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1.
Epigenetics ; 10(4): 319-28, 2015.
Article in English | MEDLINE | ID: mdl-25864488

ABSTRACT

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the ß-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between ß-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2'-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.


Subject(s)
DNA Methylation , Genome, Human , Prostatic Neoplasms/genetics , Black or African American , Antimetabolites, Antineoplastic/pharmacology , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Cell Line, Tumor , Decitabine , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Male , Organ Specificity , Promoter Regions, Genetic , Prostatic Neoplasms/epidemiology , White People
2.
Prostate Cancer Prostatic Dis ; 16(4): 292-300, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23896626

ABSTRACT

BACKGROUND: Prostate cancer (PCa) harbors a myriad of genomic and epigenetic defects. Cytosine methylation of CpG-rich promoter DNA is an important mechanism of epigenetic gene inactivation in PCa. There is considerable amount of data to suggest that DNA methylation-based biomarkers may be useful for the early detection and diagnosis of PCa. In addition, candidate gene-based studies have shown an association between specific gene methylation and alterations and clinicopathologic indicators of poor prognosis in PCa. METHODS: To more comprehensively identify DNA methylation alterations in PCa initiation and progression, we examined the methylation status of 485 577 CpG sites from regions with a broad spectrum of CpG densities, interrogating both gene-associated and non-associated regions using the recently developed Illumina 450K methylation platform. RESULTS: In all, we selected 33 promoter-associated novel CpG sites that were differentially methylated in high-grade prostatic intraepithelial neoplasia and PCa in comparison with benign prostate tissue samples (false discovery rate-adjusted P-value <0.05; ß-value 0.2; fold change >1.5). Of the 33 genes, hierarchical clustering analysis demonstrated BNC1, FZD1, RPL39L, SYN2, LMX1B, CXXC5, ZNF783 and CYB5R2 as top candidate novel genes that are frequently methylated and whose methylation was associated with inactivation of gene expression in PCa cell lines. Pathway analysis of the genes with altered methylation patterns identified the involvement of a cancer-related network of genes whose activity may be regulated by TP53, MYC, TNF, IL1 and 6, IFN-γ and FOS in prostate pathogenesis. CONCLUSION: Our genome-wide methylation profile shows epigenetic dysregulation of important regulatory signals in prostate carcinogenesis.


Subject(s)
DNA Methylation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Prostatic Intraepithelial Neoplasia/genetics , Prostatic Intraepithelial Neoplasia/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Antimetabolites, Antineoplastic/pharmacology , Azacitidine/pharmacology , Cell Line, Tumor , Cluster Analysis , CpG Islands , DNA Methylation/drug effects , Disease Progression , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Neoplasm Grading , Prostatic Intraepithelial Neoplasia/metabolism , Reproducibility of Results , Sequence Analysis, DNA
3.
Int J Obes (Lond) ; 35(8): 1095-103, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21139562

ABSTRACT

BACKGROUND: Emerging data have revealed a negative association between adiposity and muscle quality (MQ). There is a lack of research to examine this interaction among young, healthy individuals, and to evaluate the contribution of adiposity to adaptation after resistance exercise (RE). OBJECTIVE: The purpose of this investigation was to examine the influence of subcutaneous adipose tissue (SAT) on muscle function among non-obese individuals before and after RE. DESIGN: Analyses included 634 non-obese (body mass index <30 kg m(-2)) subjects (253 males, 381 females; age=23.3 ± 5.2 years). SAT and muscle mass (magnetic resonance imaging-derived SAT and biceps muscle volume), isometric and dynamic biceps strength, and MQ (strength/muscle volume), were analyzed at baseline and after 12 weeks of unilateral RE. RESULTS: At baseline, SAT was independently associated with lower MQ for males (ß=-0.55; P<0.01) and females (ß=-0.45; P<0.01), controlling for body mass and age. Adaptation to RE revealed a significant negative association between SAT and changes for strength capacity (ß=-0.13; p=0.03) and MQ (ß=-0.14; P<0.01) among males. No attenuation was identified among females. Post-intervention SAT remained a negative predictor of MQ for males and females (ß=-0.47; P<0.01). CONCLUSIONS: The findings reveal that SAT is a negative predictor of MQ among non-obese, healthy adults, and that after 12 weeks of progressive RE this association was not ameliorated. Data suggest that SAT exerts a weak, negative influence on the adaptive response to strength and MQ among males.


