection of R553X and R1162X single-base mutations in patients with cystic fibrosis and to identification of the H63D, S65C, and C282Y mutations in the hereditary hemochromatosis gene. 

Analysis of Heteroduplex DNA

                         Heteroduplex DNA is formed by pairing an unknown nucleic acid strand with a reference strand of a known sequence. Single-stranded nucleic acids with complementary sequences come together and form a double-stranded hybrid molecule in which hydrogen bonds form only between complementary base pairs. 

                          The thermal stability of DNA is related to the number of hydrogen bonds between complementary base pairs; therefore, the greater the similarity in nucleotide sequences between two samples of DNA, the more hydrogen bonds will be present in the heteroduplexes produced. Heteroduplexes generated with a mismatch in the sequence between the two strands will have a different sequence-dependent electrophoretic mobility than homoduplex DNA generated with perfectly matched sequences. 

                           Heteroduplex analysis also can be done to detect different sequences in a mixture by using different probes labeled with different colors. Multiplex analysis has been applied to the simultaneous detection of six  heterozygous mutations in BRCA1 and BRCA2, screening of the three common prothrombotic polymorphisms pl(A), factor V Leiden, and MTHFR (C677T), rapid genetic screening of hemochromatosis, genetic diagnosis of factor V Leiden, clinical diagnosis of rifampin-resistant tuberculosis strains, and analysis of complex mutational spectra of human cells in culture. 

Hybridization Technique

                         In this technique, a probe of known sequence is capable of recognizing a specific DNA sequence in a mixture of many different DNA sequences. The probe can be a synthetic oligonucleotide, a peptide nucleic acid (PNA), a DNA sequence, an RNA sequence, or DNA-based drugs such as DNA vaccines, antisense DNA, protein-binding oligonucleotides, and ribozymes. 

                          The probe is generally labeled with a fluorochrome that can be detected after the hybridization takes place. In a DNA hybridization assay, the sample DNA is heated to separate the two DNA strands and then exposed to the probe. The degree of DNA sequence identity is detected through a hybridization reaction between the DNA and the probe. 

                         The hybridization products are then resolved by CGE. This method has applications in such diverse areas as analysis of pooled genomic DNA samples, detection of mutations, screening of populations for polymorphisms, and identification of species in environmental mixtures. 

Microsatellite Instability

                          Microsatellite instability (MSI) has been shown to be relevant to various diseases, including cancers. Cells with mutations in one of the genes responsible for DNA mismatch repair (MMR) accumulate mutations at a very high rate. 

                            Because DNA mismatches are more likely to occur in DNA microsatellites, defective DNA MMR leads to the phenomenon of MSI, in which the progeny of the defective cells have  varying lengths of a given microsatellite. The microsatellite reference panel to study MSI is BAT25, BAT26, D5S346, D2S123, and D17S250. 

                            Studies of MSI are performed routinely in patients with colorectal cancer because of the defective MMR phenotype of colorectal carcinomas. The electrophoretic profile of the amplified products is compared between tumor and normal DNA from the same patient. 

Forensic DNA Typing

                         In forensic work, DNA from a biological sample and from a known reference material (most often, buccal swabs or blood) are amplified by multiplex PCR with fluorescence-labeled primers and separated by CGE. As a result, unique DNA profiles are generated from both the evidence and the known reference sample and compared to determine whether the patterns are similar. 

                         If the DNA profiles match, then the suspect cannot be excluded as a source of the questioned sample. Likewise, if the DNA profiles do not match, the suspect can be excluded as the donor of the questioned sample. The analysis of 6–10 STRs provides a random match probability of approx 1 in 5 billion. 

                         DNA typing is a useful tool in crime solving, not only for blood samples, sperm, or saliva but also for traces of DNA left on tools or pieces of clothing used in burglaries or thefts. On these kinds of samples, the sources of DNA are extremely small amounts of skin debris left after gripping tools. When a sensitive technique such as PCR coupled with CGE is used, it is possible to get a profile from these low amounts of DNA. 

                         CE provides efficient separation, resolution, sensitivity, and precision for a reliable genotyping of STR loci. Sizing precision of <0.16 nucleotide standard deviation is obtained with this system, thus allowing the accurate genotyping of variants that differ in length by a single nucleotide.

