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Short nucleotide repetitions (STRs) are commonly used as genetic markers; thus their detection and analysis constitutes a very important tool for the mapping of genetic diseases, as well as for gathering information about genetic polymorphisms at the population level. STRs can be detected with agarose- or acrylamide-based electrophoretic techniques, followed by visualization of the DNA sample with ethidium bromide, silver nitrate, or fluorophore labeling. In this work, we analyzed genomic DNA from five individuals affected with type II diabetes mellitus (T2DM) and five controls (unaffected individuals) in order to know the most precise and reproducible technique for the analysis of the existing polymorphism in the STR DG10S478 of the TCF7L2 gene. The combination of PCR with labeling of the products with the CY5 fluorophore, followed by detection on an ALFexpress sequencer, offered the required resolution to detect the variability in this STR, based solely on size analysis. Our methodology offers similar accuracy and reproducibility at lower costs than existing methods based on the sequencing of PCR products, and is a faster alternative when applied to genotyping studies.
Microsatellites or STRs are short repetitions of nucleotides in the genome. They constitute an important tool for the mapping of genetic diseases, given that they offer valuable information on the existing variability at the chromosomal level, and also due to the appearance of more exact and reproducible techniques for their detection. Currently, STR analysis is being employed in the study of the genetic factors that define complex diseases.1 Type II diabetes mellitus (T2DM) is one such disease, since its etiology involves the combination of different genetic and environmental factors that produce metabolic disorders that are expressed with a common phenotype of glucose intolerance.2 T2DM is also associated with arterial hypertension, dyslipidemias, and obesity.3 Approximately 150 million people worldwide are affected by T2DM, and this number is expected to double in the next 20 years.4 Recent research has shown the influence of at least 10 genes in the physiopathology of T2DM,5,6 including: (1) the peroxisome proliferator activated receptor gamma gene (PPAR gamma),7 a member of a superfamily that regulates adipocyte differentiation and glucose homeostasis; (2) the potassium inwardly rectifying channel, subfamily J, member 11 gene (KCNJ11), which encodes a membrane protein functioning as a potassium channel in the beta cells of the pancreas;8 (3) the calpain 10 gene (CAPN 10),9 which plays an important role in the secretion of insulin; and (4) the TCF7L2 gene, located inside the chromosome 10q 25.2 genomic region, which codes for the transcription factor 7-like 2, involved in glucose homeostasis in the intestine.
The DG10S478 STR, located in the TCF7L2 gene, has recently been reported. It contains a 4-bp repeat unit of CACA that is arrayed in one to five tandems.10 The variability of this STR has been studied in Danish, Ice-landic,11,12 German,12 African,13 English,14 and Mexican-American populations,15 showing that alleles 0, 8, and 12 represent 98% of the chromosomes in the control population, and suggesting a protective association with allele 0 compared to the other allele combinations.12
The needs of the human genome sequence project and those posed by the study of genetic polymorphisms in human populations have pushed forward the development of techniques for high-throughput sequencing analysis, resulting in turn in the detailed characterization of hundreds of STRs for Homo sapiens and other animal species, which are invaluable for the identification of genes and alleles involved in the pathogenesis of complex diseases, such as T2DM.18–20 A mainstay of the new molecular genotyping techniques is the use of automated fluorescence gel-based detection systems, such as those of Applied Biosystems (ABI),21 the LI-COR Model 4000,22 and the Pharmacia Biotech ALFexpress DNA sequencer.23 In each of these systems, DNA fragments are detected via laser-induced fluorescence of fluorescent tags as the fragments pass by a laser that scans across the gel during electrophoresis, using an internal lane standard fluorescing at a different wavelength in order to compare each sample on the same scale.
Other sequencing systems are based on the use of capillary electrophoresis (CE), which allows the optimization of the genotyping test due to the increased speed of the assay (run times are shortened from hours to minutes)24 and the possibility of fully automating the electrophoresis process, with no need to pour the gel or manually load the samples. CE systems, however, have the disadvantage of needing to dialyze PCR samples before analysis, thus lengthening assay run times.
The latest technical breakthrough in large-scale STR genotyping studies has been the use of MALDI-TOF mass spectrometers for the analysis of the DNA fragments. Here, results can be routinely reached within a few seconds. However, the technique is not without limitations, since problems during fragmentation and ionization of the samples that have yet to be overcome have limited its application to DNA fragments smaller than 150 bp.25
Many molecular genotyping techniques depend on the amplification of the loci under analysis by PCR, followed by the characterization of the products. However, the potential of PCR for nonspecific amplification of contaminant bands (increased when testing heterogeneous DNA samples) means that it is necessary to process the genotyping data with reliable and accurate computerized algorithms that compare the different size patterns obtained from the PCR products of homozygotic individuals for the same allele, in order to eliminate the possibility of an erroneous allelic classification.26
In spite of all these advances, conventional electrophoresis remains one of the most widely used tools for the characterization of the size distribution of PCR products. Horizontal electrophoretic analyses are commonly performed using agarose or low-melting agarose (LMA) gels, although denaturing vertical polyacrylamide gels are often preferred due to their higher resolution (single base-pair discrimination).16 A recent advance has been the appearance of horizontal MetaPhor agarose gel electrophoresis coupled to SYBR Green staining, which can resolve alleles in dinucleotide repeat polymorphisms.17
In order to standardize a low-cost, accurate, and reliable technique to determine the correct allelic assignment of the DG10S478 STR, we have evaluated horizontal agarose gels with ethidium bromide staining and vertical denaturing polyacrylamide gels with silver nitrate staining. Furthermore, we explored the use of a DNA ALFexpress sequencer with primers labeled with the CY5 fluorophore, in order to compare the results obtained with the conventional techniques mentioned before. With this work we made an allelic assignment of the DG10S478 STR using size analysis methods instead of sequencing the PCR products, thus reducing the cost of the assay and increasing the feasibility of this method for population studies.
