High-Speed, Multicolor Fluorescent Two-Dimensional Gene Scanning

McGrath, Sean; Bounpheng, Mangkey A.; Torres, Loyda; Calavetta, Marco; Scott, Charles; Suh, Yousin; Rines, David M.; Orsouw, Nathalie van; Vijg, Jan

doi:10.1006/geno.2001.6649

Cited by 18 publications

(9 citation statements)

References 15 publications

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“…These results by DGGE rival the theoretical maximum attained by DNA sequencing (31,44,47) and suggest that DGGE can be a viable, more economic alternative to sequencing in detecting pncA mutations. DGGE can be performed on clinical samples, such as sputum (33), and can be completed rapidly with high throughput methodology (21). DGGE also detected apparent reporting errors in PZA susceptibility testing by culture methods.…”

Section: Discussionmentioning

confidence: 99%

“…Samples were loaded onto denaturing gradient gels, 40 to 90% urea:formamide/8% acrylamide (19:1) with TAE (40 mM Tris, 20 mM acetic Acid, 1 mM EDTA, pH 8) (primer sets A to D), or 50 to 90% urea:formamide/8% acrylamide with TAE (primer set E). Electrophoresis of samples occurred at 60°C for 16 to 20 h at 100 V in an OptiAnalyzer 8000 electrophoresis tank (CBS Scientific, Solana Beach, CA) containing TAE buffer (21,43,45). After electrophoresis, gels were stained with ethidium bromide in TAE for 15 to 20 min, and DNA was visualized on a UV transilluminator.…”

Section: Methodsmentioning

confidence: 99%

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Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

McCammon¹,

Gillette²,

Thomas³

et al. 2005

Antimicrob Agents Chemother

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Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90°C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.Pyrazinamide (PZA) is a front-line drug used in the treatment of tuberculosis (TB). In a typical short-course (6-month) therapy, PZA is administered with rifampin (RIF), isoniazid (INH), and ethambutol (EMB) for the first 2 months of treatment, followed by 4 months of treatment with INH and RIF (1). During the initial acute phase, PZA and RIF are responsible for much of the killing of persisting Mycobacterium tuberculosis bacteria (23, 48). The mode of action of PZA is complex and not fully understood. It requires activation to pyrazinoic acid by a pyrazinamidase/nicotinamidase (PZase) encoded by the pncA gene (48). The activated acid form is excreted and then reabsorbed in a protonated form. It is thought that the acidification of the M. tuberculosis cells by the protonated form represents the primary mechanism by which PZA kills tubercle bacilli (48, 49). PZase inactivation is the primary mechanism for developing resistance to PZA (31,35,47,48). Any mutation in pncA that inactivates the encoded enzyme appears to be sufficient to confer resistance. Consequently, PZA resistance mutations are highly diverse and are found throughout pncA. Cumulative reports indicate that between 72 and 98% of PZAresistant isolates harbor pncA mutations (33,46,49). More recent reports, however, tend to support the upper end of this range (48). Earlier reports may have been subject to inaccurate susceptibility testing using culture methods. Susceptibility testing media must have a pH of 6 for PZA to be active against susceptible cells, and this pH is near the limit for growth of tubercle bacill...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

McCammon¹,

Gillette²,

Thomas³

et al. 2005

Antimicrob Agents Chemother

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“…In such visits, the offspring (and participating spouses) underwent similar evaluations as described (13,14,33 Mutation Screening of IGF1 and IGF1R. We screened genomic DNA isolated from blood of centenarians and controls to discover all possible genetic variations in the full coding sequence and intron/exon boundaries of the IGF1 and IGF1R genes by 2D gene scanning (35). To increase specificity, PCR amplification was designed in two steps.…”

Section: Methodsmentioning

confidence: 99%

Functionally significant insulin-like growth factor I receptor mutations in centenarians

Suh

Atzmon

Cho

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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474

