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PubMed1997

Detecting familial defective apolipoprotein B-100: three molecular scanning methods compared.

Familial defective apolipoprotein (apo) B-100 (FDB), a condition that may give rise to hypercholesterolemia, is caused by mutations around codon 3500 of the apo B gene. We have compared the ability of three molecular-scanning techniques, heteroduplex analysis, single-strand conformation polymorphism (SSCP) analysis, and denaturing gradient gel electrophoresis (DGGE), to detect these mutations in a cohort of 432 hypercholesterolemic individuals. Heteroduplex analysis and DGGE detected 11 individuals with apo B mutations, 9 of whom were heterozygous for apo B R3500Q and 2 who were heterozygous for apo B R3531C. Whereas DGGE was able to distinguish between these two mutations, heteroduplex analysis was technically simpler and gave a higher sample throughput. In contrast, SSCP analysis detected only 7 of the R3500Q and none of the R3531C heterozygotes and was the most complex of the three techniques. We believe heteroduplex analysis to be the method of choice for screening large numbers of samples for FDB.

Apolipoprotein B-100↗
PubMed1999

Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia.

Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality assessment. For polymerase chain reaction analysis, primers directed against framework (FR) 3 (FR3-A and FR3-B), FR2, and FR1 of the variable gene segments and against joining gene segments of the immunoglobulin heavy-chain gene were used. Polymerase chain reaction products were analyzed by high-resolution fingerprinting analysis using radiolabeled nucleotides, gene scanning using fluorochrome-labeled primers, and heteroduplex analysis. All of the assays generated polyclonal patterns in the reactive tissues. The sensitivity in detecting monoclonality as defined by Southern blotting varied between 60% (heteroduplex analysis with FR3 primers) and 77% (high-resolution fingerprinting analysis with FR2 primers). Comparison of the three FR3 assays revealed that gene scanning had the highest sensitivity (69%), probably because it could detect small aberrant monoclonal amplicons. False-negative results were especially frequent in follicular center lymphoma (n = 20), but also in diffuse large B-cell lymphoma (n = 18), both renowned for having mutated variable gene segments, potentially leading to primer mismatching. For example, in follicular center lymphoma, the FR3, FR2, and FR1 region primer sets detected clonality in only 35 to 40, 65, and 50%, respectively. Combining these techniques, 17 (85%) of 20 follicular center lymphoma samples showed monoclonality. Therefore, especially in follicular center lymphoma, diffuse large B-cell lymphoma, and, to a lesser extent, in other lymphomas, multiple variable gene segment primer sets must be used for a reliable assessment of clonality. Our results also suggest that gene scanning is somewhat more sensitive than other read-out systems. Heteroduplex analysis, however, is a reliable alternative within a diagnostic setting, avoiding the use of radioactivity or expensive automated sequencing equipment and fluorochrome-labeled (oligo)nucleotides.

Blotting, Southern↗
PubMed2001

Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus Section Flavi strains.

For genetic identification of Aspergillus Section Flavi isolates and detection of intraspecific variation, we developed a novel method for heteroduplex panel analysis (HPA) utilizing fragments of the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) of the rRNA gene that was PCR amplified with universal primers. The method involves formation of heteroduplexes with a set of reference fragments amplified from Aspergillus flavus, A. parasiticus, A. tamarii, and A. nomius and subsequent minislab vinyl polymer gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1, and N-1) generated by pairwise reannealing among four reference fragments. Of 90 test panels, 89 succeeded in identifying the species and 74 were identical to one of the four standard panels. Of the 16 new panels, 11 A. flavus/A. oryzae panels were identical and typed as F-2 and 4 of 5 A. nomius panels were typed as N-2 or N-3. The other strain, A. nomius IMI 358749, was unable to identify the species because no single bands were formed with any of the four reference strains. DNA sequencing revealed that our HPA method has the highest sensitivity available and is able to detect as little as one nucleotide of diversity within the species. When Penicillium or non-Section Flavi Aspergillus was subjected to HPA, the resulting bands of heteroduplexes showed apparently lower mobility and poor heteroduplex formation. This indicates that HPA is a useful identification method without morphological observation and is suitable for rapid and inexpensive screening of large numbers of isolates. The HPA typing coincided with the taxonomy of Section Flavi and is therefore applicable as an alternative to the conventional methods (Samson, R. A., E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, p. 64-97, in Introduction to Food- and Airborne Fungi, 6th ed., 2000).

Aspergillus↗
PubMed2004

Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis.

