SSCP Analysis Using an Automated Electrophoresis System

                        SSCP analysis can be performed in an automated electrophoresis system, typically an automated DNA sequencer, which monitors the mobility of the fluorescently labeled DNA fragments during electrophoresis. PCR products can be fluorescently labeled in a number of ways. Target DNA regions can be amplified by forward and reverse primers labeled with the same single fluorescent dye or two dyes of different colors. 

                           Post-PCR labeling can also be done easily and is more cost-effective because unlabeled primers are  used. Internal labeling of PCR products is another option. The automated DNA sequencer can be gel-based or, more commonly now, capillary-based . Throughput can be increased by analysis in the same lane or capillary of multiple PCR fragments labeled with multiple colors. Mobility of DNA fragments can be standardized by inclusion in the test sample of an internal mobility standard that contains DNA fragments of known size and labeled with a color different from those of the fragments being analyzed. 

                          Electrophoresis can be performed at ambient temperature or above because most automated DNA sequencers are equipped with a built-in heating device, but not a cooling unit. One exciting development is microchip electrophoresis, i.e., performance of electrophoresis in microchannels. Microchip SSCP analysis can be finished within a few  minutes, and thus greatly reduces the analysis times by more than 100-fold when compared with conventional methods. This may revolutionize molecular genetic testing in diagnostic laboratories in the future. 

Gel Composition, Buffer System, and Electrophoresis System

                         Polyacrylamide gels are defined by two parameters: %T and %C. The %T refers to the total amount in grams of acrylamide and N,N'-methylene-bisacrylamide (a common crosslinker, usually abbreviated as bis) in 100 mL solution. The %C refers to the proportion of the total monomers (acrylamide plus bis) that is the crosslinker (bis). The %C can also be expressed in another format as the ratio of acrylamide to bis; for example, 2%C is equivalent to an acrylamide:bis ratio of 49:1. 

                         The probability of detecting sequence variations in a PCR product is higher if the %C is lower (9). Low levels of crosslinking produce large pores in the gels, and thus allow efficient separation of bulky single strand conformers. We use a nondenaturing gel of 10%T/1%C as a starting point in conjunction with a conventional vertical electrophoresis system (e.g., SE600 from Hoefer) and a medium-sized gel (e.g., 16 × 14 cm). 

                         Large-sized gels of 5%T and 1−2%C are also commonly used together with conventional electrophoresis systems for manual sequencing. Another commercially available gel matrix called Mutation Detection Enhancement (MDE) Gel is also widely used for SSCP analysis. It is a polyacrylamide-like matrix and is claimed to be very sensitive to DNA conformational changes. The buffer most commonly used in SSCP analysis is Tris-borate-EDTA buffer with an alkaline pH. 

                           However, low pH buffer (e.g., Tris-MES-EDTA, pH 6.3) can still maintain very high sensitivity of detecting sequence variations for PCR fragments up to 800 bp in length.

Outline of PCR-SSCP Analysis

                        PCR is used to amplify a DNA region to be analyzed. The PCR products are diluted in a loading solution that contains formamide and indicator dyes (e.g., bromophenol blue and xylene cyanol FF). The diluted PCR products are heated to over 90°C for a few minutes to denature the products into single strands and then cooled immediately in ice water. 

                        High concentration of formamide (a chemical denaturant) in the loading solution and immediate cooling are required to keep a sizable proportion of the products in single strands. The denatured PCR products are then loaded onto a nondenaturing polyacrylamide gel (i.e., without chemical denaturants). Samples diluted in formamide are denser than the buffer and will sink to the bottom of the wells. Separation of the single strands is achieved by electrophoresis. 

                         The duration of electrophoresis depends on the gel composition, voltage applied, buffer/gel temperature, and the size and base composition of the PCR products. After electrophoresis, the DNA bands are visualized by silver staining or, less commonly, SYBR Green II. Though less popular now, PCR products can also be radioactively labeled and the bands detected by autoradiography. Banding patterns of samples are compared. 

                          The presence of different banding patterns among samples indicates that sequence variations exist in the DNA sequence amplified by the two PCR primers. Silver-stained gels can be dried in a gel dryer and the dried gels kept for permanent records if so desired.

Single Strand Conformation Polymorphism

                        Variations in DNA sequences underlie the differences among different members of the same species and also between different species. DNA sequence variations are usually known as polymorphisms if  the commonest allele is less than 0.99 in a given population. 