Genomic DNA samples (obtained from the DNA bank of the Genomics and Diagnostics division at CIGB, Havana, Cuba) were analyzed by PCR amplification. A total number of 10 samples, 5 from individuals with T2DM (E) and 5 from controls (C), were diluted in water to a final concentration of 100 ng/μL and kept on ice. We followed the instructions from the manufacturer of the GeneAmp PCR Core kit (Applied Biosystems, Foster City, CA). Briefly, the PCR reaction was carried out in a volume of 20 μL, containing 100 ng of DNA, 2 μL (10X) of Taq Polymerase buffer, 0.6 μL of each dNTP (10 mM), 1.7 μL of MgCl2 (25 mM), 25 pmoles of each DG10S478 primer, and 2 μL of 6 μM HLA-*A2 and β2-microglobulin primers, respectively (Chemical synthesis laboratories of CIGB, Heber Biotec, Havana, Cuba) and 0.1 μL Taq DNA Polymerase (5 u/μL) (see Table 1 for more information). The PCR was run using a Mastercycler Gradient cycler (Eppendorf, Hamburg, Germany), and the PCR products were stored at −20ºC until used.
Different electrophoretic protocols were performed in order to define the most sensible and reproducible method for allelic assignment of the DG10S478 STR. Table 2 summarizes the conditions used for each methodology. We used the 123-bp ladder (Sigma-Aldrich, St. Louis, MO) mixed with loading buffer orange G (1X) as a molecular-weight standard. The electrophoresis equipment used in non-automatic running conditions was the Amersham Biosciences EPS 601 (Piscataway, NJ).
We used the statistical Kodak 1D 3.6 image analysis software (Eastman Kodak, Rochester, NY) to process the images obtained from 2% (w/v) agarose gels, and the SPSS 13.0 statistical package (SPSS, Chicago, IL), together with Allelelinks 1.01 (Amersham Biosciences, Uppsala, Sweden) to process the fluorograms obtained using the ALFexpress DNA sequencer. The analysis of the pUC18 and pUC19 sequences was made using the ALFwin Sequence Analyser v2.10 software (Amersham, Buckinghamshire, UK). The calibration of DNA migration time was performed with SPSS 13.0 and Allelelinks 1.01. The calculation of the retention time for the allelic estimation is the difference between the retention time value of the specific allelic peak and the retention time value corresponding to the beginning of the fluorescent signal detection in the fluorogram. The results were correlated with known PCR size products as an internal control (different DNA patterns from the samples in analysis) and external controls (similar DNA patterns to the samples in analysis). These analyses allowed us to create an allelic pattern used to make the allelic assignment of the individuals in this study.
The DNA fragments amplified by PCR were analyzed in 2% (w/v) agarose gels. As can be seen in Figure 1, the PCR products migrated between the bands of 492 bp and 369 bp of the 123-bp ladder.
The PCR product from each individual was visualized four times in 2% (w\v) agarose electrophoresis to observe the variation in the DNA migration between the gels. Table 3 presents the first allelic definition for each individual, based on the size of PCR products and their standard deviations (SD).
On the other hand, the mixtures of different types of agaroses did not show better resolution than 2% (w\v) agarose electrophoresis gel (Figure 1). It was also difficult to obtain reproducibility at different times. However, the presence of different PCR bands higher or lower than the expected allelic range (380 bp to 400 bp) of the DG10S478 STR was revealed (Figure 2). The results of visual analysis of the band sizes for each individual using this method are shown in Table 5.
We observed that the use of 6% (w/v) denaturing polyacrylamide gel (PAGE) and silver gold staining was a better option than the two previous techniques, but even it lacked precision and reproducibility (Figure 3). In Figure 3, lanes 1, 4, 5, and 6 show the individuals potentially carrying allele X, corresponding to C1, C4, C5, and E3. Visual analysis of the band sizes for each individual using this method is shown in Table 5.
Using the ALFexpress DNA sequencer and fluorescence labeled PCR primer, it was possible to discriminate a difference of four bases in the genome (Figure 4). The reproducibility and precision of the internal and external standards used to assign alleles were carried out several times by retention time analysis using Allelelinks 1.01 and SPSS 13.0 (Table 4). This standardized method was reproducible and precise. The allelic assignments for each individual obtained from the fluorogram analysis using Allelelinks 1.01 is presented in Table 5.