Genetic Mapping

                        The almost random distribution of microsatellites and their high level of polymorphism greatly facilitated the construction of genetic maps and enabled subsequent positional cloning of several genes. A comprehensive genetic map of the human genome based on 5264 microsatellites and a genetic map of the mouse genome based on 4006 simple sequence length polymorphisms were constructed with these markers. 

                        When a trait or disease is inherited through a family and all of the affected members in the family also share a STR that is inherited together, it can be concluded that the disease and marker must be close to each other on a chromosome. 

                        This linkage is the first step in the isolation of disease-causing genes and their localization to an area of DNA. Linkage disequilibrium scanning of the human genome with many thousands of genetic markers has been used to map complex diseases such as schizophrenia and autistic disorder.

VNTR and STR

VNTR

                         Capillary gel electrophoresis has been applied to the analysis of the human apolipoprotein B gene (APOB). This gene maps to the short arm of chromosome 2, and a VNTR is located immediately downstream. 

                        The APOB VNTR alleles generally contain 25–52 repeats of a basic 16-bp unit. Larger alleles containing more than 38 repeat units are more common in people who have myocardial infarction and show a significant association with coronary disease. 

                        Capillary gel electrophoresis also has been used to determine the lung cancer risk associated with rare HRAS1 VNTR alleles. 

 STR

                        The number of tandem repeats for a STR is highly variable from individual to individual and can be as many as 15 different alleles in a certain locus. These sequences also show high levels of heterozygosity, which make them very useful as genetic markers.   

Analysis of Repetitive DNA Sequences

                         The human genome contains large numbers of repetitive DNA sequences, some of which are arrayed as tandem repeat units. Polymorphic tandem repeats occur in two general families: VNTRs and STRs. VNTR or minisatellite DNA consists of variable numbers of a repeating unit from 15 to 100 nucleotides long. 

                          The repeated unit in STR or microsatellite DNA ranges from two to six bases. Products of different lengths can be amplified by using primers complementary to conserved sequences flanking the tandem repeat regions; the length of each amplification product is directly proportional to the number of repeats. 

                            These amplification products can be resolved to individual alleles by CGE. Thus, a person can have at most two different alleles, one from each  chromosome. Laser-induced fluorescence detection of fluorescence-labeled PCR products and multicolor analysis enable the rapid generation of multilocus DNA profiles. 

Single-Strand Conformation Polymorphism

                        The SSCP method is based on the principle that the electrophoretic mobility of single-stranded DNA in a nondenaturing condition depends not only on its size but also on its sequence. Single-stranded DNA has a folded structure that is determined by intramolecular interactions. 

                         This sequence-based secondary structure affects the mobility of the DNA during electrophoresis under nondenaturing conditions. A DNA molecule containing a mutation, even a single-base substitution, will have a different secondary structure than the wild type, resulting in a different mobility shift during electrophoresis than that of the wild type. 

                           The technological aspects of CE-SSCP as well as the potential of CE-SSCP for routine genetic analysis were reviewed by Kourkine et al. in 2002. 

                            Some examples of applications of SSCP by CGE include analysis of the p53 tumor suppressor gene for DNA diagnosis of cancer, detection of mutations on the K-RAS oncogene, detection of a Mycobacterium tuberculosis– specific amplified DNA fragment, detection of mutations in the β-globin gene promoter and the lipoprotein lipase and breast cancer 2 (BRCA2) genes, and identification of multiple mycobacterial species in a sample. 

Restriction Fragment-Length Polymorphism Analysis

                          Restriction fragment-length polymorphism (RFLP) analysis is a powerful tool for the detection of known mutations and polymorphisms, which result in the loss or creation of a recognition site at which a particular restriction enzyme cuts. RFLPs are usually caused by mutation at a cutting site. 

                          DNA carrying the different allelic form will give different sizes of DNA fragments when digested with the appropriate restriction enzyme. 