The genotype of the infecting hepatitis C virus (HCV) helps determine the patient's prognosis and the duration of treatment. Heteroduplex mobility analysis (HMA) is a rapid, inexpensive method for genotyping of HCV that does not require sequencing. We developed an HMA that uses temperature gradient capillary electrophoresis (TGCE) to differentiate HCV genotypes. A 56-bp region of the HCV 5' untranslated region (UTR) that was conserved within a genotype yet whose sequence differed between genotypes was amplified for HMA-TGCE analysis. HCV amplicons of types 1, 2a, 2b, 3a, 4, and 6a were hybridized in pairs and analyzed by TGCE. Amplicons hybridized to the same subtype yielded one homoduplex peak, while hybridization of different subtypes resulted in heteroduplexes and generated multiple TGCE peaks. Heteroduplexes contain thermodynamically unstable nucleotide mismatches that reduced their TGCE mobilities compared to those of homoduplexes. Three HCV subtypes (subtypes 1a, 3a, and 4) generated unique peak patterns when they were combined with each genotype analyzed and were chosen as the reference genotypes. A blinded study with 200 HCV-infected samples was 97% accurate compared to genotyping by 5' UTR sequence analysis. The majority of discordant results were unexpected sequence variants; however, five of nine sequence variants were correctly genotyped. The assay also detected and correctly genotyped mixed HCV infections. Compared to conventional HMA, TGCE improves the resolution, with better separation of heteroduplexes and homoduplexes. All common HCV genotypes can be detected and differentiated by this HMA-TGCE assay.

Base Sequence↗
PubMed1996

HLA-DQA1 and -DQB1 genotyping by PCR-RFLP, heteroduplex and homoduplex analysis.

PCR-RFLP typing methods for DQA1 and DQB1 in conjunction with the analysis of heteroduplex and homoduplex patterns have allowed a simple method for typing all of the major DQA1 and DQB1 alleles. This method has advantages over PCR amplification with sequence-specific primers (PCR-SSP), PCR hybridization with sequence-specific oligonucleotide probes (PCR-SSO) and other PCR-RFLP strategies for typing DQ alleles. The analysis of heteroduplex and homoduplex patterns can be used in conjunction with other PCR typing systems such as PCR-SSP as a confirmatory step with little additional work. In addition, a PCR-RFLP strategy was designed for resolving the DQB1*0602 and DQB1*0603 alleles, which involved the use of a primer containing a base mutation, creating a new restriction site which distinguished the two alleles. These techniques have enabled resolution of the major homozygous and heterozygous combinations of these DQA1 and DQB1 alleles. The PCR-RFLP technique does not require the large number of oligonucleotides that are necessary for both the PCR-SSP and PCR-SSO techniques and is thus both time and cost effective for infrequent or small numbers of samples.

Alleles↗
PubMed1999

Community respiratory virus infections in patients with hematologic malignancies.

BACKGROUND AND OBJECTIVE: The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment. DESIGN AND METHODS: We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE). RESULTS: Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis. INTERPRETATION AND CONCLUSIONS: We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.

Adolescent↗
PubMed2003

An heteroduplex mobility analysis assay based on capillary electrophoresis for the study of HCV quasispecies.

The quasispecies nature of the hepatitis C virus (HCV) genome is central to the transmission, persistence and pathogenesis of the infection. Heteroduplex mobility analysis (HMA) is a simple and an inexpensive technique for the qualitative and quantitative analysis of genetic variation of viral quasispecies. An original HMA for the HVR1 region of HCV was developed, based on a semi-automated, non-radioactive capillary electrophoresis system, which allows the processing of large numbers of samples in short times, the accurate measure of mobility shifts and the quantitation of heteroduplexes. A set of 120 HVR1 clones of known sequence was used to develop the assay, which was tested on HVR1 sequences amplified directly from sera of 17 HCV-infected patients. HVR1 sequence divergence directly correlated with the heteroduplex mobility ratio (HMR) of hybrid molecules between six and 40 mismatches. Heteroduplexes between one and six mismatches were resolved, although HMRs were not proportional to base changes, likely due to an effect of type and position of the substitutions. The assay sensitivity was 1% of the total sample size. This assay may allow the application of quasispecies analysis to a wider range of clinical and basic investigations.

Adult↗
PubMed1996

Detection of congenital color vision defects using heteroduplex-SSCP analysis.

