Complete Gene Scanning by Temperature Gradient Capillary Electrophoresis Using the Cystic Fibrosis Transmembrane Conductance Regulator Gene as a Model
2005; Elsevier BV; Volume: 7; Issue: 1 Linguagem: Inglês
10.1016/s1525-1578(10)60016-5
ISSN1943-7811
AutoresLan-Szu Chou, Friederike Gedge, Elaine Lyon,
Tópico(s)Microfluidic and Bio-sensing Technologies
ResumoMany inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort. Many inherited diseases involve large genes with many different mutations. Identifying a wide spectrum of mutations requires an efficient gene-scanning method. By differentiating thermodynamic stability and mobility of heteroduplexes from heterozygous samples, temperature gradient capillary electrophoresis (TGCE) was used to scan the entire coding region of the cystic fibrosis transmembrane conductance regulator gene. An initial panel (29 different mutations) showed 100% agreement between TGCE scanning and previously genotyped results for heterozygous samples. Different peak patterns were observed for single base substitutions and base insertions/deletions. Subsequently, 12 deidentified clinical samples genotyped as wild type for 32 mutations were scanned for the entire 27 exons. Results were 100% concordance with the bidirectional sequence analysis. Ten samples had nucleotide variations including a reported base insertion in intron 14b (2789 + 2insA) resulting in a possible mRNA splicing defect, and an unreported missense mutation in exon 20 (3991 G/A) with unknown clinical significance. This methodology does not require labeled primers or probes for detection and separation through a temperature gradient eliminates laborious temperature optimization required for other technologies. TGCE automation and high-throughput capability can be implemented in a clinical environment for mutation scanning with high sensitivity, thus reducing sequencing cost and effort. Detecting single nucleotide polymorphisms is increasingly important in molecular diagnostics to link DNA variation with complex inherited diseases. With the occurrence of single nucleotide changes/substitutions in the human population greater than 1%, technology detecting any sequence alteration is especially important for large genes containing many exons and multiple mutations.1Wang DG Fan J-B Siao C-J Berno A Young P Sapolsky R Ghandour G Perkins N Winchester E Spencer J Kruglyak L Stein L Hsie L Topaloglou T Hubbell E Robinson E Mittmann M Morris MS Shen N Kilburn D Rioux J Nusbaum C Rozen S Hudson TJ Lipshutz R Chee M Lander ES Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome.Science. 1998; 280: 1077-1082Crossref PubMed Scopus (1695) Google Scholar For example, the gene that encodes cystic fibrosis transmembrane conductance regulator (CFTR) consists of 27 exons and spans a region of 188,705 bp in chromosome 7. More than 1000 mutations have been reported that are associated with cystic fibrosis, a severe autosomal recessive disorder. Common symptoms include abnormal sweat electrolytes, pulmonary disease, exocrine pancreatic insufficiency, or male infertility (congenital bilateral absence of the vas deferens).2Noone PG Knowles MR Review: “CFTR-opathies”: disease phenotypes associated with cystic fibrosis transmembrane regular gene mutations.Respir Res. 2001; 2: 328-332Crossref PubMed Scopus (133) Google Scholar, 3Ratjen F Döring G Cystic fibrosis.Lancet. 2003; 361: 681-689Abstract Full Text Full Text PDF PubMed Scopus (880) Google Scholar A panel of 25 cystic fibrosis (CF) mutations is recommended by the American College of Medical Genetics for population carrier screening.4Grody WW Cutting GR Klinger KW Richards CS Watson MS Desnick RJ Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.Genet Med. 2001; 3: 149-154Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar However, this panel is designed only for the most common mutations found in the United States population. Other sequence alteration(s) unknown whether they are pathogenic or nonpathogenic require further characterization.5Bombieri C Giorgi S Carles S de Cid R Belpinati F Tandoi C Pallares-Ruiz N Lazaro C Ciminelli BM Romey MC Casals T Pomper F Gandini G Claustres M Estivill X Pignatti PF Modiano G A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals.Hum Genet. 