                          The E4 allele on the APOE gene has been proven to be a major risk factor for late-onset familial and sporadic Alzheimer’s disease. DNA samples for APOE genotyping are prepared by PCR amplification and subsequent digestion with that HhaI restriction enzyme to yield constant fragments of 16, 18, and 35 base pairs and four typical polymorphic fragments of 48, 72, 83, and 91 base pairs, corresponding to alleles E1, E2, E3, and E4, respectively (30). 

                          The risk of developing Alzheimer’s disease for the individual having the E4/E4 genotype is about 90%, for the individual having the E3/E3 genotype, 20%; and for the individual having the E3/E4 genotype, 47%. 

Ligase Chain Reaction

                          In this method, a thermostable ligase is used to specifically bond two adjacent oligonucleotides, which hybridize to a complementary target with perfect base pairing at the junction. This approach enables single base pair differences to be detected at the ligation junction. The design of primers with distinct labels for different base substitutions allows for simultaneous detection of several changes. This technique has been applied to the screening of the 31 most frequent mutations in the cystic fibrosis gene in a multiplex oligonucleotide ligation assay.

                         Sickle cell disease refers to a collection of autosomal recessive genetic disorders characterized by a hemoglobin variant (Hb S) which differs from hemoglobin A in having a single amino acid substitution on the β-chain. Individuals who are affected with other types of sickle cell disease are compound heterozygotes, possessing one copy of the Hb S variant and one copy of a different β-globin gene variant such as Hb C or Hb β-thalassemia. CE has been applied to the detection of both hemoglobin forms Hb S and Hb C in prenatal diagnoses of sickle cell diseases. 

Allele-Specific Amplification

                        Allele-specific amplification (ASA) allows simultaneous analysis of multiple specific alleles, known point mutations, or known polymorphisms at the same locus. Specificity is achieved in a single PCR reaction by designing one or both PCR primers so that they overlap the site of sequence difference between the amplified alleles. 

                        This results in simultaneous amplification of numerous DNA fragments differing in size, which are subsequently separated by CGE. This technique was applied to the detection of K329E, the most prevalent mutation in medium-chain acyl-coenzyme A dehydrogenase deficiency. The mutant allele produces a DNA fragment that differs in size from the DNA fragment generated by the normal allele. ASA also can be used to detect multiple mutations in a locus. 

                         For example, several point mutations on the 21-hydroxylase gene of a patient with congenital adrenal hyperplasia were analyzed simultaneously by this method. Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder characterized by a decreased affinity of low-density lipoprotein (LDL) for the LDL receptor. 

                            This phenotype is a consequence of a substitution of adenine by guanine in exon 26 of the FDB gene in the region coding for the putative LDL receptor-binding domain of the mature protein. Mutation screening for FDB is performed by ASA followed by CGE. Another mutation that can be detected by ASA and CGE is the G1691A point mutation in the coagulation factor V gene, the most common genetic cause of thrombophilia. 

                              This mutation has been shown to cause resistance to cleavage by activated protein C and is associated with an increased thrombotic risk. 

Single-Base Extension or Single Nucleotide Primer Extension

                         Single-base extension (SBE) is a method used for the accurate detection of single-nucleotide polymorphism variants in transcripts. Standard labeled Sanger dideoxynucleotide terminators (ddNTP), which lack the 3' hydroxyl group needed for chain elongation, are used in the extension reaction; as a result, the primer is extended by a single nucleotide. The reaction mixture is subsequently separated by CGE. 

                         For example, Leber’s hereditary optic neuropathy was detected by annealing a primer immediately 5' to the mutation on the template and extending the primer by one fluorescently labeled ddNTP complementary to the mutation. By using two or more differently labeled terminators, both the mutant and wild-type sequences could be detected simultaneously. 

                          In addition, multiplexed SBE genotyping has been applied to the detection of known point mutations on the gene using three unique primers that probe for mutations of clinical importance on this gene and to the diagnosis of multiple endocrine neoplasia type 2 by the analysis of codons 634 and 918 on the RET proto-oncogene.

                          A different field of application is the determination of ovine prion protein allelic variants at codons 136, 154, and 171, where the four mutations responsible for amino acid changes are typed simultaneously. 

