Use of high-performance liquid chromatographic fractionation of large RNA molecules in the assay of group I intron ribozyme activity

Georgopoulos, Denise E.; Leibowitz, Michael J.

doi:10.1016/s0021-9673(99)01178-4

Cited by 14 publications

(14 citation statements)

References 11 publications

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“…Interestingly, IP-RP chromatography has been shown to allow the size-dependent fractionation of large RNA molecules [Georgopoulos and Leibowitz, 2000;Azarani and Hecker, 2001]. This came as a surprise as the retention of singlestranded DNA molecules up to at least 100 nucleotides in length has been observed to depend significantly on base composition [Oefner, 2000].…”

Section: Miscellaneous Applicationsmentioning

confidence: 99%

“…Elevated temperatures of analysis improve the resolution of single-stranded DNA and RNA molecules as they break up any existing secondary structures [Oefner, 2000;Azarani and Hecker, 2001]. The high efficiency with which RNA molecules 200-1000 nucleotides in length could be separated by ion-pair reversed-phase chromatography allowed the analysis of the intermediates and products formed in a self-splicing reaction catalyzed by a group I intron ribozyme [Georgopoulos and Leibowitz, 2000]. Azarani and Hecker [2001], on the other hand, demonstrated the successful purification of mRNA and rRNA from total RNA without any evidence of RNA degradation occurring during the chromatographic process.…”

Section: Miscellaneous Applicationsmentioning

confidence: 99%

“…The longer time required for elution with the monolithic column is a consequence of the greater gradient delay time with existing capillary HPLC instrumentation. Both chromatograms underscore the excellent size-dependent separation efficiency of poly(styrene-divinylbenzene) in ion-pair reversed-phase liquid chromatography of nucleic acids enabling a number of applications in genetics, among which are the sizing of DNA microsatellites for such purposes as human identification and parentage testing , the detection of loss of heterozygosity in tumors [Kleymenova et al, 2000], and the study of self-splicing reactions in ribozymes [Georgopoulos and Leibowitz, 2000]. Its most significant application is that of denaturing HPLC for the detection of DNA sequence variation [Oefner andUnderhill, 1995, 1998].…”

Section: Introductionmentioning

confidence: 99%

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Denaturing high-performance liquid chromatography: A review

2001

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Denaturing high-performance liquid chromatography (DHPLC) compares two or more chromosomes as a mixture of denatured and reannealed PCR amplicons, revealing the presence of a mutation by the differential retention of homo-and heteroduplex DNA on reversed-phase chromatography supports under partial denaturation. Temperature determines sensitivity, and its optimum can be predicted by computation. Single-nucleotide substitutions, deletions, and insertions have been detected successfully by on-line UV or fluorescence monitoring within 2-3 minutes in unpurified amplicons as large as 1.5 Kb. Sensitivity and specificity of DHPLC consistently exceed 96%. These features and its low cost make DHPLC one of the most powerful tools for the resequencing of the human and other genomes. Aside from its application to the mutational analysis of candidate genes, DHPLC has proven instrumental in elucidating human evolution and in the mapping of genes. Employing completely denaturing conditions, the utility of DHPLC has been extended to the genotyping of known polymorphisms by utilizing the ability of poly INTRODUCTIONThe analysis of DNA sequence variation is of fundamental importance in genetic studies [Lander, 1996]. With the increasing availability of primary sequence from genomes of human and other organisms, the implementation of sensitive, efficient, and inexpensive technologies for detecting variation that can match the pace of primary sequencing becomes critical. The least expensive method is in silico mining of publicly available databases that contain large amounts of overlapping sequence data from heterogeneous samples. The specificity of this approach which ranges from 55-82% [Buetow et al., 1999;Picoult-Newberg et al., 1999;Cox et al., 2000] is sufficient for the rapid assembly of SNPs for whole-genome linkage disequilibrium studies. But its low sensitivity of 27% [Cox et al., 2000] is insufficient for performing association studies 440 XIAO AND OEFNER on candidate genes. In addition, existing databases are heavily biased towards Caucasian-specific SNPs [Cox et al., 2001].Technologies suitable for the experimental discovery of SNPs and disease causing mutations should be capable of fully automated highthroughput analysis that ideally does not require modified PCR primers, customized specific reagent arrays, detection labels, or any sample pretreatment other than PCR. At present, numerous techniques for DNA mutational analysis are available [Cotton, 1997]. None of these methods meets this challenge. They are either formatted for manual use only and are technically challenging (e.g., denaturing gradient gel electrophoresis (DGGE), chemical cleavage) or limited by the sensitivity of detection (e.g., conformation sensitive gradient gel electrophoresis (CSGE)) [White et al., 1992;Vidal-Puig and Moller, 1994;Couch and Weber, 1996]. Other approaches such as immobilized DNA hybridization arrays and enzymatic mismatch cleavage assays, while promising, still have significant false positive signal, as well as high cost per assay ...

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Section: Miscellaneous Applicationsmentioning

confidence: 99%

Section: Miscellaneous Applicationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Denaturing high-performance liquid chromatography: A review

2001

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“…However, the reproducibility of the electrophoresis method is poor, and maintenance of the modified capillary is troublesome. LC‐employed RP chromatography mediated with ion‐pair (IP) reagent or SEC was applied to RNA separation . Although these methods can separate RNA molecules, a large amount of salts in the eluent is usually required for separation and these salts may interfere with ionization in ICP‐MS.…”

Section: Results From Analysis Of Nmij Crm 6204‐a By the Proposed Meamentioning

confidence: 99%

Separation and quantification of RNA molecules using size‐exclusion chromatography hyphenated with inductively coupled plasma‐mass spectrometry

et al. 2014

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The hyphenation of SEC with ICP-MS was successfully applied to RNA quantification. The developed method combines the separation technique for large biomolecules and element selective detection of ICP-MS. The separation of RNA molecules was performed under the SEC condition without additive reagents such as salts to prevent the adhesion of RNA molecules on the column resin. Fragments of RNA, which were commercially available as a ladder marker solution and certified reference materials, were successfully separated and analyzed by measuring ³¹P⁺ with this method. RNA was quantified with good repeatability (RSD of peak area; 2.7%, n = 3) and linearity (R² = 0.999) using a P standard solution as a calibrant. LOD and absolute detection limit of RNA were 6.7 μg/kg and 67 pg, respectively, which were equal to the values obtained by the analysis of a P standard solution. The accuracy of the proposed measurement was evaluated by measuring certified reference materials of RNA solutions for quantitative analysis (NMIJ CRM 6204-a). The results obtained by this method agreed with the certified values within uncertainty. The proposed analysis method, which demonstrates good accuracy and high precision and is free from interference by nucleotide analogues, qualifies as a method of quality control for the RNA synthesis and extraction process.

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“…IP RP HPLC has previously been used in the sequence independent sizing of duplex DNA (up to 2000 bp) under nondenaturing conditions (2). This technique has also been used under denaturing conditions for the analysis of oligonucleotides (3) and the separation of RNA (4,5) and as a technique to isolate single-stranded (ss) DNA (6). With run times of often 10 min per sample, in conjunction with direct quantification using automated data analysis software, this represents a significant improvement in analytical and preparative nucleic acid enzymology.…”

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confidence: 99%