Subject(s)
Body Composition/physiology , Muscle Contraction/physiology , Muscle, Skeletal/physiology , Resistance Training , Subcutaneous Fat/physiology , Adiposity , Adult , Body Mass Index , Female , Humans , Magnetic Resonance Imaging , Male
4.
Neurology ; 76(3): 219-26, 2011 Jan 18.
Article in English | MEDLINE | ID: mdl-21178099

ABSTRACT

OBJECTIVE: Duchenne muscular dystrophy (DMD) is the most common single-gene lethal disorder. Substantial patient-patient variability in disease onset and progression and response to glucocorticoids is seen, suggesting genetic or environmental modifiers. METHODS: Two DMD cohorts were used as test and validation groups to define genetic modifiers: a Padova longitudinal cohort (n = 106) and the Cooperative International Neuromuscular Research Group (CINRG) cross-sectional natural history cohort (n = 156). Single nucleotide polymorphisms to be genotyped were selected from mRNA profiling in patients with severe vs mild DMD, and genome-wide association studies in metabolism and polymorphisms influencing muscle phenotypes in normal volunteers were studied. RESULTS: Effects on both disease progression and response to glucocorticoids were observed with polymorphism rs28357094 in the gene promoter of SPP1 (osteopontin). The G allele (dominant model; 35% of subjects) was associated with more rapid progression (Padova cohort log rank p = 0.003), and 12%-19% less grip strength (CINRG cohort p = 0.0003). CONCLUSIONS: Osteopontin genotype is a genetic modifier of disease severity in Duchenne dystrophy. Inclusion of genotype data as a covariate or in inclusion criteria in DMD clinical trials would reduce intersubject variance, and increase sensitivity of the trials, particularly in older subjects.


Subject(s)
Muscular Dystrophy, Duchenne/genetics , Osteopontin/genetics , Polymorphism, Single Nucleotide , Child , Child, Preschool , Cross-Sectional Studies , Disease Progression , Female , Genotype , Glucocorticoids/administration & dosage , Humans , International Cooperation , Italy , Kaplan-Meier Estimate , Male , Muscular Dystrophy, Duchenne/pathology , Muscular Dystrophy, Duchenne/physiopathology , Odds Ratio , Predictive Value of Tests , Reverse Transcriptase Polymerase Chain Reaction , Severity of Illness Index
5.
Mol Psychiatry ; 7(4): 419-23, 2002.
Article in English | MEDLINE | ID: mdl-11986987

ABSTRACT

Recently, a Leu309Met mutation in WKL1 (MLC1, KIAA0027), a gene mapped to chromosome 22q13.33, was reported to co-segregate with periodic catatonia, a clinical sub-type of schizophrenia, in seven members of an extended pedigree.(1) WKL1 encodes a putative membrane protein expressed exclusively in the brain, particularly in the amygdala, nucleus caudatus, thalamus, and hippocampus.(1) We screened WKL1 for etiologic mutations in 28 probands from the United States who were given a consensus diagnosis of schizophrenia and met at least one of these criteria: (1) were from multiplex schizophrenia families where at least two schizophrenic subjects were reported to display catatonic behavior at sometime during the course of their illness; or (2) were from multiplex schizophrenia families where, in a genome scan for schizophrenia susceptibility loci, evidence for excess allele sharing among affected family members for markers in the 22q13 region was seen. In addition, 15 affected subjects from 15 German pedigrees were similarly screened for causative mutations. This German cohort exhibited the catatonia phenotype but had ambiguous linkage to 22q13 and included the mutation-positive proband as a positive control. The 43 probands were screened for base changes in WKL1: 15 SNPs in the non-coding regions of the gene, three SNPs in the 3'UTR, four synonymous coding SNPs and two non-synonymous (amino acid changing) SNPs were identified. We were able to rapidly confirm the Leu309Met nucleotide change in the positive control. No missense mutations were detected in any of the other 42 probands studied. These data exclude the role of WKL1 in schizophrenia susceptibility in the subjects studied.