Detection of Chromosomal Reorganizations

                        Chromosomal translocations and inversions are often detected in many types of cancer. Chromosomal translocations result in juxtaposition of segments from two different genes, giving rise to a fusion gene that encodes chimeric proteins with transforming activity. 

                          In general, both genes involved in the fusion contribute to the transforming potential of the chimeric oncoprotein. CE has been applied to the detection of many fusion transcripts, such as the bcr/abl transcript in chronic myeloid leukemia. On the other hand, gene activation often results from chromosomal reorganizations that relocate a proto-oncogene close to the regulatory elements of the IgH or TCR locus. 

                         This event causes deregulation of the proto-oncogene expression, which can then lead to neoplastic transformation of the cell. One of these proto-oncogenes is the BCL-2 gene, which is translocated close to the regulatory element of the IgH gene in persons with follicular Iymphoma. The different PCR products can be resolved by CGE. 

Detection of Gene Rearrangements

                          Fluorescent PCR combined with CE analysis has been used to identify monoclonal rearrangements in the T-cell receptor (TCR) and immunoglobulin heavy-chain (IgH) genes in order to detect residual disease in lymphoproliferative disorders. 

                         Normal lymphocytes rearrange the TCR and IgH genes randomly during normal development, thus, a normal polyclonal population shows many different rearrangements. Neoplastic cells exhibit a single rearrangement of the TCR or IgH gene. 

                          These rearrangements can be detected by PCR if the amplification is conducted across the sequence where the gene rearranges. Polyclonal samples show a gaussian-like distribution of the peaks, whereas monoclonal or neoplastic cases display a single predominant peak. 

                          Four or five separate peaks are consistent with oligoclonal populations and almost always are the result of immunological reactive processes to unknown antigens.

Detection of Intragene Deletion and Duplication Sequences

                         Capillary gel electrophoresis has been applied to the analysis of PCR products in the detection of small gene deletions in several genetic diseases. 

                          Examples of this application include the detection of over 98% of deletions occurring on the dystrophin gene for the diagnosis of Duchenne muscular dystrophy; an 8-bp deletion in exon 3 of the P450c21B gene in individuals affected by 21-hydroxylase deficiency, a recessively inherited disease, and the ΔF508 mutation, a 3-bp deletion in the gene CFTR that is the most frequently mutation found in individuals affected with cystic fibrosis. 

                            Another example is detection of the internal tandem duplication (ITD) in the juxtamembrane domain-coding sequence of the FLT3 gene in acute leukemias. The mutation of this receptor is important in acute myelogenous and lympho blastic leukemias because patients with this mutation generally have a poorer prognosis for survival than patients without it.

Applications of CE to Biomedicine

                        Small alterations in DNA sequences of genes lead to many human diseases, such as cancer, diabetes, heart disease, myocardial infarction, atherosclerosis, cystic fibrosis, and Alzheimer’s disease. These alterations in DNA sequence include many types of mutations and polymorphisms, such as nucleotide substitutions, deletion or insertion of some sequence, differences in a variable number of tandem repeat (VNTR) locus, and instability of microsatellite repeat sequences. 

                        CGE is a particularly suitable method for carrying out analysis of DNA fragments for diagnostic purposes. Currently available methods for analysis of mutations and polymorphisms include some modified polymerase chain reaction (PCR) techniques, restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), VNTR, and short tandem repeats (STRs) analysis, and hybridization techniques, as well as PCR analysis itself.

                            Labeled DNA fragments to be analyzed by CE are obtained by the incorporation of fluorescent-dye-labeled nucleotides or fluorescent dye-labeled primers during the PCR reaction. The range of available dyes and the  reduction in costs over recent years have allowed modification of existing tests to take advantage of the four/five-color detection system and the degree of automation offered by commercially available CE electrophoresis systems. 

                            Four-color, highresolution analysis is particularly useful for the rapid mapping of genetic traits, leading to gene discovery and eventual diagnostic testing. Automation is essential for rapid gene-based mutation analysis in clinical laboratories to screen a large number of DNA samples.