Gene deletion and hybrid gene formation result in congenital red and green color vision defects. The innumerable variations in these defects require the development of appropriate methods for detailed investigation. We used heteroduplex-single-strand conformation polymorphism (SSCP) analysis to detect the gene variations in subjects with congenital red and green color vision defects. For comparison, we also used polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) to detect these defects. In all 13 protan and 9 of the 15 deutan subjects, defects of the red or green pigment gene could be identified by either heteroduplex-SSCP or PCR-RFLP analysis. Through heteroduplex-SSCP analysis, sequence polymorphism was detected in exon 5 of the green pigment gene. One polymorphic sequence was present in 8 of 21 trichromats but in only one of 17 dichromats, showing a biased distribution. A specific variation was found in one protan by SSCP analysis. Heteroduplex-SSCP procedures are useful for objective clinical diagnostic testing and for further study of color vision variations because of their simplicity and reliability and because they provide more information.

Blotting, Southern↗
PubMed2001

Combined SSCP/duplex analysis by capillary electrophoresis for more efficient mutation detection.

SSCP and heteroduplex analysis (HA) continue to be the most popular methods of mutation detection due to their simplicity, high sensitivity and low cost. The advantages of these methods are most clearly visible when large genes, such as BRCA1 and BRCA2, are scanned for scattered unknown mutations and/or when a large number of DNA samples is screened for specific mutations. Here we describe a novel combined SSCP/duplex analysis adapted to the modern capillary electrophoresis (CE) system, which takes advantage of multicolor labeling of DNA fragments and laser-induced fluorescence detection. In developing this method, we first established the optimum conditions for homoduplex and heteroduplex analysis by CE. These were determined based on comprehensive analysis of representative Tamra-500 markers and BRCA1 fragments at different concentrations of sieving polymer and temperatures in the presence or absence of glycerol. The intrinsic features of DNA duplex structures are discussed in detail to explain differences in the migration rates between various types of duplexes. When combined SSCP/duplex analysis was carried out in single conditions, those found to be optimal for analysis of duplexes, all 31 BRCA1 and BRCA2 mutations, polymorphisms and variants tested were detected. It is worth noting that the panel of analyzed sequence variants was enriched in base substitutions, which are usually more difficult to detect. The sensitivity of mutation detection in the SSCP portion alone was 90%, and that in the duplex portion was 81% in the single conditions of electrophoresis. As is also shown here, the proposed combined SSCP/duplex analysis by CE has the potential of being applied to the analysis of pooled genomic DNA samples, and to multiplex analysis of amplicons from different gene fragments. These modifications may further reduce the costs of analysis, making the method attractive for large scale application in SNP scanning and screening.

BRCA1 Protein↗
PubMed2001

Investigating hepatitis C virus heterogeneity in a high prevalence setting using heteroduplex tracking analysis.

Hepatitis C virus (HCV) infection is very common among chronic hemodialysis patients. In the past, blood transfusion appeared to be the primary risk factor; however evidence of nosocomial HCV transmission in the hemodialysis setting has recently been reported. This report describes a molecular investigation of HCV isolates obtained from a population of 670 patients attending six different Seattle-King County based hemodialysis centers in order to identify potential common source infections. 733 serum specimens were collected from hemodialysis patients in 1992 and 1996, and were tested for HCV antibodies and RNA. Overall, 115 of 670 (17%) patients were positive for HCV RNA, and thus were considered actively infected by HCV. HCV genotype was determined in all cases by restriction fragment length polymorphism, and 93 patients were found to be infected by HCV genotype 1. HCV envelope genes were amplified from the 93 patients with genotype 1 infection, and were studied in further detail by heteroduplex tracking analysis (HTA) using genotype 1a and 1b specific probes derived from the envelope 1 (E1) and envelope 2 (E2) genes. Genetic relatedness between pairs of HCV envelope genes was estimated by calculating the degree of gel shift relative to homoduplex controls. Nucleotide sequencing and phylogenetic analysis was used to confirm genetic relatedness detected by HTA. When HTA was performed using the E1 gene probe, 12 apparently related infections were detected; 10 of 12 (83%) of these infections were confirmed as truly related using the gold standard method of nucleotide sequencing plus phylogenetic analysis. Using an E2 gene probe, 24 infections were apparently related, but only six (25%) were confirmed by sequencing. As a control, 41 envelope genes, which were unrelated by HTA, were sequenced; 0 of 41 (0%) were truly related. In summary, HTA provides a rapid and effective molecular technique for screening HCV genetic relatedness in population-based studies, and should prove valuable in future studies of HCV molecular epidemiology.

Genetic Variation↗
PubMed2006

Increase in probing depth is correlated with a higher number of Prevotella intermedia genotypes.