2000; 106: 172-178Crossref PubMed Scopus (46) Google Scholar Therefore, analyzing the entire mutation spectrum can improve correlation between genotypes and phenotypes, specifically in relation to atypical or mild forms of CF. Scanning the entire coding region of the target gene in a high-throughput format saves time and cost over full gene sequencing. Any technology used for scanning must have a high sensitivity for detecting any alteration.Current technologies available for mutation detection have been reviewed.6Kirk BW Feinsod M Favis R Kliman RM Barany F Survey and summary: single nucleotide polymorphism seeking long term association with complex disease.Nucleic Acids Res. 2002; 30: 3295-3311Crossref PubMed Scopus (170) Google Scholar, 7Kristensen VN Kelefiotis D Kristensen T Borresen-Dale AL High throughput methods for detection of genetic variation.BioTechniques. 2001; 30: 318-332PubMed Google Scholar Basically, they can be categorized into two areas. The first category detects known mutations, and methods include real-time polymerase chain reaction (PCR) coupled with melting curve analysis (allele-specific hybridization probes),8Witter CT Herrmann MG Moss AA Rasmussen RP Continuous fluorescent monitoring of rapid cycle DNA amplification.BioTechniques. 1997; 22: 130-138PubMed Google Scholar, 9Lyon E Mutation detection using fluorescent hybridization probes and melting curve analysis.Exp Rev Mol Diagn. 2001; 1: 17-26Crossref Scopus (97) Google Scholar oligonucleotide arrays,10Cronin MT Fucini RV Kim SM Masino RS Wespi RM Miyada CG Cystic fibrosis mutation detection by hybridization to light-generated DNA probe arrays.Hum Mutat. 1996; 7: 244-255Crossref PubMed Scopus (167) Google Scholar minisequencing with primer extension, and enzymatic assays such as oligo ligation assay (Applied Biosystems, Foster City, CA) and restriction fragment length polymorphism. The second category detects unknown mutations, and technologies include direct sequencing and varied electrophoretic-based assays, such as single strand confirmation polymorphism,7Kristensen VN Kelefiotis D Kristensen T Borresen-Dale AL High throughput methods for detection of genetic variation.BioTechniques. 2001; 30: 318-332PubMed Google Scholar confirmation-sensitive gel electrophoresis,11Ganguly A An update on conformation sensitive gel electrophoresis.Hum Mutat. 2002; 19: 334-342Crossref PubMed Scopus (74) Google Scholar and constant denaturant capillary electrophoresis.12Li-Sucholeiki XC Thilly WG A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA.Nucleic Acids Res. 2000; 28: E44Crossref PubMed Scopus (31) Google Scholar Both categories have advantages and limitations. For example, an assay based on allele-specific hybridization probes is sensitive but is limited to detecting a single or few mutations. Methods for full gene analysis will detect a great number of mutations, but may be less sensitive overall. Direct sequencing is the current gold standard but is costly and labor intensive for analyzing a large multiexon gene.A temperature-controlled ion-pair reverse-phase liquid chromatography has been used for unknown mutation discovery.13Liu W Smith DI Rechtzigel KJ Thibodeau SN James CD Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.Nucleic Acids Res. 1998; 26: 1396-1400Crossref PubMed Scopus (229) Google Scholar This approach separates heteroduplex mutants from homoduplex wild types by specific melting temperatures (Tm) and different hydrophobicity in an alkylated C-18 column.13Liu W Smith DI Rechtzigel KJ Thibodeau SN James CD Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations.Nucleic Acids Res. 1998; 26: 1396-1400Crossref PubMed Scopus (229) Google Scholar, 14Oefner PJ Underhill PA DNA mutation detection using denaturing high-performance liquid chromatography (DHPLC).Curr Prot Hum Genet. 