Subject(s)
Ion Channels/genetics , Mutation, Missense , Schizophrenia, Catatonic/genetics , Adolescent , Adult , Child , Family Health , Female , Genetic Predisposition to Disease , Humans , Male , United States
6.
Neurology ; 56(11): 1486-95, 2001 Jun 12.
Article in English | MEDLINE | ID: mdl-11402105

ABSTRACT

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl CpG binding protein 2 (MeCP2) gene. METHODS: One hundred sixteen patients with classical and atypical RTT were studied for mutations of the MeCP2 gene by using DHPLC and direct sequencing. RESULTS: Causative mutations in the MeCP2 gene were identified in 63% of patients, representing a total of 30 different mutations. Mutations were identified in 72% of patients with classical RTT and one third of atypical cases studied (8 of 25). The authors found 17 novel mutations, including a complex gene rearrangement found in one individual involving two deletions and a duplication. The duplication was identical to a region within the 3' untranslated region (UTR), and represents the first report of involvement of the 3' UTR in RTT. The authors also report the identification of MeCP2 mutations in two males; a Klinefelter's male with classic RTT (T158M) and a hemizygous male infant with a Xq27-28 inversion and a novel 32 bp frameshift deletion [1154(del32)]. Studies examining the relationship between mutation type, X-inactivation status, and severity of clinical presentation found significant differences in clinical presentation between different types of mutations. Mutations in the amino-terminus were significantly correlated with a more severe clinical presentation compared with mutations closer to the carboxyl-terminus of MeCP2. Skewed X-inactivation patterns were found in two asymptomatic carriers of MeCP2 mutations and six girls diagnosed with either atypical or classical RTT. CONCLUSION: This patient series confirms the high frequency of MeCP2gene mutations causative of RTT in females and provides data concerning the molecular basis for clinical variability (mutation type and position and X-inactivation patterns).


Subject(s)
Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/genetics , Gene Deletion , Repressor Proteins , Rett Syndrome/genetics , Adolescent , Adult , Child , Child, Preschool , DNA Mutational Analysis , Dosage Compensation, Genetic , Female , Gene Rearrangement , Genotype , Humans , Male , Methyl-CpG-Binding Protein 2 , Phenotype , Point Mutation , Severity of Illness Index
7.
Forensic Sci Int ; 119(1): 87-96, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11348798

ABSTRACT

Reliable amplification of short tandem repeat (STR) DNA markers with the polymerase chain reaction (PCR) is dependent on high quality PCR primers. The particular primer combinations and concentrations are especially important with multiplex amplification reactions where multiple STR loci are simultaneously copied. Commercially available kits are now widely used for STR amplification and subsequent DNA typing. We present here the use of high performance liquid chromatography (HPLC) and time-of-flight mass spectrometry (TOF-MS) methods for characterization of commercially available STR kits.