BACKGROUND: The aims of this study were to determine the genotypic diversity of Prevotella intermedia in subgingival plaque samples by using two techniques, arbitrarily primed polymerase chain reaction (AP-PCR) and heteroduplex analysis, and to assess the relationship of this diversity with increase in probing depth. METHODS: The subgingival plaque samples were obtained from 12 patients using paper points inserted into periodontal pockets (diseased sites) and healthy gingival sulci (healthy sites) of the same subjects. After isolation and identification, AP-PCR was performed for genotypic characterization of P. intermedia (80 isolates). The clinical samples with a positive result for P. intermedia were amplified by 16S rRNA-based PCR method, and the amplicons were subjected to heteroduplex analysis. RESULTS: The agreement between the two methods was very high; the AP-PCR and heteroduplex analysis showed that subjects harbored between one and five distinct genotypes of P. intermedia, with a positive association between numbers of genotypes by AP-PCR (P = 0.0042) or heteroduplex (P = 0.0099) and increase in probing depth. No matching of P. intermedia genotypes was observed between healthy and diseased sites of the same individual. Interindividual analyses demonstrated absence of identical clones and indicated a high level of genetic diversity in the species. CONCLUSION: A clear relationship was observed between a higher number of genotypes and increase in probing depth; these results suggest that environmental challenges in the periodontal pockets may modulate the microbiota by selecting genotypes best able to exploit the environment.

Adult↗
PubMed2005

Efficient SNP analysis enabled by joint application of the muTGGE and heteroduplex methods.

Gene science-based diagnoses have become an increasingly realistic option as the state of knowledge has improved regarding the genetic basis of disease. To facilitate the creation of this potential diagnostic tool, researchers have made large-scale detection of point mutations a key issue. Here, we propose an inexpensive and convenient method with a high performance level for this purpose: micro temperature gradient gel electrophoresis (muTGGE)-empowered heteroduplex analysis (muTG-HD). First, muTGGE was shown to separate double-stranded DNA containing single nucleotide polymorphism (SNP) with sufficiently high resolution when used in the mode of perpendicular TGGE. Using human c-Ki-ras and rat p53 DNA, point mutations could be unequivocally detected by muTG-HD when parallel TGGE was employed. The mutation type (such as G/C to A/T), the position of the point mutation (centre or not) and the DNA size (around 100 or 200 bp) were examined and found to be detectable. Thus, muTG-HD could detect point mutations efficiently at a much lower cost by having multiple lanes per gel.

Animals↗
PubMedDate not supplied

Clinical follow up of mexican women with early onset of breast cancer and mutations in the BRCA1 and BRCA2 genes.

OBJECTIVE: This study describes the presence of mutations in BRCA1 and BRCA2 genes in a group of Mexican women and the clinical evolution of early onset breast cancer (EOBC). MATERIAL AND METHODS: A prospective hospital-based study was performed in a sample of 22 women with EOBC (7 in clinical stage IIA, 8 in IIB, and 7 in IIIA). The patients attended a tertiary care hospital in northeastern Mexico in 1997 and were followed up over a 5-year period. Molecular analysis included: 1) a mutation screening by heteroduplex analysis (HA) of BRCA1 and BRCA2 genes and 2) a sequence analysis. RESULTS: Of 22 patients, 14 (63.6%) showed a variant band detected by heteroduplex analysis of the BRCA1 and BRCA2 genes: 8 polymorphisms, 4 mutations of uncertain significance, and 2 novel truncated protein mutations, one in BRCAI (exon 11, 3587delT) and the other in the BRCA2 gene (exon 11, 2664InsA). CONCLUSIONS: These findings support future studies to determine the significance and impact of the genetic factor in this Mexican women population.

Adult↗
PubMed2000

Mixed viral infection identified using heteroduplex mobility analysis (HMA).

It is now recognised that mixed viral infection, or infection of an individual with two or more distinct strains of a single viral species, often occurs particularly with RNA viruses. Current methods for detection of mixed infection normally involve genotyping or cloning and DNA sequencing. These methods are not always accurate or sensitive at detecting mixed infection and cannot be used for large numbers of samples. Furthermore subsequent sequence determination of the coinfecting viruses is labour intensive. This paper describes a simple, generic method based upon PCR and heteroduplex mobility analysis (HMA) that can be used to rapidly determine mixed infection with two strains of the same virus. The utility of this method is illustrated with hepatitis C virus (HCV) and TT virus (TTV) as examples. PCR-HMA detected mixed infection in 3 (8%) of 38 sera from intravenous drug users (IVDU) and 28 (30%) of 70 TTV-positive sera from Australia, China, and Vietnam. HMA can also be used to screen recombinant colonies to identify the sequences of the coinfecting viruses. The methods described here could be applied to analyse any PCR product containing two or more divergent sequences, whether derived from viruses, bacteria, or eukaryotic organisms.