                         DNA polymorphisms are widespread in many different species, particularly in humans. Examples include single nucleotide polymorphisms (SNPs), microsatellites, minisatellites, small insertions/ deletions, and large insertions/deletions. DNA polymorphisms may not haveany phenotypic effect at the protein level or at the level of the whole organism. 

                         On the other hand, they are usually called disease-causing or pathogenic mutations if they cause a change in the phenotype and results in a disease status. The frequencies of individual mutations are usually not high because of selection pressure against such less favorable base changes. It is thus important to study DNA sequence variations in various branches of biological sciences. 

Denaturing Gradient Gel Electrophoresis

                        Denaturing gradient gel electrophoresis (DGGE) is a robust method for point mutation detection that has been widely used for many years. It is a polymerase chain reaction (PCR)-based method, the principle being the altered denaturing temperature of a PCR product with a mutation compared to the wild-type product. PCR performed on DNA of an individual with a point mutation in one of two genes will lead to a mixture of different products. 

                        PCR products from both the wild-type gene and the mutated gene will be formed. These are known as the homoduplex products. The difference in melting temperature between these two products, however, is subtle. Another type of product, heteroduplexes, consisting of a wild-type strand combined with a mutant strand of DNA, will also be formed during the last cycles of the reaction. The real strength of DGGE lies in the fact that the heteroduplex PCR products will have much lower melting temperatures compared to the homoduplex PCR products, because the heteroduplexes have a mismatch. 

                        To visualize the different melting temperatures of these homoduplexes and heteroduplexes, the products should be run on an acrylamide gel with a gradient of denaturing agents: urea and formamide. These denaturing agents alone are not sufficient. In addition, the gel should be run at a high temperature, usually 60°C. During electrophoresis, the PCR products will run through the gel as double-stranded DNA until they reach the point where they start to denature. Once denatured, the PCR products could continue running through the gel as single-stranded DNA, but the fragments have to remain precisely where they denatured. To achieve this, a so-called GC clamp is attached, to prevent complete denaturing. 

                     This GC clamp is a string of 40–60 nucleotides composed only of guanine and cytosine and is attached to one of the PCR primers. PCR with a GC clamp results in a product with one end having a very high denaturing temperature. A PCR product running through a DGGE gel will, therefore, denature partially. The GC clamp remains double stranded. The fragment will form a Y-shaped piece of DNA that will stick firmly at its position on the gel.

Purification and Isolation of Nucleic Acids

                         Conventionally, nucleic acids are analyzed by gel electrophoresis for the purposes of separation, identification, and purification. However, the process of the gel-based analysis involves labor-intensive steps such as sample and gel preparations, sample loading, gel staining, and photographic processing. 

                        The DHPLC system has high resolving power and thus allows the automatic purification and isolation of nucleic acids. It has been demonstrated that dsDNA, ssDNA, and RNA can be separated, quantified, and then recovered by the fragment collector of the DHPLC system. Under nondenaturing conditions, dsDNA molecules such as PCR products and restriction fragments are separated. The isolated dsDNA fragments can then be collected for downstream applications such as sequencing and cloning. In purification and isolation of ssDNA, DHPLC can directly separate ssDNA from dsDNA under fully denaturing conditions (75°C). 

                         The purification is facilitated by using a tagged primer, which has a hydrophobic moiety such as a biotin group or fluorescein, in PCR. The hydrophobicity of the ssDNA generated by the tagged primer is increased and leads to the increased retention time in DHPLC analysis. As a result, the two ssDNA species from the dsDNA PCR products can be separated. DHPLC is a simpler and faster method of purifying ssDNA than other techniques involving a variety of analytical molecular biology procedures. Moreover, the fully denaturing conditions can be applied to the purification and quantification of mRNA from total RNA. 

                           In DHPLC analysis, RNA degradation and spurious transcription can also be detected, and hence the quality and integrity can be determined. DHPLC greatly improves the analysis and purification of RNA as compared to the conventional methods by simplifying the lengthy experimental procedures.

Individual Genotyping

                         The PE reaction in combination with completely denaturing HPLC analysis offers a simple, robust and automatic genotyping platform because a single analytic condition can be used. The accuracy and sensitivity can be increased by using fluorescent-labeling method. Equipped with the DNASep-High Throughput (HT) column and the High Sensitive Detector, the 4500HT-HS model of the WAVE System (Transgenomic) is developed for high-throughput genotyping. 