Subject(s)
Chromatography, High Pressure Liquid/methods , DNA Primers/standards , Forensic Medicine/methods , Mass Spectrometry/methods , Minisatellite Repeats/genetics , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/methods , Discriminant Analysis , Humans , Quality Control , Time Factors
8.
Hum Mol Genet ; 10(3): 189-94, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11159936

ABSTRACT

Arrhythmogenic right ventricular dysplasia type 2 (ARVD2, OMIM 600996) is an autosomal dominant cardiomyopathy, characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death. The disease locus was mapped to chromosome 1q42--q43. We report here on the physical mapping of the critical ARVD2 region, exclusion of two candidate genes (actinin 2 and nidogen), elucidation of the genomic structure of the cardiac ryanodine receptor gene (RYR2) and identification of RYR2 mutations in four independent families. In myocardial cells, the RyR2 protein, activated by Ca(2+), induces the release of calcium from the sarcoplasmic reticulum into the cytosol. RyR2 is the cardiac counterpart of RyR1, the skeletal muscle ryanodine receptor, involved in malignant hyperthermia (MH) susceptibility and in central core disease (CCD). The RyR2 mutations detected in the present study occurred in two highly conserved regions, strictly corresponding to those where mutations causing MH or CCD are clustered in the RYR1 gene. The detection of RyR2 mutations causing ARVD2, reported in this paper, opens the way to pre-symptomatic detection of carriers of the disease in childhood, thus enabling early monitoring and treatment.


Subject(s)
Arrhythmogenic Right Ventricular Dysplasia/genetics , Myocardium/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Amino Acid Sequence , Arrhythmogenic Right Ventricular Dysplasia/pathology , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 1/genetics , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , Family Health , Female , Genetic Predisposition to Disease/genetics , Humans , Male , Molecular Sequence Data , Mutation , Mutation, Missense , Pedigree , Polymorphism, Single-Stranded Conformational , Sequence Alignment , Sequence Homology, Amino Acid
9.
Circulation ; 103(4): 485-90, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11157710

ABSTRACT

BACKGROUND: Familial polymorphic ventricular tachycardia is an autosomal-dominant, inherited disease with a relatively early onset and a mortality rate of approximately 30% by the age of 30 years. Phenotypically, it is characterized by salvoes of bidirectional and polymorphic ventricular tachycardias in response to vigorous exercise, with no structural evidence of myocardial disease. We previously mapped the causative gene to chromosome 1q42-q43. In the present study, we demonstrate that patients with familial polymorphic ventricular tachycardia have missense mutations in the cardiac sarcoplasmic reticulum calcium release channel (ryanodine receptor type 2 [RyR2]). METHODS AND RESULTS: In 3 large families studied, 3 different RyR2 mutations (P2328S, Q4201R, V4653F) were detected and shown to fully cosegregate with the characteristic arrhythmic phenotype. These mutations were absent in the nonaffected family members and in 100 healthy controls. In addition to identifying 3 causative mutations, we identified a number of single nucleotide polymorphisms that span the genomic structure of RyR2 and will be useful for candidate-based association studies for other arrhythmic disorders. CONCLUSIONS: Our data illustrate that mutations of the RyR2 gene cause at least one variety of inherited polymorphic tachycardia. These findings define a new entity of disorders of myocardial calcium signaling.


Subject(s)
Ryanodine Receptor Calcium Release Channel/genetics , Tachycardia, Ventricular/genetics , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 1/genetics , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , Family Health , Female , Finland , Haplotypes , Humans , Male , Microsatellite Repeats , Mutation , Mutation, Missense , Myocardium/metabolism , Pedigree , Polymorphism, Genetic , Tachycardia, Ventricular/pathology
11.
Anal Chem ; 73(3): 620-4, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11217771

ABSTRACT

Currently, a major focus of human genetics is the utilization of single-nucleotide polymorphisms for clinical diagnostics, whole-genome linkage disequilibrium screens to identify common disease genes such as Alzheimer disease, determination of the recent evolutionary history of a species, and the process of speciation. We have examined single-nucleotide extension coupled with high-performance liquid chromatography as a method to simultaneously genotype two SNPs occurring in the coding region of the HFE gene that produce clinical effects. This assay allows concurrent genotyping of the C282Y and H63D mutations in 11 min and is 100% concordant with current testing methods for both of these mutations.