Base Sequence↗
PubMed1999

Sequence diversity in the 5'-UTR region of GB virus C/hepatitis G virus assessed using sequencing, heteroduplex mobility analysis and single-strand conformation polymorphism.

GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense RNA virus belonging to the Flaviviridae family identified recently. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect GBV-C/HGV RNA using nested primers designed to amplify 245 bp of the 5'-untranslated region (UTR). GBV-C/HGV RNA was detected in 20.7% of 101 HCV-RNA positive and 6.8% of 44 HCV-RNA negative specimens. Sequencing of the PCR products demonstrated they had between 84.3 and 100% nucleotide identity. Most of the diversity corresponded to two variable regions identified within the 5'-UTR. Phylogenetic analysis indicated that GBV-C/HGV subtypes present in Australia belonged to group 2 and were closest in evolutionary terms to isolates from the USA and Europe. All isolates were analysed using single-strand conformation polymorphism (SSCP) and heteroduplex mobility analysis (HMA) on 8% non-denaturing polyacrylamide gels. SSCP of the isolates identified a number of distinct conformation polymorphisms that corresponded with sequence-determined genetic diversity. HMA was developed to assess the amount of genetic diversity between isolates without the need for sequencing. The average difference between the predicted divergence of two isolates calculated from the mobility of the heteroduplex and the actual value (based on nucleotide sequence) was 2.3% in this sample of isolates, where the mean sequence divergence was 8.52%.

5' Untranslated Regions↗
PubMed1998

Population analysis of the collagen type IIalpha1 3' variable number of tandem repeat polymorphism by heteroduplex genotyping.

The AT-rich variable number of tandem repeat (VNTR) marker at the 3' end of the collagen type IIalpha1 (COL2A1) gene has been shown to have a complex structure with extensive sequence variations among repeat units. We analyzed this VNTR polymorphism in a large population of 972 Caucasian individuals with a genotyping procedure involving heteroduplex analysis of PCR products using polyacrylamide gel electrophoresis. Seven size alleles were identified, combining to 19 different homoduplex genotypes (heterozygosity = 0.64). These could be further dissected by analysis of heteroduplexes into 85 different heteroduplex genotypes (heterozygosity = 0.84). By systematically heteroduplexing homozygous and heterozygous individuals in vitro, characteristic heteroduplex doublet bands were generated of known allelic composition. A comparison of these doublets with heteroduplex patterns observed in the population allowed us to identify 29 alleles. The degree of correspondence with a different COL2A1 VNTR genotyping system, based on size separation of single-strand VNTR alleles [1], was investigated by the analysis of samples typed with both methods, including samples from reference CEPH pedigrees. This revealed improved genetic resolution by the heteroduplex method including discrimination of frequent alleles considered identical by the single-strand analysis. Our findings demonstrate heteroduplex analysis of the COL2A1 VNTR to be a robust and highly informative genetic marker system. The documented increased genetic variability has important implications in forensic and paternity testing, as well as in linkage and association studies relating genetic variation at this locus to disease endpoints.

Aged↗
PubMed2000

Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in multiple tissues and organs. TSC exhibits locus heterogeneity with genes at 9q34 (TSC1) and 16p13.3 (TSC2) that have 21 and 41 coding exons, respectively. The mutational spectrum at both loci is wide and previous studies have shown that 60%-70% of cases are sporadic and represent new mutations. We have formatted denaturing high performance liquid chromatography (DHPLC) for rapid screening of all coding exons of TSC1 and TSC2. DHPLC analysis detected likely disease-causing mutations in 103 of 150 unrelated cases (68%), compared with 92/150 (61%) and 87/150 (58%) for single-strand conformation polymorphism analysis (SSCP) and conventional heteroduplex analysis (HA), respectively. Capital, consumable and labour costs were determined for each exon screening procedure. Estimated costs per patient sample depended on throughput, particularly for DHPLC, where a high proportion of costs are fixed, and were pounds sterling 257, pound sterling 216 and pound sterling 242 for DHPLC, SSCP and HA, respectively, assuming a throughput of 252 samples per year, or pound sterling 354, pound sterling 233 and pound sterling 259, assuming a throughput of 126 samples per year. DHPLC had the advantages of increased sensitivity and reduced labour costs when compared with more traditional approaches to exon screening but, unless expensive DHPLC equipment is being efficiently utilised for a very high proportion of the time available, overall costs are slightly higher.

Chromatography, High Pressure Liquid↗