Capillary Gel Electrophoresis

                        There is a special situation for biopolymers such as RNA, DNA, or sodium dodecyl sulfate (SDS)–loaded proteins, which have a constant charge-to-size ratio, that is, the increase in the charge is directly related to the increase in size of the molecule. Molecules with a constant charge-to-size ratio may have very similar electrophoretic mobilities, so no electrophoretic separation occurs in free solution. In these cases, separations are performed in capillaries filled with a gel solution. In capillary gel electrophoresis (CGE), a sieving effect occurs as solutes of various sizes migrate through the gel-filled capillary toward the detector. Smaller ions are able to migrate quickly through the gel, whereas larger ions become entangled in the gel matrix, reducing their migration rate. 

                         Initially, the gels used in CGE were polyacrylamide covalently bonded to the capillary wall. These fixed gels suffered, however, from problems of shrinkage and blockage and could have relatively short lifetimes. In addition, if sample components contaminated the gel, it could not be reused and would have to be discarded. 

                          There has been a recent tendency, therefore, to use pumpable gel solutions, which can be used to fill the capillary with a noncross- linked liquid gel matrix in which pores are created by the tangling of long linear polymers. These have the advantage of being introduced into the capillary under low pressure, extending the life of the capillary. The use of liquid gels also allows replacement of the gel between injections, reducing the contamination problems encountered with fixed gels. 

Principles of Capillary Electrophoresis

                         Capillary electrophoresis (CE) separations are carried out inside a capillary tube, which usually has a diameter of 50 μm to facilitate temperature control. The length of the capillary differs in different applications, but it is typically in the region of 20–50 cm. The capillaries most widely used are fused silica covered with an external protective coating. A small portion of this coating is removed to form a window for detection purposes. 

                     The ends of the capillary are dipped into reservoirs filled with the electrolyte. Electrodes made of an inert material such as platinum are also inserted into the electrolyte reservoirs to complete the electrical circuit. The capillary is filled with running buffer, one end is dipped into the sample, and an electric field (electrokinetic injection) or pressure is applied to introduce the sample inside the capillary. Migration through the capillary is driven by application of a high-voltage current (10–30 kV). The molecules are detected as they pass through the window at the opposite end of the capillary. The most frequently used detector is laser-induced fluorescence (LIF), which detects fluorochromes attached to the DNA molecules; alternative detectors include ultraviolet (UV) absorbance and fluorescence. 

                         Given the short path, detection requires monitoring by sensitive equipment such as chargecoupled device (CCD) cameras. The detectors are interfaced with computers responsible not only for collecting and displaying the data but also for maintaining the timing between filter wheels and controling timing exposures, readouts of the CCD cameras, and run-time processing of the CCD images and spectral data. The molecules are detected as fluorescent peaks as they pass through the detector. 

                       An electropherogram, which is a plot of the detector response with time, is generated. Because the area of each peak is proportional to the concentration of the DNA molecule, integrated peak areas are routinely used for semiquantification due to their greater dynamic range than peak heights.

Fragile X Syndrome

                           Fragile X syndrome is a common genetic disease. This inherited form of mental retardation affects 1 in 4,000 males and 1 in 8,000 females (15). Males with fragile X often exhibit characteristic physical features and accompanying autistic and attention-deficit behaviors. Individual IQs are in the range 35–70 (16–18). Approximately 30% of females with full mutations are mentally retarded, and their level of retardation is, on average, less severe than that seen in males. 

                          In 1943, Martin and Bell (19) were able to link the cognitive disorder to an unidentified mode of X-linked inheritance. In 1967, Lubs (20) discovered excessive genetic material extending beyond the low arm of the X chromosome in affected males. Diagnosis was originally based on cytogenetic analysis of metaphase spreads, but less than 60% of the affected cells in affected individuals showed a positive result. With this variability in the test, the carrier status of individuals could not be determined. Interpretation of the result is further complicated by the presence of other fragile sites in the same region of the X chromosome. The fragile X gene (FMR1) is located in chromosomal band Xq27.3 and encodes an RNA-binding protein, which was initially characterized in 1991 (21–23) and contains a tandemly repeated trinucleotide sequence (CGG) end at its 5' end. 