Subject(s)
Chromatography, High Pressure Liquid/methods , HLA Antigens/genetics , Hemochromatosis/genetics , Histocompatibility Antigens Class I/genetics , Membrane Proteins , Mutation , Base Sequence , DNA Primers , Genotype , Hemochromatosis Protein , Humans , Polymerase Chain Reaction
12.
Genet Test ; 4(2): 125-9, 2000.
Article in English | MEDLINE | ID: mdl-10953951

ABSTRACT

We describe a novel molecular screening technique for hereditary hemochromatosis through which HFE genotypes at codon positions 282 and 63 are simultaneously detected. The technique combines multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) and allows automated high-throughput analysis. We used this method to genotype 43 previously characterized anonymous DNA specimens in blinded fashion and found multiplex PCR/DHPLC 100% accurate when compared with PCR/restriction enzyme digestion, yet far more efficient.


Subject(s)
HLA Antigens/genetics , Hemochromatosis/genetics , Histocompatibility Antigens Class I/genetics , Membrane Proteins , Mutation , Base Sequence , Chromatography, High Pressure Liquid/methods , Codon , DNA Primers , Genetic Carrier Screening , Hemochromatosis/diagnosis , Hemochromatosis Protein , Humans , Polymerase Chain Reaction/methods
13.
Anal Chem ; 72(4): 858-64, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10701274

ABSTRACT

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.


Subject(s)
Chromatography, High Pressure Liquid/methods , DNA/genetics , Microsatellite Repeats , Alleles , DNA/analysis , DNA/blood , Genetic Markers/genetics , Humans , Polymerase Chain Reaction
14.
J Biomol Tech ; 11(2): 67-73, 2000 Jun.
Article in English | MEDLINE | ID: mdl-19499039

ABSTRACT

Hereditary hemochromatosis is one of the most common hereditary disorders in Caucasians.The disease is linked to two single-nucleotide polymorphisms (SNPs) in the HFE gene.The two point mutations result in a change of Cys to Tyr at position 282 and His to Asp at position 63 in the resultant protein.We have developed a single-nucleotide extension (SNE) assay for hereditary hemochromatosis genetic testing, which employs capillary electrophoresis to simultaneously detect the SNE products generated from the two SNP sites. An upstream or a downstream primer adjacent to the possible mutation site is designed and extended one nucleotide further at the 3' end, complementary to the nucleotide at the possible mutation site.The extended nucleotide is one of four fluorescently labeled dideoxynucleotide triphosphates that also act as terminators. Analysis of the extended products by laser-induced fluorescence capillary electrophoresis (LIF-CE) directly reflects the identity of the possible mutation site. Using one primer upstream or downstream from the possible mutation site, three genotypes at one mutation site can be distinguished. Using both upstream and downstream primers provides a second level of specificity and increases the accuracy of the genetic test. The protocol can also be applied to the study of other SNP analyses and to simultaneous detection of multiple mutation sites.

15.
J Chromatogr B Biomed Sci Appl ; 732(2): 365-74, 1999 Sep 24.
Article in English | MEDLINE | ID: mdl-10517359

ABSTRACT

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye-DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THOI locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.


Subject(s)
Benzoxazoles/analysis , DNA/analysis , Electrophoresis, Capillary/methods , Intercalating Agents/analysis , Quinolinium Compounds/analysis , Spectrometry, Fluorescence/methods , Alleles , Genotype , Lasers , Reference Standards , Tandem Repeat Sequences/genetics
16.
Anal Chem ; 70(21): 4514-9, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9823710

ABSTRACT

Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration.