1998; 7: 10.1-10.12Google Scholar Two research articles have been published for screening the CFTR gene using denaturing high-performance liquid chromatography and suggest this technology is suitable for large gene analysis with high accuracy.15Le Marechal C Audrezet MP Quere I Raquenes O Langonne S Ferec C Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling.Hum Genet. 2001; 108: 290-298Crossref PubMed Scopus (112) Google Scholar, 16Ravnik-Glavac M Atkinson A Glavac D Dean M DHPLC screening of cystic fibrosis gene mutations.Hum Mutat. 2002; 19: 374-383Crossref PubMed Scopus (46) Google Scholar However, a limitation of denaturing high-performance liquid chromatography is that it requires intensive optimization to determine the best resolving temperature for each individual exon (amplicon) in a large gene. This limitation may decrease throughput for a high volume test. Recently, a technology using an automated temperature gradient capillary array electrophoresis (TGCE) provides another option for a rapid analysis of a large multiexon gene. This technology is similar to a method previously described17Gelfi C Righetti PG Cremonesi L Ferrari M Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients.Electrophoresis. 1994; 15: 1506-1511Crossref PubMed Scopus (73) Google Scholar but instead of using a denaturing agent, samples are resolved in a capillary array with a proprietary polymer matrix. This technology differentiates heteroduplex mutants from homoduplex wild types based on different mobility in a specific polymer matrix under a temperature gradient. Using a temperature gradient reduces Tm optimization for individual exons. Movement of different species of DNA is then captured by a charge-coupled device camera as image files for data analysis.18Gao Q Yeung ES High-throughput detection of unknown mutations by using multiplexed capillary electrophoresis with poly(vinylpyrrolidone) solution.Anal Chem. 2000; 72: 2499-2506Crossref PubMed Scopus (79) Google Scholar, 19Li Q Liu Z Monore H Culiat CT Integrated platform for detection of DNA sequence variants using capillary array electrophoresis.Electrophoresis. 2002; 23: 1499-1511Crossref PubMed Scopus (101) Google ScholarThe purpose of this study was to implement an automated analysis format using TGCE (model SCE 2410; SpectruMedix Inc., State College, PA) for mutation scanning of the CFTR gene. Exon-specific primers were designed so the entire coding regions as well as intron-exon boundaries of the 27 exons were optimally amplified with a standard PCR protocol. After amplification, PCR products were slowly cooled and subjected to the automated TGCE analysis. This technique showed 100% agreement with sequencing results for distinguishing between heterozygous mutants and wild types. We also detected one nucleotide alteration not previously reported [Cystic Fibrosis Genetic Analysis Consortium (CFGAC) database (http://www.genet.sickkids.on.ca/)]. It is desirable to detect single-base alteration in a wide range of DNA fragments, in terms of sizes and GC contents, using an automated high-throughput format. The method described in this study is capable of detecting single (or multiple) base alterations in varied length PCR products (175 bp to 834 bp). This technology has a great potential to be implemented in a high-throughput environment for mutation scanning of large multiexon genes.Materials and MethodsSample Collection for TGCE ScanForty-two previously characterized samples including 14 samples from Coriell Repository (Camden, NJ) and 28 deidentified clinical samples with known CF genotypes for 29 specific mutations were used to compare peak patterns