                           The disease is caused by the absence of a functional FMR1 gene product (24). A small number of individuals classified as fragile X cytogentically have expansion at the nearby FRAXE locus, which also contains an unstable CGG repeat (25–28). The normal distribution of the repeat in the unaffected population varies from 6 to 50. Affected individuals are classified into one of two major groups; premutations of approx 50–200 repeats and full mutations with more than 200 repeats. Some alleles with approx 45–55 copies of the repeat are unstable and expand from generation to generation; others are stably inherited (29–31). 

                             The larger the size of the female premutation, the greater the risk of expansion during meiosis (32). Individual male or females carrying a premutation are unaffected (20,29,30). Males pass on the mutation relatively unchanged to all their daughters, all of whom are unaffected. 

Southern Blotting in the Diagnosis of Human Disease

                       Southern blotting has been applied to the diagnosis of many human diseases  at the molecular level. These genetic diseases are caused by point mutations, gene rearrangements, or the amplification of genes or specific sequences within the genome. 

                         These methods have in common that restriction-digested genomic DNA is size-separated in agarose gel electrophoresis, transferred onto the membrane, and hybridized with gene-specific probes. Restriction fragment length polymorphism (RFLP) analysis was one of the early methods to diagnose point mutations implicated in genetic diseases. This method was based on the observation that if a point mutation changes a restriction fragment recognition sequence, it is possible to detect this change by Southern blotting analysis in which the affected restriction enzyme is used to cut genomic DNA before analysis. 

                         The change in the size of detected fragments with a gene-specific probe signals the presence of mutation in the analyzed gene. For example, this method has been applied to the diagnosis of hemophilia A, which is the most common inherited bleeding disorder, affecting approx 1 in 5,000 males worldwide. Hemophilia A is an X-linked, recessively inherited bleeding disorder that results from a deficiency of procoagulant factor VIII (FVIII). Affected males suffer from joint and muscle bleeds and easy bruising, the severity of which is closely correlated with the level of activity of coagulation factor VIII (FVIII:C) in their blood. 

                          Gitschier et al. demonstrated using Southern blotting that it was possible to diagnose the disease in 42% of affected families (10). Having identified the BclI polymorphism, X-linked inheritance was demonstrated in three generations of a Utah family. DNA from a family member was restricted with BclI, electrophoresed on a 0.8% agarose gel, and blotted on to nylon membrane and probe with a complementary sequence within the factor VIII gene labeled with radioactivity. Twelve bands were observed by autoradiography. Eleven hybridizing bands remained constant, and one varied in position at either approx 0.9 or 1.1 kb in size. 

Transcription and translation

                  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.

                   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. 

                    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.

Damage

                   DNA can be damaged by many sorts of mutagens, which change the DNA sequence. Mutagens include oxidizing agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light and X-rays. The type of DNA damage produced depends on the type of mutagen. For example, UV light can damage DNA by producing thymine dimers, which are cross-links between pyrimidine bases.

Quadruplex structures

                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.

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. 

  

                  In both prokaryotes and eukaryotes, antisense 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.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. 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.

What is Grooves

                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.

DNA- Properties

             DNA is a long polymer made from repeating units called nucleotides. As first discovered by James D. Watson and Francis Crick, the structure of DNA of all species comprises two helical chains each coiled round the same access, and each with a pitch of 34 Ångströms  and a radius of 10 Ångströms .  According to another study, when measured in a particular solution, the DNA chain measured 22 to 26 Ångströms wide, and one nucleotide unit measured 3.3 Å  long. Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides. For instance, the largest human chromosome, chromosome number 1, is approximately 220 million base pairs long.

                      In living organisms, DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together. These two long strands entwine like vines, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a base, which interacts with the other DNA strand in the helix. A base linked to a sugar is called a nucleoside and a base linked to a sugar and one or more phosphate groups is called a nucleotide. If multiple nucleotides are linked together, as in DNA, this polymer is called a polynucleotide.

Deoxyribonucleic acid

              

                 DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms with the exception of some viruses. 

                  The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints, like a recipe or a code, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. 

                This information is read using the genetic code, which specifies the sequence of the amino acids within proteins. The code is read by copying stretches of DNA into the related nucleic acid RNA, in a process called transcription.