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Rather than being a passive, haphazard process of wear and tear, lifespan can be modulated actively by components of the insulin/ insulin-like growth factor I (IGFI) pathway in laboratory animals. Complete or partial loss-of-function mutations in genes encoding components of the insulin/IGFI pathway result in extension of life span in yeasts, worms, flies, and mice. This remarkable conservation throughout evolution suggests that altered signaling in this pathway may also influence human lifespan. On the other hand, evolutionary tradeoffs predict that the laboratory findings may not be relevant to human populations, because of the high fitness cost during early life. Here, we studied the biochemical, phenotypic, and genetic variations in a cohort of Ashkenazi Jewish centenarians, their offspring, and offspring-matched controls and demonstrated a gender-specific increase in serum IGFI associated with a smaller stature in female offspring of centenarians. Sequence analysis of the IGF1 and IGF1 receptor (IGF1R) genes of female centenarians showed overrepresentation of heterozygous mutations in the IGF1R gene among centenarians relative to controls that are associated with high serum IGFI levels and reduced activity of the IGFIR as measured in transformed lymphocytes. Thus, genetic alterations in the human IGF1R that result in altered IGF signaling pathway confer an increase in susceptibility to human longevity, suggesting a role of this pathway in modulation of human lifespan.IGF1 receptor ͉ human longevity ͉ genetic variation

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“…Samples were loaded onto denaturing gradient gels, usually 40 to 90% ureaformamide-8% acrylamide (19:1) with TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8) or 20 to 90% urea-formamide-8% acrylamide with TAE. Samples were electrophoresed at 60°C for 16 to 18 h at 100 V in an OptiAnalyzer 8000 electrophoresis tank (CBS Scientific, Solana Beach, CA) containing TAE buffer (13). After electrophoresis, gels were stained with ethidium bromide in TAE for 15 to 20 min, and DNA was visualized on a UV transilluminator.…”

Section: Methodsmentioning

confidence: 99%

Detection of rpoB Mutations Associated with Rifampin Resistance in Mycobacterium tuberculosis Using Denaturing Gradient Gel Electrophoresis

McCammon¹,

Gillette²,

Thomas³

et al. 2005

Antimicrob Agents Chemother

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Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.Drug resistance is a major concern in the global tuberculosis epidemic. Multidrug resistance, defined as resistance to at least isoniazid and rifampin (RIF), is greater than 10% in many countries and appears to be more widespread than previously documented (25,26). Resistance to RIF is almost exclusively associated with mutations in the rpoB gene that encodes the ␤-subunit of RNA polymerase (17, 28). Over 70 distinct rpoB mutations have been reported for RIF-resistant Mycobacterium tuberculosis isolates worldwide (7,17,20,28). Approximately 95% of RIF-resistant isolates harbor mutations in the rifampin resistance-determining region (RRDR), an 81-bp region within rpoB that spans codons 507 to 533. Mutations at the serine 531, histidine 526, and aspartate 516 codons have been observed in approximately 86% of RIF-resistant isolates and therefore represent hot spots within the RRDR (17, 28). Alterations outside of the RRDR have also been reported for the N-terminal, CII, and CIII regions of rpoB (6, 28) (Fig. 1).Molecular techniques are receiving increased scrutiny as alternatives to traditional drug susceptibility testing for M. tuberculosis (22). Molecular techniques can detect DNA alterations in hours or days, while assaying for drug resistance by culture methods takes weeks. Although the clustering of most mutations in the RRDR simplifies molecular analysis, the heterogeneity of mutations represents a challenge in detecting DNA alterations as a predictor of RIF resistance. These genetic considerations affect b...

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High-Speed, Multicolor Fluorescent Two-Dimensional Gene Scanning

Cited by 18 publications

References 15 publications

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

Functionally significant insulin-like growth factor I receptor mutations in centenarians

Detection of rpoB Mutations Associated with Rifampin Resistance in Mycobacterium tuberculosis Using Denaturing Gradient Gel Electrophoresis

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