of heterozygous samples to wild types using TGCE scanning (Table 1). Subsequently, 12 clinical specimens previously submitted to ARUP Laboratories (Salt Lake City, UT) were deidentified (according to an institutional review board-approved protocol) and used for full gene-scanning analysis (27 exons). These samples were previously genotyped as negative for a panel of 32 CF mutations by oligo ligation assay (Celera Diagnostics LLC., Alameda, CA).Table 1Mutation Samples with Known Genotypes Scanned by TGCE*Samples were compared with their respective wild-type control (confirmed by sequencing).ExonMutation†All genotypes were heterozygous except homozygous sample 663delT/663delT, ΔF508/ΔF508, and G542X/G542X.Amplicon size (bp)Location of mutation from 5′ end (bp)Base changeDetection‡Number of samples scored (+) by TGCE/number of samples tested. Homozygous samples were detected only after mixing with a wild-type sample.3G85E234124G to A1/13394delTT234132del TT1/14R117H27083G to T2/24I148T270176T to C3/3Intron 4621 + 1 G/T270233G to T1/15663delT/663delT18675del T0/1Intron 5711 + 1 G/T186124G to T1/17R334W345208C to T1/17R347P345248G to C1/19A455E263155C to A2/210I506V292168A to G1/110ΔI507292171del ATC2/210ΔF508292174del TTT2/210ΔF508/ΔF508292174del TTT0/110F508C292175T to G1/110V520F292210G to T1/1Intron 101717–1 G/A17550G to A1/111G542X17590G to T2/211G542X/G542X17590G to T0/111G551D175118G to A3/311R553X175123C to T3/311R560T175145G to C2/2132184delA834356del A1/1Intron 14b2789 + 5G/A192102G to A1/1Intron 163120 + 1G/A216111G to A1/119R1162X32268C to T1/1193659delC322111del C1/120W1282X206154G to A1/121N1303K250175C to G2/2Total exon/intronOverall accuracy1793%* Samples were compared with their respective wild-type control (confirmed by sequencing).† All genotypes were heterozygous except homozygous sample 663delT/663delT, ΔF508/ΔF508, and G542X/G542X.‡ Number of samples scored (+) by TGCE/number of samples tested. Homozygous samples were detected only after mixing with a wild-type sample. Open table in a new tab Primer Design, DNA Amplification, and TGCE PreconditioningGenomic DNA of all specimen samples collected for this study was extracted by the MagNA Pure LC instrument (Roche Diagnostics, Indianapolis, IN) and 2 μl of each extracted DNA was used for PCR. Primers (except exon 9) specific to each exon were designed ∼20 to 100 bp (if applicable) upstream and downstream of each exon using Primer3 software20Rozen S Skaletsky HJ Primer3 on the WWW for general users and for biologist programmers.in: Krawetz S Misener S Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa2000: 365-386Google Scholar so partial intron sequences and intron/exon boundaries will also be scanned. The primer pair specific to exon 9 was adapted from a published sequence15Le Marechal C Audrezet MP Quere I Raquenes O Langonne S Ferec C Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling.Hum Genet. 2001; 108: 290-298Crossref PubMed Scopus (112) Google Scholar to exclude a TG/T repeat polymorphic region in intron 8 that forms heteroduplexes in nearly all samples. The complete designed primers are listed in Table 2.Table 2Primer Sets Used in This Study to Amplify Entire 27 Exons of the CFTR Gene Using the Same PCR Condition (Tm = 55°C, 30 Cycles)ExonForward primer (5′ to 3′)Reverse primer (5′ to 3′)1CAGCACTCGGCTTTTAACATACACACGCCCTCCTCT2TCCAAATCTGTATGGAGACCTGAATTTCTCTCTTCAACTAAACA3CAACTTATTGGTCCCACTTTCACCTATTCACCAGATTTCG4TTGTAGGAAGTCACCAAAGCTACGATACAGAATATATGTGCCA5TTGAAATTATCTAACTTTCCATTTTCGCCTTTCCAGTTGTATAAT6aGCTGTGCTTTTATTTTCCAGACTAAAGTGGGCTTTTTGAA6bCTTAAAACCTTGAGCAGTTCTCAATATTGAAATTATTGGAACAAC7AGATCTTCCATTCCAAGATCTGCAGCATTATGGTACATTA8AAGATGTAGCACAATGAGAGTATAAAGAAAACAGTTAGGTGTTTAGAGCAA9*Primers adapted from Le Marechal et