The most accurate technique for the analysis of genetic variability at the population level is DNA sequencing. However, since the large-scale use of DNA sequencing for this purpose requires sophisticated and expensive technologies that are not always available, the research community often resorts to less costly although also less accurate alternatives, such as the use of electrophoretic methods.
In this work, we have made an exploratory study using different electrophoretic methods and signal visualization techniques in order to determine the most precise and reproducible protocols for the analysis of the existing polymorphism in the DG10S478 STR of the TCF7L2 gene in 10 individuals.
Firstly, we standardized the PCR conditions to amplify the DG10S478 STR region located in intron 3 of the TCF7L2 gene. Figure 1 shows the PCR product of the DG10S478 STR. The 2% (w/v) agarose electrophoresis gels did not resolve the allelic differences of the studied population. These visual results were quantitatively confirmed by the application of the Kodak 1D software for image analysis and for allelic assignment of the PCR products (Table 3). Although the quantitative signal analysis showed the existence of an overlap between the size values of the PCR products from different individuals that precluded the differentiation of allele 0 from the different types of allele X, it provided us a first approximation for the allelic assignment of STR DG10S478 by individual (Table 3).
In order to increase the resolving power from the 2% (w/v) agarose electrophoresis gels, we decided to evaluate two other electrophoretic methodologies. The first alternative we tested was the use of a mixture of 3% (w/v) agarose and 1% (w/v) LMA. These results are shown in Figure 2. Using this mixture, we obtained a better separation between the different PCR products, which allowed the visualization of small differences in size between the DNA bands (Figure 2, lines 2, 3, and 4). Although this method still suffers from the disadvantage of poor sharpness of the signal due to the time it takes to complete the electrophoretic run (approximately 2 h at 120 V, room temperature), the analysis gave us a second approximation to the allelic assignment of the DG10S478 STR for each individual (Table 5).
The second alternative evaluated was the use of 6% (w/v) PAGE followed by silver nitrate staining. This technique, which has been mentioned in the literature for the determination of STR polymorphisms,28 has several advantages over the previous methodology: (a) the denaturation of DNA allows for better defined and sharper band signals due to the elimination of secondary conformations, and (b) the silver staining signal is more stable and sensitive than ethidium bromide staining (Figure 3 and Table 5).28 This electrophoresis method allowed visualization of the presence of two alleles in the analyzed samples. The disadvantages of this technique are the difficulty of its execution and the time consumed.
The three electrophoretic methodologies discussed above suffer from the disadvantage of not being easily amenable to automation; thus, the quality and reliability of their results vary heavily depending on external factors such as temperature, humidity, voltage variability, operator manipulation, running time, quality, and reproducibility of the preparation of the gels and the later visualization and digital capture of the resulting image. Therefore, these techniques are not the best solution for the discrimination of the addition of very small numbers of bases to an STR. Taking into account these disadvantages, we explored the possibility of using primers labeled with Cy5 for the amplification of STRs from the TCF7L2 and HLA-*A2 genes, followed by analysis of the fluorescent PCR products in the semi-automatic sequencer ALFexpress II.
The fluorograms were processed using Allelelink 1.01, which is assigns to the fluorescent signals a value that corresponds with allelic size, by interpolation from the retention time of known DNA sequences as standards. Further we processed the fluorograms using SPSS 13.0. This analysis generates an allele assignment through the use of external controls, such as sequences from pUC18 and pUC19.
The regression coefficient for the linear regression of the retention times of the peaks from the standards was R2 = 0.99, indicating an accurate assignment of the corresponding allelic size to each individual. The standard allelic pattern was obtained by mixing the PCR product obtained from homozygotic individuals for each allele (0, 4, 8, 12, and 16 bp additions) and a PCR product from the HLA-*A2 gene. The inclusion of the allelic standard increased the accuracy and reproducibility of the results, allowing a consistent detection and assignment of the possible allelic differences in the population (Figure 4 and Tables 4 and and5).5). After comparing the results from the different electrophoretic techniques to those obtained from the application of the ALFexpress II sequencer, for some individuals there is correspondence in allelic assignment (Table 5).
The assignment of allelic sizes by the combination of a semiautomatic technology such as the ALFexpress II sequencer combined with the use of Allelelink 1.01 constitutes, to the best of our knowledge, a cheaper and at least as reliable alternative to the direct sequencing of PCR products for genotyping STRs in complex diseases such as T2DM.
We wish to thank Drs. Marta Dueñas Porto, Hamlet Camacho-Rodriguez, Maria Elena Fernández de Cossio, and Dania M. Vazquez (CIGB, Havana, Cuba), and Dr Roberto Frías (Havana University, Cuba), for their assistance. Furthermore, we appreciate the important contributions to the manuscript by Sorange Hernández and Alejandro Martín (CIGB, Havana, Cuba).