Subject(s)
Alleles , Fluorescent Dyes/chemistry , Intercalating Agents/chemistry , Tandem Repeat Sequences , Adenine/analysis , Base Pairing , Homozygote , Humans , Polymerase Chain Reaction , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Thymine/analysis
17.
Electrophoresis ; 19(1): 108-18, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9511871

ABSTRACT

Polymerase chain reaction (PCR) amplified alleles need to be isolated and purified before carrying out additional analysis to confirm sequence, number of repeats and microvariants within a short tandem repeat (STR) locus. Also, PCR amplification of tetranucleotide repeat loci, used in DNA typing assays, often result in heteroduplex formation, adding to the complexity of analysis. Sequencing reactions require single specific target DNA for reliable sequencing analysis. Alkylated poly(styrene-divinylbenzene) columns at elevated temperature and gradient elution conditions increase the efficiency of separation to allow for the purification of PCR products. Using the separation technique of ion-pairing reverse-phase (IPRP) high performance liquid chromatography (HPLC), molecular biologists can separate and purify DNA fragments without alteration to the double-stranded DNA sequencing properties. In this study, the IP-RP chromatography technique has been demonstrated by separation of alleles of the short tandem repeat loci of TH01, vWA31, F13A01 and FES/ FPS. Alleles differing in size range of 12 to 4 base pairs were separated by IPRP/HPLC and individual alleles were peak-captured, then cycle-sequenced. These HPLC fractions required no additional steps prior to cycle sequencing. Capillary electrophoresis (CE) was used to sequence the alleles. Furthermore, CE offers advantages over traditional slab methods via automation and higher applied voltages. Interestingly, unlike traditional gel electrophoresis, samples were introduced into the sieving matrix by electrokinetic injection, which allows for multiple injections from a single sample, a key feature for method development. Applied voltage was 320 V per centimeter using a nonderivatized fused silica capillary with an interior diameter of 50 microm and a total length of 47 centimeters. The total analysis time including capillary filling and pre-electrophoresis was less than 30 min for a 220-bp fragment. A sequencing rate of 530 bp/h was achieved using these conditions. By combining the techniques of HPLC separation and CE sequencing, the results confirmed the sequence and number of nucleotide repeats for each STR loci. An average sequencing efficiency of 97% was achieved. Additionally, this method defined the absence of a 9.3 microvariant for a TH01 heterozygous individual previously typed as a 9, 9.3/10 using slab gel electrophoresis. The techniques described can be applied to other DNA purification and isolation problems.


Subject(s)
Alleles , Chromatography, High Pressure Liquid/methods , Electrophoresis, Capillary/methods , Repetitive Sequences, Nucleic Acid , Base Sequence , DNA/isolation & purification , Molecular Sequence Data
18.
J Chromatogr B Biomed Appl ; 683(1): 109-14, 1996 Aug 09.
Article in English | MEDLINE | ID: mdl-8876445

ABSTRACT

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised due to the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column; hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time and cost. One particularly effective clean-up system, membrane dialysis, was automated using a robotic workstation.


Subject(s)
Electrophoresis, Capillary/methods , Polymerase Chain Reaction/methods , Automation , DNA/analysis
19.
Appl Theor Electrophor ; 6(1): 11-4, 1996.
Article in English | MEDLINE | ID: mdl-9072075

ABSTRACT

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised by the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column, hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing, and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time, and cost.


Subject(s)
DNA/isolation & purification , Electrophoresis, Capillary/methods , Membranes, Artificial , Polymerase Chain Reaction/methods
20.
J Chromatogr A ; 680(2): 525-40, 1994 Oct 07.
Article in English | MEDLINE | ID: mdl-7981833

ABSTRACT

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.


Subject(s)
DNA Restriction Enzymes , DNA/analysis , Electrophoresis, Polyacrylamide Gel/methods , Polymerase Chain Reaction , Bacteriophage phi X 174/genetics , Base Sequence , Capillary Action , DNA, Viral/analysis , Deoxyribonucleases, Type II Site-Specific , Electrophoresis, Polyacrylamide Gel/statistics & numerical data , Humans , Molecular Sequence Data , Molecular Weight , Phosphorylation , Plants/genetics , Reproducibility of Results
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