al.15TGGGGAATTATTTGAGAAAGCTTCCAGCACTACAAACTAGAAA10GCGTGATTTGATAATGACCTTGGGTAGTGTGAAGGGTTC11AGATTGAGCATACTAAAAGTGACTGCTTGCTAGACCAATAATTAG12CCAGGAAATAGAGAGGAAATGCATACCAACAATGGTGAACA13GCTAAAATACGAGACATATTGCATTCTGTGGGGTGAAATACC14aGGTGGCATGAAACTGTACTATACATCCCCAAACTATCTTAAT14bATGGGAGGAATAGGTGAAGACAAAGTGGATTACAATACATACA15TGCCAAATAACGATTTCCTAGTGGATCAGCAGTTTCATTT16TTGAGGAATTTGTCATCTTGTGACTTCAACCCTCAATCAAA17aCACTGACACACTTTGTCCACATCGCACATTCACTGTCATA17bATTTGCAATGTTTTCTATGGTGCTTAGCTAAAGTTAATGAGTTC18TTCATTTACGTCTTTTGTGCGGTATATAGTTCTTCCTCATGC19GTGAAATTGTCTGCCATTCTCAAGCAGTGTTCAAATCTCA20TGATCCCATCACTTTTACCTTTTCTGGCTAAGTCCTTTTG21AGAACTTGATGGTAAGTACATGCATTTCAGTTAGCAGCCTTA22TCTGAACTATCTTCTCTAACTGCAATGATTCTGTTCCCACTGT23TTCTGTGATATTATGTGTGGTATTAAGAATTACAAGGGCAATGA24CAGATCTCACTAACAGCCATTTGTCAACATTTATGCTGCTC* Primers adapted from Le Marechal et al.15Le Marechal C Audrezet MP Quere I Raquenes O Langonne S Ferec C Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling.Hum Genet. 2001; 108: 290-298Crossref PubMed Scopus (112) Google Scholar Open table in a new tab PCR was performed in a 50 μl reaction using the High Fidelity PCR master kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions. This enzyme mixture contains both TaqDNA polymerase and Tgo 3′ to 5′ exonuclease proofreading polymerase. The use of a high-fidelity PCR enzyme mixture was to minimize polymerization errors during PCR thus improving detection efficiency for genetic diversity.21Malet I Belnard M Agut H Cahour A From RNA to quasispecies: a DNA polymerase with proofreading activity is highly recommended for accurate assessment of viral diversity.J Virol Methods. 2003; 109: 161-170Crossref PubMed Scopus (53) Google Scholar PCR was performed in a PTC-200 thermocycler (MJ Research, Waltham, MA). One standard PCR protocol was used to amplify all 27 exons of the CFTR gene simultaneously. PCR cycling conditions were 5 minutes at 95°C, 30 cycles of 94°C for 30 seconds, 55°C for 1 minute, 72°C for 1 minute, then 72°C for 5 minutes, and cooling to 4°C. After PCR, heteroduplexes were formed as recommended by the vender (SpectruMedix Inc.). PCR products were heated 5 minutes at 95°C, cooled slowly to 50°C in 1°C/minute intervals, held at 50°C for 20 minutes, and cooled to 25°C at the rate of 2.5°C/minute. To obtain the best peak resolution, treated PCR products were diluted with either 1× or 10× PCR buffer (Applied Biosystems, Foster City, CA) to ensure unsaturated fluorescent intensity and suitable salt contents, and injected into a TGCE equipped with 24 capillaries (model SLE 2410, SpectruMedix Inc.). Other parameters requiring optimization before scanning included injection time, range of temperature gradient, and ramping rate. Two injection conditions were tested, 3 kV for 20 seconds and 5 kV for 30 seconds. Five different temperature gradient ranges for capillary electrophoresis (CE) were also tested: 40 to 50°C, 50 to 55°C, 50 to 60°C, 55 to 60°C, and 60 to 65°C. The ramp period was always 21 minutes. The optimized dilution factor (1:4), injection time (3 kV for 20 seconds), and temperature gradient (50 to 55°C) were used for the rest of the study.CFTR Full Gene-Scanning SetupBased on the basic setup (24-capillary format) in our instrument, the full gene scanning of one patient sample requires one plate (24 exons), plus an additional three wells because of the 27 exons of the CFTR gene. Multiplexing several exons is feasible to fit a 24-well format, but requires further optimization to avoid interaction between multiplexed amplicons. In our design, each plate contained two exons for 12 patient samples. Thus the 27 exons of the 12 deidentified samples (324 PCR products) were scanned within three runs (14 plates; 6 plates per one complete run). These 12 samples constituted our wild-type controls, although 10 of 12 samples had variations in at least one exon. Ideally, peaks of additional samples will be compared to these original samples, thus reducing the need to run wild-type controls with every run.Data AnalysisData were analyzed using the Revelation 2.4 image analysis software (SpectruMedix Inc.). For graphical illustration, analyzed data (by Revelation 2.4) were exported to Microsoft Excel using time (seconds) as the x axis and fluorescent intensity as the y axis. After scanning and data analysis, for each individual exon, at least two samples possessing a single-sharp peak were assumed to be the wild types (negative control) and were sequenced for confirmation. The rest of the samples were compared to the confirmed wilt-type peaks for each exon. Samples possessing multiple peaks (2 to 4) or with any differences in peak shape when compared to the negative control were scored as positive. Positive samples were sequenced for the specific exon to identify the base alteration(s).SequencingPCR samples showing an alteration by TGCE were sequenced using dideoxy terminator sequencing reactions (Applied Biosystems Inc.). In each reaction, 5 μl of Big Dye Terminator Ready Reaction mixture, 3 μl of undiluted and purified PCR product, and 4 μl of 0.8 pmol/μL primer (forward or reverse) were mixed and injected into the automated DNA sequencer (ABI Prism 3100 genetic analyzer, Applied Biosystems). Results were analyzed using both ABI Sequencing Analysis and Sequencher software (The BioCommons, Seattle, WA) to locate and identify alterations. The confirmed alteration(s) were compared to a current CF database (CFGAC) (http://www.genet.sickkids.on.ca/) for identification. The identified nucleotide changes were then classified into three categories: 1) known mutations with reported clinical significance, 2) known variants without clinical significance, and 3) sequence variants with unknown clinical significance (usually missense or intronic mutations). For alterations not previously reported, the laboratory must decide the classification, with deleterious (or suspected deleterious) mutations compromising insertions/deletions (frame shift), nonsense mutations, and predicted splice site mutations; or alterations with unknown significance include missense mutations and some intronic mutations.ResultsPeak ResolutionTGCE detects sequence alteration(s) based on the differentiation of different heteroduplex mobilities in a specifically designed polymer matrix. In theory, heteroduplexes move slower than homoduplexes because of the formation of bubble-like structures of strands with a single mismatch under a specific temperature (Tm). In addition to heteroduplex formation, the length of PCR amplicon, the GC content, the location and types of single nucleotide polymorphisms (A/T, A/C, A/G, C/T, C/G, or G/T) also affect the mobility of formed heteroduplexes under a partially denatured condition (temperature gradient). Thus, parameters that affect heteroduplex mobility in a capillary array will affect the performance of the TGCE. Parameters that need to be optimized for TGCE scanning include temperature gradient, sample injection time, and PCR buffer (salt concentration) used to dilute amplicons. Figure 1 shows the temperature gradient effect on peak resolution. The best temperature range for both heterozygous R117H (exon 4, 270 bp) (Figure 1; A to C) and the hard-to-discriminate mutation G551D heterozygous (exon 11, 175 bp) (Figure 1; D to F) is 50 to 55°C. This temperature range was used for all amplicons.Another factor that can reduce peak resolution is saturated fluorescent signal (>60,000) (Figure 2 A and B). To avoid this, we decreased the sample injection time from the factory default (5 kV for 30 seconds) to 3 kV for 20 seconds for better resolution. Even with decreased injection time, some amplicons required dilution to avoid saturated signal. However, the salt and Mg+2 contents in the buffer may affect peak resolution. Figure 3 demonstrates the dilution effect using different buffers (1× and 10×) on peak resolution. In this figure, the hard-to-discriminate G551D heterozygous shows better resolution in both 1 to 2 and 1 to 4 dilutions using 1× PCR buffer (Figure 3 A and B). However, with higher salt and Mg+2 contents, peak resolution was disrupted and cannot be used for comparison (Figure 3 C and D). For consistency, all 27 amplicons of the CFTR gene were diluted 1 to 4 (using 1× PCR buffer) and injected at 3 kV for 20 seconds.Figure 2Different sample injection times affect peak resolution. A: R117H heterozygous (exon 4, 270 bp) without dilution and injected at 5 kV for 30 seconds; B: R117H heterozygous (exon 4) without dilution and injected at 3 kV for 20 seconds.