                          The PE/DHPLC genotyping platform has been used in the genotyping of the mutations of the hemochromatosis gene and the β-globin gene for the diagnosis of hereditary hemochromatosis and β-thalassemia respectively. Two mutation sites (C282Y and H63D) of the hemochromatosis gene were simultaneously genotyped in a multiplex format including the PCR, PE reactions and DHPLC detection. 

                            For β-thalassemia, two different studies demonstrated the successful simultaneous genotyping of five common mutations within the β-globin gene. Amplicons containing the five common mutations were amplified, and the mutations distinguished by multiplex PE reactions followed by DHPLC analysis. 

                              This approach would help the development of diagnostic mutation panels for the diagnosis of β-thalassemia and other genetic diseases showing extensive allelic heterogeneity.

 

Mutation Detection

                        The most significant function of DHPLC is mutation detection. Partially denaturing HPLC is used to detect unknown sequence variations. Heteroduplexes are generated before analysis. This step is essential for the detection of X-linked mutations in males and homozygous mutations. The screening  throughput can be increased by mixing several test samples with 1 reference sample. 

                          As has been mentioned above, the ideal size of PCR products is 150–450 bp for detection of unknown sequence variations. Long DNA fragments tend to have more than 1 melting domain and require several column temperatures for complete screening of the fragment. 

                          There are occasions in which several sequence variations are found within a small DNA region in different chromosomes (i.e., in different individuals). These are well illustrated by the diverse mutations in the CFTR and HBB genes. Mutations in CFTR result in cystic fibrosis whereas mutations in HBB give rise to β-thalassemia or sickle cell disease. Distinct mutations in a PCR product usually give consistent distinct chromatograms. 

                           Therefore, partially denaturing HPLC can also be used to genotype known sequence variations, particularly known mutations, once their corresponding distinct chromatograms have been established. However, it is still possible that different mutations may share indistinguishable chromatograms. 

DHPLC Analysis

                        Regardless of the operation mode, the DNA samples are placed in the cooled 96-well autosampler compartment of the DHPLC system. It is controlled by the software from the manufacturer of the system (e.g. the WAVEMAKER software for the WAVE DNA Fragment Analysis System from Transgenomic). 

                         All the analytical parameters are entered into the program, including the sample information (sample identity and volume to be injected), application types, column temperature (mode of HPLC), and the linear gradient profile (the percentage of acetonitrile in the mobile phase). A linear gradient of 1.8–2.0% per  minute at a flow rate of 0.9 ml/min is usually used. In general, the start- and end-points of the gradient are adjusted according to the size of the DNA fragments. 

                          For the detection of unknown mutation, the optimal column temperature and the linear gradient profile are calculated by the algorithm of the software. The gradient profile for each injection includes the column regeneration and equilibrium steps prior to the next injection. The eluted DNA fragments are shown as peaks in the chromatogram. The retention time and the intensity of the peaks (height or area) are determined by the software. 

Primer Extension Reactions

                         For allelic discrimination, the amplicons containing the target polymorphic site serve as templates for PE reactions. Prior to the PE reactions, the amplicons are purified by treatment at 37°C with exonuclease I and shrimp alkaline phosphatase in order to remove the unincorporated single strand oligo primers and deoxynuclotides respectively.

                         The enzymes are then inactivated at 80°C. After purification, the PCR products are mixed with Thermo Sequenase (GE Healthcare) and appropriate ddNTPs. The PE reactions are carried out in a thermal cycler with a universal thermal cycling condition that includes denaturation at 96°C, annealing at 43°C followed by extension at 60°C.

                         The single strand extended products are analyzed by DHPLC under completely denaturing condition.

Heteroduplex Formation

                         For mutation detection, the PCR product from a test sample is mixed with a homozygous reference PCR product in a 1:1 (v/v) ratio.

                         The mixed DNA fragments are denatured at 95°C and then cooled slowly for reannealing of the DNA strands by gradually lowering the temperature at a rate of 1°C per 20 seconds from 95°C to 25°C.

                          A single base pair difference between the test and reference fragments will produce two heteroduplexes and two homoduplexes. The mixture of DNA samples are then analyzed by DHPLC under partially denaturing conditions.