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Effect of dilution with PCR buffers (1× buffer containing 1.5 mmol/L Mg+2; 10× buffer containing 15 mmol/L Mg+2) on peak resolution using amplicon G551D heterozygous (exon 11) as an example. A: Dilution (1 in 2) using 1× PCR buffer; B: 1 in 4 dilution using 1× PCR buffer; C: 1 in 1.25 dilution using 10× PCR buffer; D: 1 in 2 dilution using 10× PCR buffer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutation Scanning of the CFTR GeneIn this study, no confirmed peak patterns for wild-type sequences of each exon were initially available, but samples possessing a single peak for each specific exon after TGCE scanning were considered as wild types and sequenced for confirmation. After sequencing confirmation, these samples were used as wild-type controls for peak comparison in the further studies.Because of limited sample availability of rare CF mutations, detection of all 1291 reported mutations for accuracy study using TGCE is not feasible. Therefore, to test the accuracy of the TGCE protocol that we developed, we used 42 genotyped samples with 29 specific genotypes representing 27 mutations (Table 1). Each amplicon was injected in duplicate into the automated TGCE. Peak patterns were identical between duplicates (data not shown). Frame numbers of the same amplicon that appeared in the electropherogram were slightly different (±∼40 frames) between duplications. This is a normal phenomenon because each capillary acts independently.After comparison with each wild-type control, all heteroduplex mutations were identified correctly. Figure 4 demonstrates detection of different locations of a single base alteration in exon 4 fragment. In this 270-bp fragment, heterozygous R117H (G/T) is located 83 bp from the 5′ end, heterozygous I148T (T/C) is located in the middle of the fragment, and heterozygous 621 + 1 (G/T) is located at the end of the fragment (37 bp from the 3′ end) (Figure 4 A to D; Table 1). Heterozygous 621 + 1 (G/T) had the least distinct split-peak pattern when compared to heterozygous R117H and I148T (Figure 4; B to D). Different heterozygous 621 + 1 (G/T) samples showed similar patterns, with less peak resolution than the other exon 4 mutations (Figure 4; D to F). The reason for less peak resolution of heterozygous 621 + 1 (G/T) is not clear. A possible explanation is the nearest neighbor structure that helps to stabilize the mismatch, which results in similar mobility between the homoduplexes and heteroduplexes or the location close to the 3′ end. Another example of reduced resolution is heterozygous A455E in exon 9. A shoulder instead of a small peak was observed in heterozygous A455E when compared to the wild-type control (Figure 5 A and B). To clarify the detection limit of the TGCE, further studies should focus on the thermodynamic effect of amplicon length and type of single nucleotide polymorphisms on heteroduplex formation using either artificial templates or engineered plasmids as a study model.Figure 4TGCE scan of samples containing single base changes in exon/intron 4 of the CFTR gene. A: Wild-type; B: R117H heterozygous (exon 4); C: I148T heterozygous (exon 4); D: 621 + 1 G/T heterozygous sample 1 (intron 4); E: 621 + 1 G/T heterozygous sample 2 (intron 4, from Coriell Repository); F: 621 + 1 G/T heterozygous sample 3 (intron 4, from Coriell Repository).View Large Im
Referência(s)